direct pharmacological targeting of a mitochondrial ion ... et al cc.pdf · direct pharmacological...

Article

Direct Pharmacological Ta
rgeting of a MitochondrialIon Channel Selectively Kills Tumor Cells In Vivo
Graphical Abstract

Highlights

d Inhibition of a mitochondrial K+ channel (mitoKv1.3) alters

mitochondrial function

d Two mitochondria-targeted mitoKv1.3 inhibitors induce

death of chemoresistant cells

d The inhibitors reduce tumor size of melanoma and pancreatic

adenocarcinoma in vivo

d Immune and cardiac functions are preserved upon

application of mitoKv1.3 blockers

Leanza et al., 2017, Cancer Cell 31, 516–531April 10, 2017 ª 2017 Elsevier Inc.http://dx.doi.org/10.1016/j.ccell.2017.03.003

Authors

Luigi Leanza, Matteo Romio,

Katrin Anne Becker, ..., Erich Gulbins,

Cristina Paradisi, Ildiko Szabo

[email protected] (E.G.),[email protected] (C.P.),[email protected] (I.S.)

In Brief

Leanza et al. show that two inhibitors that

selectively target the mitochondrial

potassium channel Kv1.3, which is often

overexpressed in malignant cells, alter

mitochondrial function, leading to ROS-

mediated death of malignant cells in vitro

and in vivo without overt effect on

normal cells.

Cancer Cell

Article

Direct Pharmacological Targetingof a Mitochondrial Ion Channel SelectivelyKills Tumor Cells In VivoLuigi Leanza,1,9 Matteo Romio,2,9 Katrin Anne Becker,3 Michele Azzolini,4,5 Livio Trentin,6 Antonella Manago,1

Elisa Venturini,3 Angela Zaccagnino,7 Andrea Mattarei,2 Luca Carraretto,1 Andrea Urbani,1 Stephanie Kadow,3

Lucia Biasutto,4,5 Veronica Martini,6 Filippo Severin,6 Roberta Peruzzo,1 Valentina Trimarco,6 Jan-Hendrik Egberts,7

Charlotte Hauser,7 Andrea Visentin,6 Gianpietro Semenzato,6 Holger Kalthoff,7 Mario Zoratti,4,5 Erich Gulbins,3,8,*Cristina Paradisi,2,* and Ildiko Szabo1,5,10,*1Department of Biology, University of Padova, viale G. Colombo 3,2Department of Chemical Sciences, University of Padova, via F. Marzolo 1

35121 Padova, Italy3Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany4Department of Biomedical Sciences, University of Padova5CNR Institute of Neuroscience

viale G. Colombo 3, 35121 Padova, Italy6Department of Medicine, Hematology and Immunological Branch, University of Padova, and Venetian Institute for Molecular Medicine

(VIMM), via G. Orus 2, 35129 Padova, Italy7Institute for Experimental Cancer Research, Medical Faculty, CAU, Kiel, and Department of Surgery, UKSH, Campus Kiel,

Arnold-Heller-Strasse 3 (Haus 17), 24105 Kiel, Germany8Department of Surgery, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0558, USA9Co-first author10Lead Contact*Correspondence: [email protected] (E.G.), [email protected] (C.P.), [email protected] (I.S.)

http://dx.doi.org/10.1016/j.ccell.2017.03.003

SUMMARY

The potassium channel Kv1.3 is highly expressed in the mitochondria of various cancerous cells. Here weshow that direct inhibition of Kv1.3 using two mitochondria-targeted inhibitors alters mitochondrial functionand leads to reactive oxygen species (ROS)-mediated death of even chemoresistant cells independently ofp53 status. These inhibitors killed 98% of ex vivo primary chronic B-lymphocytic leukemia tumor cells whilesparing healthy B cells. In orthotopic mouse models of melanoma and pancreatic ductal adenocarcinoma,the compounds reduced tumor size bymore than 90%and 60%, respectively, while sparing immune and car-diac functions. Our work provides direct evidence that specific pharmacological targeting of a mitochondrialpotassium channel can lead toROS-mediated selective apoptosis of cancer cells in vivo, without causing sig-nificant side effects.

INTRODUCTION

Mitochondrial functions and bioenergetics have become central

to our understanding of pathological mechanisms as well as for

the development of therapeutic strategies against cancer: direct

pharmacological targeting ofmitochondriamay trigger apoptosis

Significance

Mitochondria are important oncological targets due to their cruthat simultaneously exploits both the high expression of the potcancer cells and the characteristic altered redox state of malignchemoresistant malignant cells by two mitochondria-targetedfects observed in melanoma and pancreatic ductal adenocarcdepression, cardiac toxicity, or histological alteration of healthyadvance in the pharmacological treatment of some high-impa

516 Cancer Cell 31, 516–531, April 10, 2017 ª 2017 Elsevier Inc.

independently of upstream signal transduction elements that are

frequently impaired in cancers (Fulda et al., 2010). Oxidative

phosphorylation linking electron transfer to ATP synthesis re-

quires an electrochemical gradient across the inner mitochon-

drial membrane (IMM). K+ transport modulates the tightness of

coupling between mitochondrial respiration and ATP synthesis

cial role in apoptosis. Our work identifies a therapeutic toolassium channel Kv1.3 in themitochondria of various types ofant cells, thereby leading to the selective elimination of evenKv1.3 inhibitors. Importantly, the strong tumor-reducing ef-inoma preclinical models are not accompanied by immunetissues. These findings thus offer the perspective of amajor

ct, poor-prognosis cancers.

and contributes to the regulation of matrix volume, in addition to

influencing the mitochondrial membrane potential (DJm) and

DpH, calcium transport, reactive oxygen species (ROS) produc-

tion, and mitochondrial dynamics (Szabo and Zoratti, 2014).

Kv1.3 is expressed inmany organs (Comes et al., 2013), partic-

ularly in the CNS and in immune cells where it regulates prolifer-

ation (Cahalan and Chandy, 2009). Several types of cancer cells

also produce high levels of the protein (Comes et al., 2013; Ar-

cangeli et al., 2009), including melanoma, leukemia, and pancre-

atic tumors. High expression of K+ channels in the plasma mem-

brane (PM)might promote tumor cell proliferation, migration, and

metastasis (Pardo and Stuhmer, 2014). Inhibition of PM Kv1.3 by

membrane-impermeant toxins leads to decreased proliferation

and a slight reduction in tumor volume (Jang et al., 2011; Leanza

et al., 2012).

Kv1.3 has been shown to be expressed and active in both the

PM and the IMM (mitoKv1.3) of lymphocytes (Szabo et al., 2005),

hippocampal neurons (Bednarczyk et al., 2010), and various tu-

mor cells (Gulbins et al., 2010; Leanza et al., 2012). While PM

Kv1.3 is required for cell proliferation, mitoKv1.3 participates in

apoptosis. A physical interaction between Bax and mitoKv1.3

has been demonstrated in apoptotic cells, leading to inhibition

of Kv1.3 activity at nanomolar concentrations of Bax (Szabo

et al., 2008, 2011). The interaction triggers apoptotic events,

including membrane potential changes (i.e., hyperpolarization

followed by depolarization because of permeability transition

onset), ROS production, and cytochrome c release.

The most potent toxin inhibitors of Kv1.3 are margatoxin and

ShK, which occlude the channel (Beraud and Chandy, 2011).

Small organic inhibitors were also discovered, which act by bind-

ing in the inner pore or interfacing between Kv1.3 subunits (Zimin

et al., 2010). Differently from peptide inhibitors, they permeate

biomembranes. Among them is Psora-4 (Figure 1A), a 5-phenyl-

alkoxypsoralen (Vennekamp et al., 2004), and its derivative

PAP-1 (Figure 1A), which is 23- to 125-fold selective for Kv1.3

over other Kv1 channels and shows a >1,000-fold lower affinity

for HERG (Kv11.1) and other channels (Schmitz et al., 2005),

and has been proposed for use against autoimmune diseases

(Beeton et al., 2006). Clofazimine is another membrane-perme-

ant Kv1.3 inhibitor, which affects tumor size in vivo (Leanza

et al., 2012). However, recent in vitro and in silico studies suggest

the possibility of additional cytotoxicity mechanisms (Koval

et al., 2014; Patil, 2013).

RESULTS

Synthesis and Stability in Blood of Psoralen DerivativesHere, we report the development of two psoralen derivatives that

accumulate in negatively chargedmitochondria (Dcm=�180mV)

due to the presence of a lipophilic, positively charged triphenyl-

phosphonium group (TPP+) (Smith et al., 2011; Yan et al., 2016)

and thereby trigger apoptosis. We synthesized two molecules

from the natural compound bergapten (Figure S1A and STAR

Methods), i.e., PAPTP and PCARBTP (Figure 1A), in which the

TPP+-containing chain is linked to the molecule by a chemically

stable C-C bond (PAPTP) or to the PAP-1 core via a carbamic

ester bond O-C(O)-N (PCARBTP). As expected (Smith et al.,

2011), the TPP+ moiety efficiently drives rapid uptake of the

compounds into isolated mitochondria (Figures S1B and S1C).

PCARBTP is liable to undergohydrolysis in physiological settings,

releasing PAPOH, which differs fromPAP-1 only for the presence

of a hydroxyl group (Figure 1A). Upon incubation in fresh mouse

blood at 37�C, PAPTP was quantitatively recovered unaltered af-

ter 4 hr (data not shown)while PCARBTPunderwent complete hy-

drolysis toPAPOHwithin 1 hr (Figure 1B). Thus, PCARBTP indeed

represents a ‘‘prodrug’’ of PAPOH in which the hydroxyl group

has been reversibly protected. PAPOH is one of themajormetab-

olitesofPAP-1 in vivo and inhibitsKv1.3withahalf-maximal inhib-

itory concentration (IC50) of 6.5 nM (Hao et al., 2011).

Mitochondriotropic PAP-1 Derivatives Induce ApoptosisIn Vitro Exclusively in Cancer Cells Expressing Kv1.3To verify the specificity toward Kv1.3 of the compounds

described here, we performed experiments using Jurkat T cells

in which Kv1.3 expression was abolished using small interfering

RNA (siRNA). Both derivatives caused cell death onlywhenKv1.3

was expressed (Figures 1C and S1D), similarly to the intrinsic

apoptosis inducer staurosporine (Szabo et al., 2008). In some

experimental settings, inhibitors of the multi-drug resistance

pumps (MDRi) were used to prevent active export of the com-

pounds from the cells. These data suggest that the PAP-1 deriv-

atives efficiently block the Kv1.3 channel in intact cells even

without MDRi.

The IC50 of PAPTP for the Kv1.3 current measured in Jurkat

T lymphocytes by patch-clamp experiments was higher than

that of PAP-1 by a factor of 15 (Figure 1D). The IC50 of PCARBTP

was substantially increased with respect to PAP-1 but remained

selective for Kv1.3, since it did not inhibit Kv1.1 and Kv1.5, two

related members of the Kv family, up to 30 mM (Figure S1E). In

cells PCARBTP, at least in part, are degraded to PAPOH upon

accumulation in mitochondria, which reduces cell survival simi-

larly to PAP-1 + MDRi (Figures 2A and 2B).

PAPTP, PCARBTP, and PAPOH + MDRi reduced cell viability

and efficiently killed B16F10 cells, even at 1 mM concentration

(Figures 2A and 2B). In contrast, the membrane-impermeant

Kv1.3 inhibitor margatoxin (Figure 2B) was without effect. Inhib-

itor effectiveness correlated with Kv1.3 expression (Figures 2B

and S2A). Primary human fibroblasts, expressing low levels of

Kv1.3, were resistant (Figure S2B).

PCARBTP and PAPTP Kill Only Pathological but NotHealthy Ex Vivo Primary Human B CellsThe inhibitors were then tested on primary pathological CD19+/

CD5+ B cells isolated from patients with chronic lymphocytic leu-

kemia (B-CLL), previously shown to expressmitochondrial Kv1.3

(Leanza et al., 2013), and on B lymphocytes obtained from

healthy volunteers. The PAP-1 derivatives induced more than

50% apoptosis already at 1 mM concentration, even in the

absence of MDRi (Figure 2C) and independently both from the

expression of ZAP70 or CD38 and of somatic hypermutation or

mutation of p53 (data not shown), while PAP-1 required a dose

of 20 mM + MDRi. PAPTP at 10 mM even triggered apoptosis in

up to 85% of B-CLL cells when cultured together with mesen-

chymal stromal cells (MSCs) that protect leukemia cells from

apoptosis induced by chemotherapeutic compounds (Pillozzi

et al., 2011) (Figure 2D). MSCs that lack Kv1.3 were not affected

(Figures S2C and S2D). Cells with higher expression of Kv1.3

showed more apoptosis upon treatment, suggesting that

Cancer Cell 31, 516–531, April 10, 2017 517

C

A

0

20

40

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untre

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PAP

TP 1

μM

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TP 1

0 μM

MD

Ri+

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TP 1

μM

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TP 1

0 μM

PC

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10 μM

PC

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PC

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1 μ

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MD

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10 μM

Sta

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% o

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l dea

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ScramblesiRNA Kv1.3Kv1.3

GAPDH

D

100 ms

200 pA

1E-4 1E-3 0,01 0,1 1 10 100 1000 10000 1000000,0

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peak

curr

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mV

Concentration (nM)

B

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0 50 100 150 200

% to

tal

Time (min)

PAPOHPCARBTP

Figure 1. PAP-1 Derivatives Induce Apoptosis by Acting on Kv1.3

(A) Structure of psoralen and psoralen derivatives PAP-1, PAPOH, Psora-4, bergapten, PAPTP, and PCARBTP. Hydrolysis of PCARBTP yields the active

molecule PAPOH.

(B) Hydrolysis of PCARBTP in mouse blood, as determined by high-performance liquid chromatography (HPLC) analysis. Values are percentage of the initial

PCARBTP concentration ± SD (error bars are smaller than symbols) (n = 3).

(C) Human Jurkat leukemic T cells were transfected with either control siRNA (scramble) or siRNA against Kv1.3. Inset: western blot for Kv1.3 (50 mg protein/lane).

Forty-eight hours following transfection, the cells were treated as indicated for 24 hr in the presence or absence of MDRi (CSH 4 mM). Cell death was assessed

by annexin-V staining and flow-cytometry analysis. Staurosporine (1 mM) was used as positive control. Shown are mean values of percentage of dead cells ± SD

(n = 5). Differences between scramble and Kv1.3 siRNA-transfected cells are statistically significant for all inhibitor-treated samples (p < 0.05).

(D) Normalized peak currents measured at +70 mV at the indicated concentrations of PAPTP. Mean values ± SD (n = 4–11). Curve fitting using the Origin Program

set yielded an IC50 value of 31 nM. Jurkat cells were kept at �50 mV pipette potential in the whole-cell configuration and peak current was elicited by voltage

pulses to depolarizing voltage (+70 mV) at 45-s intervals. Inset: representative whole-cell current traces elicited by stepping the voltage from�50 mV (holding) to

values ranging from �90 to +90 mV (in 20-mV steps) under control conditions (upper part) and following addition of 100 nM PAPTP.

See also Figure S1.

expression of Kv1.3 sensitizes B-CLL cells to Kv1.3 inhibitors

(Figures S2D and S2E).

PAP-1 Derivatives Directly and Efficiently AffectMitochondrial FunctionWe investigated how the PAP-1 derivatives affected mitochon-

drial function in intact cells. Block of the IMM Kv1.3 is expected

to lead to an initial hyperpolarization followed by ROS release

and a secondary depolarization and swelling due to the opening

518 Cancer Cell 31, 516–531, April 10, 2017

of the PTP (Szabo et al., 2008). PAPTP, but neither margatoxin

nor partial or complete depolarization of the PM, induced a rapid

mitochondrial swelling and fragmentation of the mitochondrial

network in intact B16F10 cells, but only in cells expressing

Kv1.3 (Figures 3A, S3A, and S3B). Primary human fibroblasts,

expressing low levels of Kv1.3, did not respond (Figure 3B). Early

events leading to swelling consisted in mitochondrial hyperpo-

larization (Figure 3C), in accordance with the block of depolariz-

ing Kv1.3-mediated potassium influx into the matrix by the

A

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PAP-1 PAPOH PAPTP PCARBTP

***

*** ****** ***

*** ***

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Concentration (μM)

PAP-1PAPTPPCARBTP

Healthy B cells B-CLL cells

*** ***

***

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***

PCARBTP 1 μM

PCARBTP 10 μM

PAPTP 1 μM

PAPTP 10 μM

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Kv1.3GAPDH

MDRi+PAPOH

μM 20 10PCARBTP

1 1 10 10

ScramblesiRNA Kv1.3

WT

*** *** ***

C MgTx10

Figure 2. PAP-1 Derivatives Efficiently Kill B16F10 Melanoma Cells and Pathological B-CLL Cells, While Leaving B Cells from Healthy Sub-

jects Unaffected

(A) Cell viability of B16F10 cells following treatment for 24 hr was measured (MTT assay). Values are reported as mean percentage of viable cells normalized with

respect to untreated cells (n = 15); *p < 0.05, ***p < 0.01.

(B) Cell death assayed by annexin-V binding upon 24-hr treatment with the indicated compounds on B16F10 cells, transfected with Alexa 555-labeled siRNA

targeting Kv1.3 or control siRNA (scramble) (n = 4). Percentage of apoptotic cells was determined by counting fluorescein isothiocyanate (FITC)-labeled annexin-

V-positive cells versus total number by microscopic analysis. ***p < 0.01.

(C) Killing of B cells derived from CLL patients (n = 19) and from healthy donors (n = 6) by PAP-1 derivatives, with or without MDRi, after treatment for 24 hr.

***p < 0.01.

(D) Cell death of B-CLL cells co-cultured with mesenchymal stromal cells (MSCs) upon treatment with the indicated compounds (n = 5). The B-CLL cells were

co-cultured with MSC for 6 days and then treated with the compounds for 24 hr. In each panel, error bars represent ±SD. *p < 0.05, ***: p < 0.01.

See also Figure S2.

inhibitors, followed by an increased ROS level (Figures 3D and

3E), activation of PTP, and dissipation of Dcm (Figures 3C and

3F). Similar results were obtained in B-CLL cells within 30 min

following addition of the compounds (Figures S3C and S3D).

The loss of Dcm was not a consequence of the accumulation

of the positively charged TPP+ moiety, since addition of 10 mM

TPP+ alone did not cause depolarization (Figure S4A). Cyclo-

sporine A, a molecule widely used to prevent opening of the

PTP (Bernardi et al., 2015), prevented inhibitor-induced loss of

Dcm (Figure 4A) but not hyperpolarization or ROS release (Fig-

ure S4B), indicating that Kv1.3 inhibitor-induced mitochondrial

changes involve permeability transition. Increased ROS level

and PTP-related depolarization (but not the initial hyperpolar-

ization) was abolished by pretreatment of the cells with the anti-

oxidant N-acetylcysteine (NAC) (Figure S4C), indicating that

hyperpolarization-linked ROS release triggered PTP-mediated

depolarization. Partial or complete depolarization of the PM did

not correlate with loss of mitochondrial membrane potential or

ROS release (Figures S4D and S4E). Direct mitochondrial action

of the inhibitors was further indicated by relative matrix acidifica-

tion (Figure 4B). Similar results were obtained with PCARBTP

(Figures 3, 4, S3, and S4), although with slower kinetics (e.g.,

swelling was visible after 15 min following addition of the inhibi-

tor). Finally, these mitochondrial changes were associated with

a hallmark of apoptosis, namely cytochrome c release (Fig-

ure S4F). While mitochondrial function and morphology was


A

30 min 8 min

siRNA scramble

30 min 30 min

untreated PAPTP 10 μM PCARBTP 10 μM

siRNA Kv1.3

untreated PAPTP 10 μM PCARBTP 10 μM

30 min 15 min

EAntimycin A 1 μMuntreated PAPTP 10 μM PCARBTP 10 μM

30 min 10 min 20 min 10 min

Mito

sox

Mito

track

er g

reen

BValinomycin 10 μM untreated PAPTP 10 μM PCARBTP 10 μM

MDRi+PAP-1 20 μM

DC

FFCCP 2 μMuntreated PAPTP 10 μM PCARBTP 10 μM

30 min 10 min 20 min 10 min

Mitotracker green

TMR

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0 5 10 15 20 25 30

Time (min)35

FCCP 2 μM

compound compound

untreatedMgTx 1 μM

PAPTP 1 μMPCARBTP 1 μM

0 5 10 15 20 25 30

Time (min)35 0 5 10 15 20 25 30

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Antimycin A 1 μM

compound

(legend on next page)


severely and rapidly affected (Figure 4C), nomorphological alter-

ations were induced by treatment with the inhibitors in other

organelles as assessed by transmission electron microscopy

(Figure S4G), further indicating that the observed apoptosis

was indeed intimately linked to PAPTP/PCARBTP-inducedmito-

chondrial malfunction and not to, for example, ER stress (e.g.,

Park et al., 2015).

In agreement with the above results, the inhibitors significantly

reduced maximal respiration by adherent B16F10 cells (Figures

4D and S4H), consistent with opening of PTP. Both mitochon-

driotropic derivatives, tested at low concentration to avoid cell

death during analysis, analogously to 20 mM PAP-1 + MDRi,

significantly reduced the respiratory response to the uncoupler

FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone)

when added either before (Figure S4H) or after (Figure 4D) oligo-

mycin, which blocks the ATP synthase. The cells responded

to antimycin A, an inhibitor of complex III, indicating that the

changes we observed were related to respiratory chain func-

tion. The effect of PAPTP was more pronounced than that of

PCARBTP, likely due to a higher concentration of the active

Kv1.3-inhibiting molecule at the mitochondria. As a control, we

excluded that the applied compounds directly affected the func-

tion of the respiratory chain at the level of themajor ROS produc-

tion sites, i.e., complexes I and III, and of ATP synthesis, i.e.,

complex V (Figure S4I). ATP production by mitochondria was

completely abolished within 6 hr as assessed by measuring the

ATP content in the presence of 2-deoxyglucose, which inhibits

glycolysis (Figure 4E).

These results demonstrate that the aforementionedmitochon-

driotropic Kv1.3 inhibitors intimately affect mitochondrial func-

tion in intact cells, with kinetics compatible with their action on

mitochondrial channels mediating fast ion fluxes.

In Vivo Effects in an Animal Model of MelanomaTo test the compounds in vivo, we employed an orthotopic

mouse B16F10 melanoma model and treated mice with the de-

rivatives PCARBTP and PAPTP or with PAP-1 at post-injection

days 5, 7, 9, and 11. Both mitochondriotropic derivatives ex-

hibited a drastic effect on tumor volume. PAPTP was especially

Figure 3. Direct Effects of PAP-1 Derivatives PAPTP and PCARBTP on

(A) Mitochondrial network of B16F10 cells visualized usingMito Tracker Green (50

(scramble), and following treatment with PAPTP or PCARBTPmitochondrial morp

independent experiments. Please note swollenmitochondria upon addition of PAP

0.5 mm (enlarged images, lower row).

(B) As in (A) but human fibroblastswere used. Valinomycin, known to causemitoch

time point 0 (upper images) and after 30 min (lower row). Images are representa

(C) Mitochondrial membrane potential was followed using 5 nM TMRM on B16F1

off so as to allow further uptake following hyperpolarization. For the first 2 min

minimize the danger of bleaching. Nigericin (potassium/proton exchanger) was us

the presence of PAPTP and PCARBTP. Results are shown as mean ± SD fro

analyzed.

(D) As in (C) but using MitoSOX, the signal of which correlates with mitochondrial

following their addition (i.e., when hyperpolarization takes place) while a stronger R

10 to 25 min after addition of the compounds), i.e., when depolarization was meas

(E) Mitochondrial ROS production was assayed by MitoSOX; antimycin A was us

Mitochondria were marked with Mito Tracker Green (upper row).

(F) Mitochondrial membrane potential changes upon treatment with the indicated

points. FCCP induced complete depolarization and was used as positive contro

bars, 25 mm.

See also Figure S3.

effective already when used alone at 5 nmol/g body weight (gbw)

(Figure 5A). PAPTP, when co-administered with cisplatin, was

able to improve the effect of cisplatin, leading to a reduction of

tumor volume by more than 90% (Figure 5B).

To prove in vivo the hypothesis that Kv1.3 inhibitors selectively

kill cancer cells due to their ability to induce an excessive ROS

production, thus passing a critical threshold in cancerous but

not in normal cells (Gorrini et al., 2013; Ralph et al., 2010; Sab-

harwal and Schumacker, 2014), we performed in vivo experi-

ments on animals that had been pretreated with NAC (Qin

et al., 2015). In vitro experiments indicated that membrane-per-

meant superoxide dismutase or catalase, NAC, or mitochondria-

targeted ROS scavenger MitoTEMPO prevented the apoptosis-

inducing effect of PAPTP and PCARBTP (Figure S5A). NAC did

not chemically interact with the compounds used in this study

(Figure S5B). Although NAC may have multiple effects, the

finding that the tumor-reducing effect of Kv1.3 inhibitors was

abolished by ROS scavenging points to a link between Kv1.3

inhibition, increased ROS level, and apoptosis also in vivo

(Figure 5C).

Most chemotherapeutic agents currently in use affect

fast-proliferating normal cells, thereby inducing a substantial

decrease of the immune system cell number. Importantly, our

derivatives had no impact on the number (Figure 5D) or the rela-

tive composition of the immune cell repertoire (Figure 5E) in

thymus, spleen, inguinal lymph nodes, and blood. Cisplatin at

the lower concentration used here had no effect on immune cells

in the spleen (Figure S5C), but had also only a minor effect on the

tumor, showing that the PAP-1 derivatives are clearly superior at

a dose that is not toxic. The inhibitors caused a maximum 2-fold

decrease in the cell number for T lymphocytes, macrophages,

and neutrophils in the tumor tissue itself in comparison with

that found in untreated mice, while B lymphocyte number was

not altered at all (Figure S5D). PAPTP did not affect mtDNA sta-

bility in human primary fibroblasts (Franzolin et al., 2015): mtDNA

copy number per nuclear genome was 518.47 ± 44.79 for un-

treated primary human fibroblasts and 519.37 ± 17.55 for those

cultured for 6 days in the presence of a sublethal dose of PAPTP

(n = 3, mean ± SD).

Mitochondria

0 nM). Cells were transfectedwith either siRNA targeting Kv1.3 or control siRNA

hologywas assessed by confocal microscopy. Images are representative of six

TP and PCARBTP to Kv1.3-expressing cells. Scale bars, 5 mm (upper row) and

ondrial swelling, was used as positive control. Morphologywas assessed at the

tive of three independent experiments. Scale bars, 15 mm.

0 cells for the indicated time. In these experiments the probe was not washed

following addition, images were acquired every 20 s and then every 2 min to

ed as positive control. Depolarization occurred without addition of uncoupler in

m three biological replicates where fluorescence of 15 cells/condition were

ROS production. Note that the compounds increased ROS level within 10 min

OSproductionwas observed (up to 3.5-fold increase) in a later time frame (from

ured. Mean normalized values ± SD from three biological replicates are shown.

ed as positive control (n = 3). Scale bars, 25 mm.

compounds as assayed by staining with TMRM (lower row) at the reported time

l (n = 3). Mitochondria were marked by Mito Tracker Green (upper row). Scale


B

0

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PAP-1

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io (4

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compoundsodiumacetate

Time (min)

D

Oligomycin

Compound

FCCP

Antimycin

0

40

80

120

160

200

0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119

Time (min) Time (min)

untreatedPAP-1MDRi+PAP-1

untreatedPCARBTPPAPTP

E

0

20

40

60

80

100

untreated oligo 10 μM

% o

f ATP

leve

lsM

ouse

mel

anom

a B

16F1

0 ce

lls

MDRi +PAP-1 20 μM

PAPTP PCARBTP

DMEM 5.5 mM 2-DG

1 μM 10 μM 10 μM1 μM

***

***

*** *** ***

**

A untreated + CsA

PAPTP 10 μM + CsA

PCARBTP 10 μM + CsA

0 m

in30

min

10 m

in+

FCC

P

0 m

in10

min

0 m

in20

min

C untreated

MgTx 1 μM

PAPTP 1 μM PCARBTP 1 μM

PAPTP 10 μM PCARBTP 10 μM

1 μm 1 μm 1 μm

1 μm1 μm1 μm

4%

OC

R/1

.5x1

0 B

16F1

0 ce

lls Oligomycin

Compound

FCCP Antimycin

(legend on next page)


Finally, pharmacokinetic propertieswereaddressed. Thecom-

pounds were found to accumulate in the liver at 2 hr after treat-

ment (Figures 5F and 5G) in contrast to PAP-1, which was found

prevalently in the kidney at this time point (Figure 5H). PCARBTP

as an intactmolecule could not be detected. Instead, the product

of hydrolysis of its carbamoyl link, PAPOH, was present. All com-

pounds were rapidly eliminated from the organism, decreasing

to low-nanomolar concentrations after 8 hr from administra-

tion. The PAP-1 derivatives were not detectable in the brain

and heart in our experimental setting, but were present in the tu-

mor tissue at slightly higher concentration than in the blood and

other tissues (except liver) (Figures 5F and 5G).

Importantly, none of the derivatives affected healthy tissues as

assessed by immunohistochemistry (Figure S6A). TUNEL assay

showed the lack of inhibitor-induced apoptosis in healthy tissues

(Figure 6A). In contrast, tumor tissue was characterized by the

presence of apoptotic cells (Figure S6B). PAP-1, besides inhibit-

ing Kv1.3, decreases the activity of Kv1.5 as well, although with a

23-fold higher IC50 (Schmitz et al., 2005). Kv1.5 carries the ultra-

rapid delayed rectifier current (IKur) in the heart; therefore, we

excluded that the PAP-1 derivatives affect cardiac function (Fig-

ures 6B and S6C). The unchanged electrocardiogram indicates

that none of the Kv channels (Kv1.5, Kv11.1), which are also

important for the functionality of the heart, are affected by the in-

hibitors at the concentrations used. Effector memory T cells

(TEM) (CD3+/CCR7�), important against viral infection and also

for immune surveillance in tumors (e.g., Chimote et al., 2017),

are known to express high levels of Kv1.3 (Cahalan and Chandy,

2009) even in their mitochondria (Leanza et al., 2013). Primary

ex vivo TEM cells either from B-CLL patients (Figures 6C and

6D) or from healthy subjects (Figures S6D–S6F) were resistant

to treatment with PAP-1 derivatives. Pathological B-CLL cells

from the same individuals underwent apoptosis (Figure 6D).

The resistance of TEM cells can be attributed to a significantly

lower basal ROS production than in B-CLL cells (Figures S6E

and S3D). Indeed, synergy between ROS level and Kv1.3 inhibi-

tion was further indicated by the finding that in TEM cells applica-

tion of a sublethal concentration of a mitochondria-targeted

pro-oxidant (Q7BTPI) (Sassi et al., 2012) led to sensitization of

these cells to PAPTP (Figures S6E and S6F). Instead, treatment

with Q7BTPI and PAPTP did not induce apoptosis in leukemic

K562 cells that do not express Kv1.3 (Figures S6G and S6H).

In Vivo Effects in an Animal Model of Human PancreaticDuctal AdenocarcinomaTo further extend the possible therapeutic potential of the

aforementioned compounds, we tested the in vivo effect on

Figure 4. PAPTP and PCARBTP Decrease Respiration(A)Mitochondrial membrane potential of B16F10 cells pretreated for 1 hr with cyclo

added as a control in the same experiment (n = 3). Scale bars, 25 mm.

(B)Measurement of variation ofmitochondrialmatrix pH inB16F10cells expressing

the 535-nm fluorescence emission ratio after alternate excitation at 405 and 488 n

experiments. A decrease in the ratio indicates acidification. PAP-1: 20 mM+MDRi;

(C) Representative transmission electron microscopy images of B16F10 cells

mitochondria, as indicated by arrowheads, with profoundly altered ultrastructure

(D) Oxygen consumption rate (OCR) of B16F10 cells measured in the presenc

experiments are shown. PAP-1: 20 mM; MDRi (CSH 4 mM); PAPTP and PCARBT

(E) ATP content of B16F10 cells 6 hr after treatment in the presence of 2-deoxyg

See also Figure S4.

pancreatic ductal adenocarcinoma (PDAC) using an orthotopic

xenograft model, which more closely recapitulates the human

disease (Herreros-Villanueva et al., 2012). PDAC is one of the

most aggressive types of tumors, being the fourth leading

cause of cancer mortality.

Various human PDAC lines express Kv1.3 (Zaccagnino et al.,

2016). Expression of Kv1.3 in Colo357 cells was confirmed by

western blot (Figure 7A). Both inhibitors efficiently acted on

Colo357 cells (Figures 7A and 7B) and also killed more than

90% of five other PDAC lines, all characterized by p53mutations

and chemoresistance (Figures 7C and 7D) (Sipos et al., 2003).

The membrane-impermeant Kv1.3 inhibitors margatoxin and

ShK did not affect survival (Figure S7A). The effects of PAPTP

and PCARBTP correlated with Kv1.3 expression in Colo357

and BxPC-3 cells as assessed using siRNA against Kv1.3 (Fig-

ures 7E and S7B–S7D). A correlation was found between sensi-

tivity of other PDAC lines to the inhibitors and Kv1.3 expression

(Figure S7E). Non-tumoral pancreatic duct epithelial cells and hu-

man umbilical vein endothelial cells were largely resistant to the

treatment (Figure 7D), further indicating that cytotoxicity con-

cerns only cancerous cells. Cell cycle in Colo357 was not altered

by low, sublethal doses of the compounds (not shown). Hypoxia,

typically found in solid tumors andalso inPDAC, did not affect the

apoptosis-inducing ability of the inhibitors.Metabolic reprogram-

ming frommitochondrial aerobic respiration to aerobic glycolysis

is a hallmark of many types of cancer. Galactose is not used effi-

ciently as glycolytic substrate; therefore, the cells need to switch

their metabolism to produce all of their energy from oxidative

phosphorylation for survival. The switch of the medium did not

change in vitro efficacy of the compounds (Figures 7A and 7B).

We then treated severe combined immunodeficient (SCID)

beige mice bearing orthotopically xenotransplanted human

pancreatic cancer Colo357 cells (Zaccagnino et al., 2016). A sta-

tistically significant reduction of tumor weight occurred with both

compounds, in particular by more than 60% in the PCARBTP-

treated mice (Figure 7F).

On the basis of the above experiments, we propose the work-

ingmodel shown in Figure 8A for the PAP-1 derivatives regarding

the mitochondrial events, while Figure 8B illustrates that Kv1.3

inhibitor-induced cell death depends on both the level of Kv1.3

expression and the basal redox state.

DISCUSSION

Our data indicate that direct inhibition of a well-defined target,

mitoKv1.3, by specific, mitochondria-targeted inhibitors is a

promising strategy against cancers expressing this channel.

sporin A (CsA; 4 mM) and then treatedwith PAP-1 derivatives. FCCP (2 mM)was

mito-SypHer (Manago et al., 2015b).Changes in pHcorrespond to variations in

m. Results are expressed as mean 488/405-nm ratios ± SEM of three different

PAPTP and PCARBTP: 10 mM.Na-acetate (3mM)was used as positive control.

fixed after 20 min of incubation with the indicated compounds. Please note

and disorganized cristae in the presence of PAPTP/PCARBTP.

e of the indicated compounds. Mean values ± SD from three representative

P: 3 mM; oligomycin: 1 mg/mL; FCCP: 300 nM; antimycin: 1 mM.

lucose (n = 4, mean ± SD). **p < 0.01, ***p < 0.001.


A B C

0

10

20

30

40

50

60

untreated PAPTP PCARBTP

% o

f cel

ls

Spleen

untreated PAPTP PCARBTP0

10

20

30

40

50

60

% o

f cel

ls

iLN

0

10

20

30

40

50

60


% o

f cel

ls

Blood

+ +CD4 /CD3+ +CD8 /CD3

+ +CD19 /MCHII+ +CD11b /F4/80

+ +CD4 /CD3+ +CD8 /CD3

+ +CD19 /MCHII+ +CD11b /F4/80

+ +CD4 /CD3+ +CD8 /CD3

+ +CD19 /MCHII+ +CD11b /F4/80

F G H

untre

ated

PAP

TP

PC

AR

BTP

untre

ated

PAP

TP

PC

AR

BTP

untre

ated

P AP

TP

PC

AR

BTP

untre

ated

PAP

TP

PC

AR

BTP

iLNsSpleenThymus Blood

0

2

4

6

8

7TO

TAL

cells

(x 1

0)

0

1

2

3

4

1

5TO

TAL cells (x 10

)

D

*** ***

***

***

0

1000

2000

3000

0

1000

2000

3000

0

1000

2000

3000

3Tu

mor

vol

ume

(mm

)

untreated PAP-1 PAPTP PCARBTP untreated Cisplatin Cisplatin + PAPTP

NAC NAC + PAP-1

NAC + PAPTP

NAC + PCARBTP

3Tu

mor

vol

ume

(mm

) 3Tu

mor

vol

ume

(mm

)

E

0

20

40

60

80

100


% o

f cel

ls

Thymus- -CD4 /CD8+ +CD4 /CD8

+ -CD4 /CD8- +CD4 /CD8

50 2 hr4 hr8 hr

nmol

/ g

tissu

e

Brain

40

30

20

10

0

50

nmol

/ g

tissu

e

40

30

20

10

0

50

nmol

/ g

tissu

e

40

30

20

10

0Heart Liver Spleen Kidney Blood Brain Heart Liver Spleen Kidney Blood Brain Heart Liver Spleen Kidney Blood

2 hr4 hr8 hr

2 hr4 hr8 hr

untreated PAPTP

Tumor Tumor

Figure 5. In Vivo Tumor-Reducing Effects in an Orthotopic Melanoma Model

(A) Tumor volume in mice treated with PAP-1 (20 nmol/gbw), PAPTP (5 nmol/gbw), or PCARBTP (10 nmol/gbw) (n = 8 each) and in untreated mice (n = 16). The

compounds were injected intraperitoneally on days 5, 7, 9, and 11 after tumor cell injection and tumor volume was assessed 16 days after tumor cell inoculation

(***p < 0.001).

(B) Tumor volume in mice treated with cisplatin (1.7 nmol/gbw) alone (n = 9) or in combination with PAPTP (n = 4; ***p < 0.001).

(C) Mice were treated with the antioxidant NAC (N-acetylcysteine, 0.7 mg/g mouse) 1 hr before every injection of the compounds (n = 4 each). In (A) to (C) box plots

represent 25th and 75th percentiles, with midlines indicating the median values and points within the boxes indicating the mean values. Whiskers extend to the

lowest/highest values of the data sample.

(D) Lymphocyte and macrophage subpopulations were measured by flow cytometry in thymus, spleen, inguinal lymph nodes (ILN), and blood frommice treated

with PAP-1 derivatives (mean ± SD, n = 4 each) as specified for (A).

(E) Different immune cell subpopulations were identified by flow cytometry using antibodies against the indicated marker antigens (mean ± SD, n = 3 each).

(F–H) PAPTP (F), PAPOH (G), and PAP-1 (H) weremeasured in the indicated organs 2, 4, and 8 hr after intraperitoneal injection. In tumor tissue the concentration of

the inhibitors was determined at 2 and 4 hr. PCARBTP was detectable in the form of its hydrolytic product, PAPOH. HPLC analysis was as reported in STAR

Methods (n = 3, mean ± SD).

See also Figure S5.


0.000

0.075

0.150

0.225

0.300

RR (s) PR (s) QRS (s) QTc (s)

Vehicle + DMSO PAPTPPAP-1 PCARBTP

Inte

rval

dur

atio

n (s

)

B

D

0

20

40

60

80

100

120

untreated

% o

f cel

l dea

th

PAPTP 1 μM

+ -Healthy T from B-CLL patients (CD3 /CCR7 )+ +Healthy T from B-CLL patients (CD3 /CCR7 )

+ +B-CLL (CD19 /CD5 )

PAPTP 10 μM

PCARBTP 1 μM

PCARBTP 10 μM

C


BR

AIN

HE

AR

TLIV

ER

DNAse

SP

LEE

NK

IDN

EY

A

+ -CD3 /CCR7

+ +CD3 /CCR7- +CD3 /CCR7

- -CD3 /CCR7

Figure 6. PAP-1 Derivatives Do Not Induce

Apoptosis in Healthy Organs, Lack Cardiotoxicity,

and Do Not Kill Human Primary TEM

(A) TUNEL assay on indicated organ slides. DNase treat-

ment was used as positive control. Shown are represen-

tative images of three similar slides indicating lack of

toxicity in different tissues including heart, liver, and brain.

Scale bars, 100 mm.

(B) Effect of the indicated molecules in comparison

with vehicle during electrocardiogram recording in anes-

thetized mice (isoflurane). RR and PR intervals, as well as

QRS duration and corrected QT interval (QTc), were taken

into account. All values are expressed in seconds and

have been obtained from a 30-min recording after injection

(n = 3 ± SD). No significant variation was found among the

four groups (two-way ANOVA).

(C) Subpopulations of isolated residual T lymphocytes

from B-CLL patients identified by fluorescence-activated

cell sorting (FACS) analysis using FITC-labeled anti-CCR7

and PE/Cy7-conjugated anti-CD3 antibodies.

(D) Mean values ± SD of dead CCR7- or CCR7+ T cells with

respect to the total number of CD3+ T cells, and mean

values ± SD of dead CD5+/CD19+ B-CLL cells with respect

to the total number of CD19+ B cells from the same in-

dividuals 24 hr after treatment (n = 3).

See also Figure S6.


1E-4 1E-3 0,01 0,1 1 100

20

40

60

80

100

%of

cell

sur v

ival

PCARBTP Concentration µM

normal oxygen hypoxic galactose medium

1E-4 1E-3 0,01 0,1 1 100

20

40

60

80

100

%of

surv

ival

PAPTP Concentration µM

normal oxygen hypoxic galactose medium

A

F

**

C

0

10

20

30

40

50

60

70

80

90

100

control MDRi + PAP-1 20 µM

PAPTP 10 µM

% o

f MTT

abs

orba

nce

BxPC-3PANC-1CAPAN-1AsPC-1MiaPaCa-2

E

0

20

40

60

80

100

120

MDRi+PAP-1 20 µM

PAPTP10 µM

PCARBTP10 µM

% o

f cel

l dea

th

Scramble siRNA Kv1.3

untreated

** *** ***

***

Tum

or w

eigh

t (g)

untreated PAPTPPCARBTP0.0

0.2

0.4

0.6

Kv1.3

GAPDH

Jurka

t

Colo35

7

B

D

0

20

40

60

80

100

120

0 1 10 20 MDRi+1 MDRi+10 MDRi+20

% o

f MTT

abs

orba

nce

PCARBTP concentration (µM)

BxPC-3PANC-1AsPC-1MiaPaCa-2

Capan-1

HPDEHuvec

*** *** *** ***

% o

f MTT

abs

orba

nce

% o

f MTT

abs

orba

nce

IC50 = 2 µMIC50 = 3.7 µM

Figure 7. PCARBTP and PAPTP Significantly Reduce Pancreatic Tumor Weight in an Orthotopic PDAC Model

(A) Dose-response curve of cell viability for PAPTP for Colo357 (n = 3, mean ± SD). Inset shows expression of Kv1.3 in Colo357 cells. Whole-cell lysate of Jurkat

lymphocytes was used as a control (50 mg protein/lane).

(B) As in (A), for PCARBTP (n = 3, mean ± SD). In (A) and (B) the effect of the compounds was determined with cells cultured under hypoxic conditions (less than

residual 1% oxygen concentration).

(C and D)MTT assay performed on five PDAC lines treated with PAP-1 (C) or PCARBTP (D) as indicated (n = 12 for each cell line, mean ± SD; ***p < 0.001). HPV16-

E6E7-immortalized human pancreatic duct epithelial cells (HPDE) and human umbilical vein endothelial cells (Huvec) are non-tumoral lines.

(E) Colo357 cells were transfected with siRNA against Kv1.3 or with control siRNA (scramble). Shown are mean values of percentage of dead cells ± SD (n = 4;

**p < 0.01; ***p < 0.001). Percentage of apoptotic cells was determined at the microscope by counting FITC-labeled annexin-positive cells.

(F) Tumor weight of mice treated with the indicated compounds for 20 days (untreated: n = 6; PCARBTP: 10 nmol/gbw, n = 6; PAPTP: 5 nmol/gbw, n = 6) *p < 0.05

(t test). Box plots represent 25th and 75th percentiles, with midlines indicating the median values and points within the boxes indicating themean values. Whiskers

extend to the lowest/highest values of the data sample.

See also Figure S7.


Mito Kv1.3

Mito Kv1.3

PM Kv1.3

PM Kv1.3

MgTx, Shk, PAP-1PAPTP, PCARBTP

PAPTP, PCARBTP (PAPOH)PAP-1

HEALTHY CELL(low basal ROS level)

MgTx, Shk, PAP-1

Inhibition of proliferation

PAPTP, PCARBTP

PAPTP, PCARBTP(PAPOH)

MALIGNANT CELL(high basal ROS level)

Inhibition of proliferation



DEATH

MgTx, Shk, PAP-1 MgTx, Shk,

PAP-1

PAP-1

PAPTP, PCARBTP PAPTP, PCARBTP

ROS

SURVIVAL

RO

S

PAP-1

PAP-1

PCARBTP

PAPTP

Kv1.3+K ROS

PTP

ROSBax

Bax

Cyt c

apoptotic cascade

Outer membrane

Inner membrane

ΔΨ increasem

(hyperpolarization)

ΔΨ dem

(depolarization) crease

PAPTPPAP-OH

A

B

Figure 8. Proposed Mechanism of Action

of the Mitochondriotopic Derivatives in

Healthy Cells Versus Malignant Cells

(A) Kv1.3 inhibition in IMM causes hyperpolar-

ization. Hyperpolarization-induced increase of

ROS level at mitochondria triggers PTP opening as

well as detachment of cytochrome c from the outer

surface of the IMM according to the literature. The

detached cytochrome c is released due to cristae

remodeling and matrix swelling and/or via Bax

oligomers, according to the literature, and triggers

the apoptotic cascade, leading to apoptosis.

Further details are given in the text.

(B) PAPTP and PCARBTP, by rapid accumulation

in the mitochondria, induce cell death by triggering

a series of events via mitoKv1.3 inhibition that

leads to substantially increased ROS level in ma-

lignant cells expressing higher level of Kv1.3 with

respect to healthy cells. The resulting oxidative

stress above a critical threshold selectively kills the

cancer cells, which are characterized by a high

basal ROS level. In contrast to membrane-im-

permeant Kv1.3 inhibitors margatoxin (MgTx) and

ShK and to PAP-1, the compounds described

here act prevalently on the mitochondrial channel.

The active moiety responsible for the action of

PCARBTP is PAPOH, which is released from the

prodrug following its accumulation at mitochon-

dria. In summary, the apoptotic effect of Kv1.3-

inhibiting compounds takes place when (1) Kv1.3

is expressed and (2) the basal ROS production is

relatively high, so a synergistic action exists be-

tween Kv1.3 inhibition and the altered redox state,

which is typical of cancer cells (Sabharwal and

Schumacker, 2014). Thus, apoptosis induced by

the compounds does not only depend on the level

of Kv1.3 expression but also on the basal redox

state. See the text for further details.

The mitochondriotropic drugs are effective against primary tu-

mor cells from B-CLL patients as well as melanoma and PDAC

cells. This is in vivo experimental evidence that targeting a mito-

chondrial channel by a specific inhibitor may strongly reduce tu-

mor size without drastic side effects. Our findings also elucidate

C

the physiological consequences of the

specific inhibition of a mitoK+ channel.

While targeting PM ion channels has

been tested in various cancer models

(Leanza et al., 2015), many of the channel

modulators used have pleiotropic effects

and in most cases their specificity was

not proved. This is likely also the case

for clofazimine, reported to inhibit Kv1.3

(Ren et al., 2008) and reduce tumor

growth (Cholo et al., 2012; Leanza et al.,

2012). In contrast, PAP-1 is a highly spe-

cificmembrane-permeant Kv1.3 inhibitor.

We designed and exploited two mito-

targeted PAP-1 derivatives to demon-

strate that: (1) mitoKv1.3 is functionally

active in these cells, since its inhibition

drastically alters organelle function; (2) it

is the mitochondrial channel whose inhibition is sufficient to

selectively induce apoptosis of cancer cells characterized by

elevated ROS production; and (3) the rapid mitochondrial accu-

mulation of the compounds (and the low affinity of the prodrug

PCARBTP for Kv1.3) might account for the modesty of the

ancer Cell 31, 516–531, April 10, 2017 527

impact on immune cells. Previous work showed that intratumoral

injection of the membrane-impermeant highly specific Kv1.3

inhibitor margatoxin slowed tumor growth by reducing PM

Kv1.3-dependent proliferation (Jang et al., 2011). In contrast,

the inhibitors used in this study act on the mitochondrial Kv1.3,

actively kill tumor cells, and can be applied by i.p. injection.

The conjugation of a TPP+moiety to PAP-1 to give PAPTP only

caused an increase of the IC50 for Kv1.3 activity in patch-clamp

experiments from 2 to 30 nM. PCARBTP was less effective as an

inhibitor. Variations of the PAP-1 structure have already been

shown to reduce Kv1.3 blocking potency (Bodendiek et al.,

2009). However, PCARBTP behaves as a prodrug: PAPOH is ex-

pected to be recovered at the site of action, i.e., mitochondria

(Azzolini et al., 2015). If most PAPOH was released before

PCARBTP accumulation into mitochondria, one would not

expect drastic short-term effects on this organelle, since the un-

charged PAPOH would not concentrate there. The observed

changes in mitochondrial physiology thus indicate that at least

part of PCARBTP reaches the IMM. Like other psoralens with

large substituents such as PAP-1, our derivatives are presum-

ably too bulky to intercalate into DNA and thereby cause muta-

tions and cytotoxicity. Accordingly, mtDNA content was stable

over a 6-day culturing of healthy cells with PAPTP. PAP-1

has been shown not to exert UV phototoxicity (Schmitz et al.,

2005). In our study, as a precaution, all operations involving the

compounds were nonetheless performed in semi-darkness.

The inhibitors act by inducing intrinsic apoptosis via the same

chain of events prompted by mitoKv1.3 block by Bax: stopping

the depolarizing K+ influx causes IMM hyperpolarization, with

ensuing increased ROS level, PTP activation, swelling, loss of

Dcm, loss of cytochrome c, and further ROS release (Szabo

et al., 2008). Our data demonstrating that mitochondrial swelling,

loss of Dcm, and significantly augmented ROS level occur in

intact cells (provided the PTP inhibitor cyclosporine A is not

present) indicate that PAPTP and PCARBTP ‘‘replace’’ Bax

and trigger the same downstream effects. The mitochondrial

effects of the compounds are responsible for cell lethality, since

already a 1 mM concentration of either compound is sufficient to:

(1) cause death of B16F10melanoma and B-CLL cells; (2) induce

an instantaneous mitochondrial hyperpolarization followed by

an increase in the MitoSOX fluorescence signal (indicative of

mitochondrial ROS production, prevented by MitoTEMPO), a

PTP-dependent depolarization at a later time point, and cyto-

chrome c release; and (3) induce swelling and profound alteration

of mitochondrial ultrastructure in intact cells. A rapid ultrastruc-

tural change was observed only for mitochondria, while other

organelles harboring Kv1.3 such as Golgi and nucleus (Jang

et al., 2015; Zhu et al., 2014) as well as ER remained unaltered.

Furthermore, margatoxin and PM depolarization did not induce

these effects. The mitochondria-targeted specific inhibitors

thus allowed us to gain insights into the consequences of IMM

mitoKv1.3 inhibition for the physiology of mitochondria in cancer

cells. Whether other IMM K+ channels share such an important

bioenergetic function is uncertain, since neither specific pharma-

cological tools nor appropriate genetic models are available

(Szewczyk et al., 2010). An early matrix acidification occurred

upon mitoKv1.3 inhibition, in accordance with the finding that in-

hibition of the ATP-dependent K+ channel results in acidification

(Akopova et al., 2014). The observed immediate increase of


respiration followed by a dramatic decrease in respiration and

ATP levels is compatible with opening of the PTP, which induces

ROS production in vivo (Zorov et al., 2000) and in vitro via the

mechanism of ROS-induced ROS release (Zorov et al., 2014),

by triggering a specific conformational change of respiratory

chain complex I (Batandier et al., 2004; Kweon et al., 2004). As

in other cases, since the acute inhibition of mitoKv1.3 triggers a

series of events that do not take place when the channel is not

expressed, lack of the channel or its inhibition does not lead to

equivalent outcomes. For example, either glucose- or inhibitor-

induced K(ATP) channel closure has been shown to lead to insu-

lin secretion in rodent b islet cells, whereas the lack of a functional

channel resulted in greatly reduced rather than increased

glucose-induced insulin release (Miki et al., 1998).

The selectivity of our compounds for cancer cells versus

healthy cells, including those of the immune system, might be

ascribed to synergy between different factors: (1) the channel

is highly expressed in cancer cells in comparison with non-malig-

nant cells (Arcangeli et al., 2009; Leanza et al., 2013); (2) mito-

chondria in cancer cells have a hyperpolarized IMM (Hockenb-

ery, 2010); and (3) cancer cells are characterized by an altered

redox state (e.g., Sabharwal and Schumacker, 2014) and mole-

cules (such as our derivatives) able to increase oxidative stress

above a critical threshold may selectively kill them (Ralph et al.,

2010; Trachootham et al., 2009). Apoptosis induced by the com-

pounds depends not only on the level of Kv1.3 expression but

also on the basal redox state. The observed selectivity is of

utmost importance for a potential clinical use. In particular, tu-

mor-reactive cytotoxic T and TEM cells important for immune sur-

veillance (Dudley et al., 2002) are not significantly affected, and

the immune system in the tumor host remains intact.

In summary, we demonstrate here that direct modulation of

mitoKv1.3 is advantageous for multiple reasons: (1) p53 is not

mandatory for the induction of apoptosis; (2) apoptosis induction

is independent of membrane receptor/kinase-dependent intra-

cellular signaling and of the metabolic state; and (3) the com-

pounds are active on cells resistant to other compounds targeting

proliferating cells (see section on PDAC). The in vivo experiments

indicate that the Kv1.3 inhibitors may be used to selectively elim-

inate cancer cells, independently of their origin, provided they

express mitoKv1.3. Improvementsmay be obtained via optimiza-

tion of dosage and delivery. Further work is required to verify

whether pharmacological targeting of other K+ channels highly

expressed in the mitochondria of cancer cells (Leanza et al.,

2014) can represent an efficient and general strategy.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL MODEL AND SUBJECT DETAILS

B Human Studies

B Animal Studies

d METHOD DETAILS

B Chemistry

B Kinetic Experiments

B HPLC/UV Analyses

B Cell Culturing and Reagents

B Downregulation of Kv1.3 Expression by siRNA

B Isolation of B Lymphocyte from Human Blood and

Mesenchymal Stromal Cell Cultures

B Cell Viability and Cell Death Assays

B Western Blot

B Determination of Immune Cell Subpopulations

B Oxygen Consumption Assay and Activity of Respira-

tory Chain Complexes

B Mitochondrial Morphology, ROSProduction andMem-

brane Potential

B In Vivo Experiments and Immunohistochemistry

B Pharmacokinetic Analysis

B Electrophysiology

B Electrocardiography

B Mitochondrial DNA (mtDNA) Quantification

d QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION

Supplemental Information includes seven figures and can be found with this

article online at http://dx.doi.org/10.1016/j.ccell.2017.03.003.

AUTHOR CONTRIBUTIONS

Conceptualization, I.S., C.P., E.G., M.Z., and H.K.; Investigation and Formal

Analysis, L.L., M.R., K.A.B., M.A., A. Manago, E.V., A.Z., A. Mattarei., L.C.,

A.U., S.K., L.B., V.M., F.S., R.P., and V.T.; Resources, G.S., L.T., A.V.,

J.-H.E., and C.H.; Visualization, L.L., I.S., K.A.B., A.Z., A.U., and L.C.; Writing –

Original Draft, I.S., E.G., C.P., andM.Z.; Supervision, I.S., E.G., G.S., L.T., H.K.,

and C.P.; Project Administration, I.S., E.G., and C.P.; Funding Acquisition, I.S.,

E.G., C.P., M.Z., and L.L.

ACKNOWLEDGMENTS

We thank Prof. Wulff for critical reading of the manuscript and Profs. N. Pre-

varskaya, A. Arcangeli, H. Wulff, P. Bernardi, G. Hajnoczky. S. Piccolo, and

L. Scorrano for useful discussion. The authors also thank J. Tepel, B. Linder

and G. Alp for help with the PDAC experiments and Prof. M. Mongillo for the

use of the electrocardiograph. The authors are grateful to A. Tosatto, S.

Grancara, C. Rampazzo, B. Linder, and R. Quintana-Cabrera for help with

some experiments and to the TEM service of the Department of Biology.

The authors thank the Italian Association for Cancer Research (AIRC) for

financial support (AIRC IG grants 15544 to I.S. and 15397 to L.T.). L.L. is

recipient of a young researcher grant of the University of Padova

(no. GRIC12NN5G) and is grateful to EMBO for a short-term fellowship

(ASTF 233-2014). This study was supported by Deutsche Forschungsge-

meinschaft (DFG) grants GU 335/13-3 and GU 335/30-1 to E.G. H.K. is

also grateful to DFG and H.K., A.Z., and I.S. to Iontrac Marie-Curie Training

Network. M.Z., L.B., and I.S. acknowledge support by the Italian Ministry of

University and Education (PRIN 20107Z8XBW_004 to M.Z. and L.B.; PRIN

2015795S5W to I.S.) and by the CNR Project of Special Interest on Aging.

This work was supported also by grants of Regione Veneto on chronic lym-

phocytic leukemia to L.T. and G.S.

Received: May 25, 2016

Revised: February 3, 2017

Accepted: March 7, 2017

Published: April 10, 2017

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

Anti Human CCR7 fluorescein isothiocyanate (CCR7-FITC) R&D System Cat# FAB197F; RRID: AB_2259847

Anti Human CD3 phycoerythrin-cyanin 7 (CD3-PEcy7) Becton Dickinson Cat# 557851; RRID: AB_396896

Anti Human CD19 allophycocyanin (CD19-APC) Becton Dickinson Cat# 555415; RRID: AB_398597

Anti Human CD5 fluorescein isothiocyanate (CD5-FITC) Becton Dickinson Cat# 561896; RRID: AB_10894588

Anti Kv1.3 extracellular Alomone labs Cat# APC-101; RRID: AB_2040149

Anti Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

(clone 6C5)

Millipore Cat# MAB374; RRID: AB_2107445

Anti-Mouse CD3 molecular complex (clone 17A2) BD Biosciences Cat# 740268

Anti-Mouse CD4 FITC (clone GK1.5) eBioscience Cat# 11-0041-81; RRID: AB_464891

Anti-Mouse CD8a Purified (clone 53-6.7) eBioscience Cat# 14-0081-82; RRID: AB_467087

Anti-Mouse CD19 PE (clone eBio1D3) eBioscience Cat# 12-0193-81; RRID: AB_657661

Anti-Mouse MHC Class II I-Ab APC (clone AF6-120.1) eBioscience Cat# 17-5320-80; RRID: AB_2573211

Biotin Rat Anti-Mouse CD25 (clone 7D4) BD Biosciences Cat# 550529; RRID: AB_2125455

Anti-Mouse/Rat FoxP3 PE (clone FJK-16s) eBioscience Cat# 72-5775; RRID: AB_469978

Anti-Mouse F4/80 Antigen Alexa Fluor 488 (clone BM8) eBioscience Cat# 53-4801-80; RRID: AB_469914

Anti-Mouse CD11b Biotin (clone M1/70) eBioscience Cat# 13-0112-82; RRID: AB_466359

Anti-Mouse CD11c FITC (clone N418) eBioscience Cat# 11-0114-81; RRID: AB_464939

TruStain fcX� (anti-mouse CD16/32) Antibody (clone 93) Biolegend Cat# 101319; RRID: AB_1574973

Rat anti Mouse CD204 Alexa Fluor 488 (clone 2F8) Bio-Rad Cat# MCA1322A488; RRID: AB_324818

Anti-Mouse Ly-6G (Gr-1) APC (clone RB6-8C5) eBioscience Cat# 17-5931-81; RRID: AB_469475

PE Streptavidin BD Biosciences Cat# 554061; RRID: AB_10053328

APC Streptavidin BD Biosciences Cat# 554067; RRID: AB_10050396

Biological Samples

Sheep red blood cells (SRBC) (Leanza et al., 2013)

B-CLL (Leanza et al., 2013)

Human B cells (Leanza et al., 2013)

Chemicals, Peptides, and Recombinant Proteins

Cyclosporine H Sequoia Cat# SRP046746c

PAP-1 Sigma Aldrich Cat# P6124

Staurosporine Sigma Aldrich Cat# S4400

Lipofectamine 2000 Thermo Scientific Cat# 11668027

Annexin V FITC Roche Cat# 11828681001

Annexin V alexa 568 Roche Cat# 03703126001

Accutase Sigma Aldrich Cat# A6964

Ficoll-Hypaque GE Healthcare Bio-Sciences AB Cat# 17-1440-03

Bergapten (5-Methoxypsoralen) Carbosynth Cat# FM05395

MitoSOX� Red Mitochondrial Superoxide Indicator Thermo Fisher Scientific Cat# M36008

Tetramethylrhodamine Methyl Ester (TMRM) Thermo Fisher Scientific Cat# T668

MitoTracker� Green FM Thermo Fisher Scientific Cat# M7514

Critical Commercial Assays

RosetteSep isolation kit for B cells STEMCELL Technologies Cat# 15064

CellTiter 96� AQUEOUS One solution Promega Cat# G3581

Puregene Core kit B Qiagen Cat# 158388

(Continued on next page)

e1 Cancer Cell 31, 516–531.e1–e10, April 10, 2017

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

Experimental Models: Cell Lines

Mouse B16F10 melanoma ATCC Cat# CRL-6475; RRID: CVCL_0159

Human Jurkat T-lymphocytes ATCC Cat# TIB-152; RRID: CVCL_0367

AsPC-1 ATCC Cat# CRL-1682; RRID: CVCL_0152

BxPC3 ATCC Cat# CRL-1687; RRID: CVCL_0186

Capan-1 ATCC Cat# HTB-79; RRID: CVCL_0237

MIA PaCa-2 ATCC Cat# CRL-1420; RRID: CVCL_0428

PANC-1 ATCC Cat# CRL-1469; RRID: CVCL_0480

Human metastatic Colo357 pancreas adenocarcinoma Morgan et al., 1980 N/A

HPV16-E6E7 - immortalized human pancreatic duct

epithelial cells (HPDE)

Ouyang et al., 2000 N/A

Experimental Models: Organisms/Strains

C57BL/6J mice Charles River laboratories Strain code #027

SCID beige (C.B.-17. Cg-Prkdcscid Lystbg/Crl) mice Charles River laboratories Strain code #250

Oligonucleotides

Hs_KCNA3_1 Flexi tube siRNA 3’-alexa Fluor 555 Qiagen Cat# SI00034762

All star negative control siRNA Alexa Fluor 555 Qiagen Cat# 1027294

Software and Algorithms

BD Vista software BD Bioscience

BD CellQuest Pro software BD Bioscience

Clampfit 8.1 software Molecular Devices

LabChart 7 Pro software ADInstruments

CONTACT FOR REAGENT AND RESOURCE SHARING

Requests for further information and for reagents may be directed to, and will be fulfilled by the corresponding author Ildiko Szabo

([email protected]).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Human StudiesFor the human studies a written informed consent was obtained from all patients, prior to sample collection, according to the Decla-

ration of Helsinki. The ethical approval for our study was obtained from the local ethic committee ‘‘Regione Veneto on chronic lym-

phocytic leukemia’’. Both CLL patient and healthy control groups were formed by equal numbers of 50 to 70-years-old male and

female subjects. Isolated B cells from 11 healthy subjects and from 31 B-CLL patients were analyzed andmesenchymal stromal cells

(MSC) were from 5 CLL patients.

Animal StudiesAnimal experiments and care compliedwith the institutional guidelines of institutional authorities, were approved by Italian authorities

(both the local Ethic Committee OPBA (Organismo preposto al benessere animale) at University of Padova and Italian Ministry

for Health (CEASA number 54/2011) as well as the Animal Care and Use Committee of the Bezirksregierung D€usseldorf

(AZ 84-02.04.2015.A374) and of Kiel (V312-7224.121-7 (123-10/11), Germany. Experiments were carried out with the supervision

of the Central Veterinary Service of the University of Padova (in compliance with Italian Law DL 116/92, embodying UE directive

86/609), Duisburg-Essen andKiel. Two to sixmonths oldmale or female C57BL/6Hmiceweighing 18-26 gwere obtained fromHarlan

and used both for the orthotopic in vivo melanoma model as well as for the pharmacokinetic and ECG experiments. Four weeks old

female SCID beige (C.B.-17. Cg-Prkdcscid Lystbg/Crl) mice weighing 14-19 g were obtained from Charles River and used for the

orthotopic in vivo model of PDAC.

METHOD DETAILS

ChemistryThe mitochondriotropic compounds PCARBTP and PAPTP were synthetized as shown in Scheme 1.

Cancer Cell 31, 516–531.e1–e10, April 10, 2017 e2

Scheme 1. Synthesis of Mitochondriotropic Derivatives PCARBTP and PAPTP

Starting materials and solvents were reagent grade chemicals purchased from Aldrich, Sigma-Aldrich, TCI, Fluka, Riedel-de Haen

(Seelze, German), Prolabo (Fonyenay sous Bois, France), Carbosynth (Compton, Berckshire, UK), and were used as received.1H-NMR and 13C-NMR spectra were recorded with a Bruker AC 250F spectrometer operating at 250 MHz for 1H-NMR and

62.9 MHz for 13C-NMR, or with a Bruker 300 UltraShield spectrometer operating at 300 MHz for 1H-NMR and 75 MHz for13C-NMR, or with a Bruker 500 UltraShield spectrometer operating at 500 MHz for 1H-NMR and 126 MHz for 13C-NMR. Chemical

shifts (d) are given in ppm, and the residual solvent signal was used as an internal standard. TLCs were run on silica gel supported

on plastic (Macherey-Nagel Polygram�SIL G/UV254, silica thickness 0.2 mm) and were visualized by UV detection. Flash chroma-

tography was performed on silica gel (Macherey-Nagel 60, 230-400 mesh) under compressed air pressure. HPLC/ESI-MS analyses

and mass spectra were performed with a 1100 Series Agilent Technologies system, equipped with a binary pump (G1312A) and an

MSD SL Trap mass spectrometer (G2445D SL) with ESI source. ESI-MS spectra were obtained from solutions in acetonitrile, eluting

with a water:acetonitrile = 1:1 mixture containing 0.1% formic acid.

HPLC/ESI-MS analysis was used to confirm the purity (>95%) of isolated intermediates and products. Fluorescence/UV-Vis

spectra were recorded at 25�Cwith a Perkin-Elmer LS-55 spectrofluorimeter. The case of a fluorescent mitochondriotropic quercetin

derivative clearly demonstrated that the TPP+ moiety drives the molecule to mitochondria but not to other intracellular membranes

such as ER or nucleus (Mattarei et al., 2008; Sassi et al., 2012).

The various steps are described in detail in the following paragraphs.

4-Hydroxy-7H-furo-[3,2-g]benzopiran-7-one (2)

A BBr3 solution (10 mmol, 5 eq) was slowly added at room temperature and under nitrogen to a stirred bergapten (2 mmol, 1 eq) so-

lution in anhydrous dichloromethane (20 mL). After 100 min, the mixture was washed with saturated aqueous NaHCO3 (100 mL) and

extracted with ethyl acetate (3 x 300 mL). The combined organic layers were dried over MgSO4 and the solvent was removed

under vacuum to obtain 2 as an off–white solid (100% yield). 1H-NMR (250 MHz, CDCl3): d = 8.11 (d, J = 9.8 Hz, 1H; CH), 7.58

(d, J = 2.4 Hz, 1H; CH), 7.10 (t, 1H; CH), 6.93 (dd, J = 2.4, 1.0 Hz, 1H; CH), 6.26 (d, J = 9.8 Hz, 1H; CH), 4.49 (t, J = 5.8 Hz,

2H; CH2), 3.66 (t, J = 6.0 Hz, 2H; CH2), 2.21-1.93 ppm (m, 4H; CH2CH2);13C-NMR (62.9 MHz, CDCl3): d = 161.1 (CO), 158.2,

152.6, 148.6, 144.8, 139.1, 113.0, 112.6, 106.5, 105.0, 93.9, 71.9, 44.5, 29.1, 27.4 ppm; ESI-MS (ion trap): m/z: 203, [M+H+].

4-(4-clorobutoxy)-7H-furo-[3,2-g]benzopyran-7-one (3)

Compound 2 (3.5 mmol, 1 eq in 25 mL), Cs2CO3 (5.2 mmol, 1.5 eq) and 1-bromo-4-chlorobutane (5.2 mmol, 1.5 eq) were suspended

in anhydrous DMF (25mL) and stirred under inert atmosphere at 50�Covernight. After this time, ethyl acetate (100mL) was added and

the mixture was extracted with 0.5 M HCl (3 x 170 mL). The aqueous phase was extracted with dichloromethane (2 x 70 mL), the

combined organic layers were dried over MgSO4, filtered and the solvent was removed under reduced pressure. The crude product

was purified by flash chromatography using dichloromethane/ethyl acetate (98:2) as eluent to afford 3 as a white solid (87% yield).1H-NMR (250 MHz, CDCl3): d = 8.11 (d, J = 9.8 Hz, 1H; CH), 7.58 (d, J = 2.4 Hz, 1H; CH), 7.10 (t, 1H; CH), 6.93 (dd, J = 2.4, 1.0 Hz, 1H;

CH), 6.26 (d, J = 9.8 Hz, 1H; CH), 4.49 (t, J = 5.8 Hz, 2H; CH2), 3.66 (t, J = 6.0 Hz, 2H; CH2), 2.21-1.93 ppm (m, 4H; CH2CH2);13C-NMR

(62.9 MHz, CDCl3): d = 161.1 (CO), 158.2, 152.6, 148.6, 144.8, 139.1, 113.0, 112.6, 106.5, 105.0, 93.9, 71.9, 44.5, 29.1, 27.4 ppm;

ESI-MS (ion trap): m/z: 293, [M+H+].

4-(4-iodobutoxy)-7H-furo[3,2-g]benzopyran-7-one (4)

Compound 3 (1.7 mmol, 1 eq) and NaI (17 mmol, 10 eq) were added to anhydrous acetone (25 mL) under nitrogen. The suspension

was stirred at 70 �C overnight. After addition of ethyl acetate (30 mL), the mixture was washed with deionized water (1 x 150 mL) and


the organic phase was dried over MgSO4. The crude product obtained after removal of the solvent under reduced pressure was pu-

rified by flash chromatography using dichloromethane/ethyl acetate (97:3) as eluent. Pure 4 was obtained as a light yellow powder

(80% yield). 1H-NMR (250MHz, CDCl3): d = 8.11 (d, J = 9.8 Hz; 1H, CH), 7.58 (d, J = 2.4 Hz; 1H, CH), 6.93 (dd, J = 2.4, 1.0 Hz; 1H, CH),

6.26 (d, J = 9.8 Hz; 1H, CH), 4.47 (t, J = 5.8 Hz; 2H, CH2), 3.29 (t, J = 6.5 Hz; 2H, CH2), 2.18 – 1.91 ppm (m; 4H, CH2CH2);13C-NMR (62.9

MHz, CDCl3): d = 161.1 (CO), 158.2, 152.6, 148.6, 144.8, 139.1, 113.0, 112.6, 106.5, 105.0, 93.9, 71.5, 30.8, 29.9, 5.9 ppm; ESI-MS

(ion trap): m/z: 385, [M+H+].

4-(4-(4-hydroxyphenoxy)butoxy)-7H-furo[3,2-g]benzopyran-7-one (PAPOH, 5)

Hydroquinone (4.5 mmol, 15 eq) was added under stirring to a mixture of 4 (0.3 mmol, 1 eq) and Cs2CO3 (0.6 mmol, 2 eq) in DMF

(15 mL). The mixture was allowed to react overnight in the dark and under stirring at 45 �C. After addition of ethyl acetate (90 mL),

the mixture was extracted with 0.5 M aqueous HCl (5 x 50 mL). The organic phase was dried over MgSO4, filtered and the solvent

was removed under reduced pressure. The crude product was purified by flash chromatography using chloroform/acetone (90:10) as

eluent. Pure 5was obtained as a cream-white solid (84% yield). 1H-NMR (250 MHz, CDCl3): d = 8.10 (dd, J = 9.8, 0.6 Hz), 7.75 (d, J =

2.4 Hz), 7.16 (dd, J = 2.4, 1.0 Hz), 7.07 – 6.98 (m), 6.67 (d, J = 2.2 Hz), 6.12 (d, J = 9.8 Hz), 4.55 (t, J = 6.0 Hz), 3.93 (t, J = 6.0 Hz), 2.08 –

1.80 (m). 13C-NMR (62.9 MHz, CDCl3): d = 160.8, 159.1, 153.7, 153.1, 152.2, 150.1, 146.2, 140.0, 116.6, 116.3, 113.9, 113.2, 107.2,

106.4, 93.8, 73.5, 68.6, 27.5, 26.7 ppm. ESI+-MS (ion trap): m/z: 367, [M+H+].

4-nitrophenyl (3-chloropropyl)carbamate

3-chloropropylamine hydrochloride (2.31 mmol, 1 eq) was added to a 10 mL anhydrous dichloromethane solution of 4-(dimethyla-

mino)pyridine (4.6 mmol, 2 eq). The resulting solution was added dropwise to a stirred solution of bis-paranitrophenyl carbonate

(2.5 mmol, 1.1 eq) in dry THF (20 mL) under nitrogen at room temperature. After 3 hr, ethyl acetate (150 mL) was added and the

mixture was extracted with aqueous 0.5 M HCl (3 x 75 mL). The combined aqueous layers were extracted with dichloromethane

(1 x 80 mL) and the organic fraction was dried over MgSO4 and the solvent removed at reduced pressure. The crude product was

purified by flash chromatography using dichloromethane/ethyl acetate (98:2), which afforded a yellow solid (87% yield). 1H NMR

(300 MHz, acetone) d = 8.28 (d, J = 9.1 Hz, 2H), 7.44 (d, J = 9.1 Hz, 2H), 7.19 (s, 1H), 3.74 (t, J = 6.5 Hz, 2H), 3.43 (dd, J = 12.8,

6.5 Hz, 2H), 2.10 (dd, J = 13.2, 6.6 Hz, 2H) ppm. 13C NMR (75 MHz, CDCl3) d = 157.48, 154.22, 145.49, 125.75, 123.05, 43.09,

39.27, 33.33, 29.84 ppm.

4-(4-((7-oxo-7H-furo[3,2-g]benzopyran-4-yl)oxy)butoxy)phenyl (3-chloropropyl) carbamate (6)

An acetonitrile (20 mL) solution of 4-nitrophenyl (3-chloropropyl)carbamate, prepared as described in the following paragraph,

(1.1 mmol, 2 eq), 5 (0.5 mmol, 1 eq) and 4-(dimethylamino)pyridine (1.1 mmol, 2 eq) was stirred under nitrogen at 50�C for 48 hr. After

addition of aqueous 0.5 M HCl (150 mL), the mixture was extracted with chloroform (3 x 100 mL). The combined organic layers

were dried over MgSO4 and filtered. After removal of the solvent at reduced pressure, the crude product was purified by flash chro-

matography using chloroform/Et2O/petroleum ether (60:10:30) as eluent. Pure 6 was obtained as a white powder (75% yield).1H NMR (500 MHz, CDCl3) d = 8.10 (d, J = 9.8 Hz, 1H), 7.56 (d, J = 2.3 Hz, 1H), 7.10 (s, 1H), 7.01 (d, J = 9.0 Hz, 2H), 6.93

(dd, J = 2.3, 0.8 Hz, 1H), 6.84 (d, J = 9.0 Hz, 2H), 6.23 (d, J = 9.8 Hz, 1H), 4.52 (t, J = 6.0 Hz, 2H), 4.04 (t, J = 5.8 Hz, 2H), 3.62

(t, J = 6.3 Hz, 2H), 3.41 (q, J = 6.4 Hz, 2H), 2.15 – 1.90 (m, 6H) ppm. 13C NMR (126 MHz, CDCl3) d = 161.40, 158.33, 156.19,

155.26, 152.73, 148.95, 144.93, 144.67, 139.42, 122.59, 114.99, 113.22, 112.56, 106.71, 105.22, 93.89, 77.16, 72.53, 67.75,

42.36, 38.69, 32.32, 26.98, 25.92 ppm. ESI-MS (ion trap): m/z 488 [M+H]+.

4-(4-((7-oxo-7H-furo[3,2-g]benzopyran-4-yl)oxy)butoxy)phenyl (3-iodopropyl) carbamate (7)

Compound 6 (0.4 mmol, 1 eq) was dissolved under nitrogen in anhydrous acetone (30 mL) satured with NaI. The suspension

was maintained at 70 �C under stirring and in the dark overnight. After addition of ethyl acetate (100 mL), the mixture was washed

with water (4 x 75 mL). The combined aqueous layers were extracted with dichloromethane (1 x 50 mL). After drying over MgSO4

and filtration, the solvent mixture was removed under reduced pressure. The crude product was purified by flash chromatography

using chloroform/methanol (95:5) as eluent to afford 7 as a light yellow solid (75% yield). 1H NMR (300 MHz, CDCl3) d = 8.11

(d, J = 9.8 Hz, 1H), 7.57 (d, J = 2.3 Hz, 1H), 7.11 (s, 1H), 7.02 (d, J = 8.9 Hz, 2H), 6.94 (d, J = 2.0 Hz, 1H), 6.84 (d, J = 8.9 Hz, 2H),

6.24 (dd, J = 9.8, 2.6 Hz, 1H), 4.52 (t, J = 5.8 Hz, 2H), 4.04 (t, J = 5.6 Hz, 2H), 3.35 (q, J = 6.3 Hz, 2H), 3.22 (t, J = 6.8 Hz, 2H), 2.19 –

1.91 (m, 6H) ppm. 13C NMR (75 MHz, CD2Cl2) d = 161.41, 158.37, 156.24, 155.25, 152.78, 150.24, 148.99, 144.93, 144.70, 139.42,

122.60, 116.21, 115.64, 115.05, 113.30, 112.63, 106.79, 105.23, 93.97, 77.16, 72.59, 67.80, 41.80, 33.17, 27.03, 25.96, 2.81 ppm.

(3-(((4-(4-((7-oxo-7H-furo[3,2-g]benzopyran-4-yl)oxy)butoxy)phenoxy)carbonyl) amino) propyl) triphenylphosphonium

iodide (PCARBTP, 8)

Amixture of 7 (0.3mmol, 1 eq) and triphenylphosphine (6mmol, 20 eq) was heated at 95�Cunder nitrogen, in the dark, for 3 hr. A small

amount of dichloromethane (5 mL) was added and the product was precipitated with diethyl ether (150 mL). The solvent was

decanted and the product was filtered under vacuum and washed with Et2O (5 x 15 mL). After solvent removal under reduced pres-

sure, pure 8was obtained as a white powder (0.22 mmol, 82%). 1H NMR (300 MHz, CDCl3) d = 8.11 (d, J = 9.8 Hz, 1H), 7.72 (m, 15H),

7.56 (d, J = 2.1 Hz, 1H), 7.36 (d, J = 4.3 Hz, 1H), 7.08 (s, 1H), 6.96 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 6.22 (d, J = 9.8 Hz, 1H),

4.51 (t, J = 5.7 Hz, 2H), 4.01 (t, J = 5.4 Hz, 2H), 3.73 (dd, J = 15.5, 13.0 Hz, 2H), 3.55 (d, J = 4.9 Hz, 2H), 2.20 – 1.80 (m, 6H) ppm.13C NMR (75 MHz, CD2Cl2) d = 161.33, 158.33, 155.96, 155.64, 152.73, 149.00, 144.92, 139.44, 135.33, 133.73, 133.60, 130.78,

130.62, 122.65, 118.65, 117.51, 114.86, 113.28, 112.57, 106.73, 105.26, 93.86, 77.16, 72.61, 67.78, 65.89, 26.98, 25.93 ppm.

ESI-MS (ion trap): m/z: 713, [M-Iodine+H+].


4-(4-(4-(3-chloropropyl)phenoxy)butoxy)-7H-furo[3,2-g]benzopyran-7-one (9)

Compound 4 (1.5 mmol, 2.5 eq), Cs2CO3 (1.2 mmol, 2 eq) and 4-(3-chloropropyl)phenol (0.6 mmol, 1 eq) were stirred under inert at-

mosphere in anhydrous DMF (20mL) overnight at 50 �C in the dark. After addition of ethyl acetate (250mL), themixture was extracted

with aqueous 0.5MHCl (5 x 70mL) and the combined aqueous layers were extracted with dichloromethane (1 x 100mL). After drying

over MgSO4 and filtering, the organic solvent was removed under reduced pressure. The crude product was purified by flash chro-

matography using dichloromethane/petroleum ether (95:5) as eluent. Pure 9 was obtained as a solid white powder (87% yield).1H NMR (300 MHz, CD2Cl2) d (ppm) = 8.12 (d, J = 9.8 Hz, 1H), 7.60 (t, J = 2.1 Hz, 1H), 7.18 – 7.04 (m, 3H), 6.98 (dd, J = 5.4,

2.4 Hz, 1H), 6.87 – 6.74 (m, 2H), 6.29 – 6.11 (m, 1H), 4.50 (dt, J = 17.8, 5.6 Hz, 2H), 4.05 (t, J = 5.7 Hz, 2H), 3.53 (t, J = 6.5 Hz, 2H,

C-Cl), 3.32 (t, J = 6.4 Hz, 2H), 2.75 – 2.65 (m, 2H), 2.17 – 1.91 (m, 4H).

4-(4-(4-(3-iodopropyl)phenoxy)butoxy)-7H-furo[3,2-g]benzopyran-7-one (10)

Compound 9 (0.9 mmol, 1 eq) and NaI (12 mmol, 13 eq) were added to anhydrous acetone (25 mL) under an inert atmosphere. The

suspension wasmixed and heated in the dark at 70 �Covernight. After addition of ethyl acetate (150mL) themixture waswashedwith

deionized water (1 x 150 mL). The aqueous layer was extracted with dichloromethane (1 x 70 mL) and the organic phase, after drying

over MgSO4, was concentrated under reduced pressure. The crude product was purified by flash chromatography using dichloro-

methane/petroleum ether/ethyl acetate (50:45:5) as eluent to afford 10 as a light yellow solid (65% yield). 1H NMR (500 MHz, CD2Cl2)

d (ppm) = 8.15 (d, J = 9.8 Hz, 1H), 7.62 (dd, J = 4.5, 2.4 Hz, 1H), 7.11 (t, J = 6.9 Hz, 3H), 6.99 (d, J = 8.5 Hz, 1H), 6.87 – 6.75 (m, 2H), 6.22

(dd, J = 19.1, 9.8 Hz, 1H), 4.52 (dt, J = 28.7, 5.9 Hz, 2H), 4.05 (t, J = 5.9 Hz, 2H), 3.31 (t, J = 6.7 Hz, 2H), 3.17 (t, J = 6.9 Hz, 2H, C-I), 2.65

(t, J = 7.3 Hz, 2H), 2.14 – 1.96 (m, 4H). 13C NMR (126MHz, CD2Cl2) d (ppm) = 160.77, 158.29, 157.36, 152.83, 149.06, 144.91, 139.16,

132.69, 129.48, 114.37, 113.20, 112.55, 106.71, 105.17, 93.54, 93.47, 72.71, 67.43, 35.23, 30.86, 26.90, 25.94, 6.48, 6.16.

(3-(4-(4-((7-oxo-7H-furo[3,2-g]benzopyran-4-yl)oxy)butoxy)phenyl)propyl)triphenyl phosphonium iodide (PAPTP, 11)

A mixture of 10 (0.6 mmol, 1 eq) and triphenylphosphine (5.6 mmol, 10 eq) in HPLC-grade toluene (20 mL) was stirred under nitrogen

and in the dark overnight at 120�C. After removal of the solvent under reduced pressure the residue was taken up in dichloromethane

(2mL) and precipitatedwith diethyl ether (150mL). The solvent was decanted and the product was filtered under vacuumandwashed

with Et2O (6 x 50 mL); residual solvent was removed under reduced pressure to afford 11 as a light yellow powder (65% yield).1H NMR (300 MHz, CDCl3) d (ppm) = 8.11 (d, J = 9.8 Hz, 1H), 7.98 – 7.59 (m, 9H), 7.57 (d, J = 2.4 Hz, 1H), 7.16 – 7.04 (m, 1H),

6.98 – 6.92 (m, 1H), 6.76 (d, J = 8.6 Hz, 1H), 6.14 (dd, J = 22.1, 9.8 Hz, 1H), 4.56 (dt, J = 26.2, 5.8 Hz, 1H), 4.01 (t, J = 5.7 Hz, 1H),

3.96 – 3.81 (m, 1H), 3.66 (td, J = 12.8, 8.2 Hz, 1H), 2.94 (t, J = 7.2 Hz, 1H), 2.17 – 1.78 (m, 2H) ppm. 13C NMR (126 MHz, CDCl3)

d =161.24, 158.27, 157.43, 152.63, 148.95, 144.94, 139.43, 135.15, 133.81, 133.69, 133.61, 132.07, 130.60, 130.50, 129.99,

129.23, 128.66, 128.60, 118.36, 117.68, 114.55, 113.19, 112.38, 106.61, 105.23, 93.73, 72.56, 67.36, 65.83, 34.80, 34.66, 30.93,

26.94, 25.93, 24.64, 22.07, 21.67, 15.26 ppm. ESI-MS (ion trap): m/z: 654, [M-Iodine+H+].

Kinetic ExperimentsHydrolysis in Aqueous Solutions

The chemical stability of the compounds described here was tested in aqueous media approximating gastric (0.1 N HCl, NormaFix)

and intestinal (0.1 M PBS buffer, pH 6.8) pH values. A 5 mM solution of the compound was prepared from a 5 mM stock solution in

DMSO, and incubated at 37�C for 24 h. Samples (2 mL) were withdrawn at different times and analyzed by HPLC-UV as detailed

below. Hydrolysis products were identified by comparison with compounds of known identity.

Hydrolysis in Blood

Mouse were anesthetized and blood was withdrawn from the jugular vein, heparinized and transferred into tubes containing EDTA.

Blood samples (1 mL) were spiked with compound (5 mM; dilution from a 5 mM stock solution in DMSO), and incubated at 37�C for

4 hr (the maximum period allowed by blood stability). Aliquots were taken after 10 min, 30 min, 1 hr, 2 hr and 4 hr before HPLC-UV

analysis. 4,4’-dihydroxybiphenyl was added as internal standard to a carefully measured blood volume (25 mM final concentration).

Blood was then stabilized with a freshly-prepared 10 mM solution of ascorbic acid (0.1 vol) and acidified with 0.6 M acetic acid

(0.1 vol); after mixing, an excess of acetone (4 vol) was added, followed by sonication (2 min) and centrifugation (12,000 g, 7 min,

4�C). The supernatant was finally collected and stored at -20�C. Acetone was allowed to evaporate at room temperature using a Uni-

vapo 150H (UniEquip) vacuum concentrator centrifuge before analysis, and up to 40 mL of acetonitrile were added to precipitate re-

sidual proteins. After centrifugation (12,000 g, 5 min, 4�C), cleared samples were directly used for HPLC-UV analysis (Azzolini

et al., 2014).

HPLC/UV AnalysesHPLC/UV analyses were carried out with a 1290 Infinity LC System (Agilent Technologies) using a reverse-phase column (Zorbax

Extend-C18, 1.8 mm, 50 x 3.0 mm i.d.; Agilent Technologies) and a UV diode array detector (190-500 nm). Solvents A and B were

water containing 0.1% trifluoroacetic acid (TFA) and acetonitrile, respectively. The gradient for B was as follows: 10% (0.5 min)

then from 10% to 100% in 4.5 min; the flow rate was 0.6 mL/min. The eluate was preferentially monitored at 286 and 320 nm.

The column compartment was maintained at 35�C.

Cell Culturing and ReagentsB16F10 cells (ATCC) were grown in Minimum Essential Media (MEM, Thermo Fisher Scientific) supplemented with 10 mM HEPES

buffer (pH 7.4), 10% (v/v) fetal bovine serum (FBS), 100U/mL penicillin G, 0.1mg/mL streptomycin and 1%non-essential amino acids


(100X solution; Thermo Fisher Scientific). Lymphocytes (Jurkat and B cells) were grown in RPMI-1640 (Thermo Fisher Scientific), sup-

plemented as MEM. A panel of pancreatic cancer cell lines representing different phases of tumor progression was used: AsPC1,

BxPC3, Capan-1, MIA PaCa-2 and PANC-1 were provided by ATCC. AsPC1 and BxPC3 were cultured in RPMI-1640 supplemented

with 10%FBS ‘‘GOLD’’ (PAA Laboratories/GEHealthcare Life Sciences), 1mMGlutaMAX and 1mMsodiumpyruvate (Thermo Fisher

Scientific). MIA PaCa-2 and PANC-1 were cultured in DMEM (4.5 g/L D-glucose) supplemented with 10% FBS ‘‘GOLD’’, 1mM

GlutaMAX and 1 mM sodium pyruvate. Capan-1 cells were grown in IMEM supplemented with 20% FBS ‘‘GOLD’’, 1 mM GlutaMAX

and 1mMsodium pyruvate. The human cell line of metastatic pancreas adenocarcinoma, Colo357, was obtained fromDr. R. Morgan

(Denver, CO) (Morgan et al., 1980) and was cultured in a complete growth medium composed of RPMI-1640, 10% FCS (PAN-

Biotech), 1 mM GlutaMAX and 1 mM sodium pyruvate. The HPV16-E6E7 - immortalized human pancreatic duct epithelial cells

(HPDE), kindly provided by Dr. Ming-Sound Tsao (Ontario Cancer Institute, Toronto, Ontario, Canada) (Ouyang et al., 2000) were

used as a model for benign pancreatic ductal epithelium. The complete HPDE growth medium was a mixture of 50% RPMI 1640,

supplemented with 10% FCS and 1 mM GlutaMAX and 50% keratinocyte medium SFM (Thermo Fisher Scientific) supplemented

with 0.025% bovine pituitary extract, 2.5 mg/L epidermal growth factor (Thermo Fisher Scientific). Hypoxic condition was obtained

by reducing oxygen percentage to less than 1% by inflating nitrogen in a modular incubator chamber (Billups-Rothemberg, USA).

Metabolism was altered by growing the cells (seeded 3000/well) for three days in DMEM lacking glucose but supplemented with

galactose, before treatment with the indicated compounds.

Downregulation of Kv1.3 Expression by siRNAThe sequences for the siRNA targeting human Kv1.3 were coupled to Alexa Fluo 555 (Hs_KCNA3_1 Flexi tube siRNA for Kv1.3 and All

star negative control siRNA as scramble/control; Qiagen). 80,000 adherent cells/well (B16F10, Colo357, BxPC-3) were seeded into a

12well plate in 1mL of the growthmedium. After 24 h, the cells were transiently transfected with 2 mg siRNA/well using Lipofectamine

2000, as suggested by the supplier. Cells growing in suspension (Jurkat) were transfected by electroporation (Leanza et al., 2012).

After 48 h from transfection, cells were treated for 24 hr with the various compounds as indicated. Cell death, evaluated by the binding

of FITC-labelled Annexin V, as well as the successful transfection with Alexa555-coupled siRNA were determined using a DMI 4000

Leica fluorescence microscope or FACS analysis.

Isolation of B Lymphocyte from Human Blood and Mesenchymal Stromal Cell CulturesPeripheral blood mononuclear cells (PBMCs) from the patients were isolated by density-gradient centrifugation using the Ficoll-Hy-

paque (F/H) technique (Amersham Biosciences; Buckinghamshire, UK) as previously described (Leanza et al., 2013). The samples

were checked for purity by flow cytometry; if the percentage of cells other than CD19+ B cells exceeded 5%, the purification proced-

ure was repeated. For healthy donors, non-manipulated peripheral blood B cells were isolated from the PBMCs by negative selection

using the RosetteSep isolation kit for B cells (STEMCELL Technologies; Vancouver, Canada). To obtain distinct populations of B- and

T-cells we used the separation method of sheep red blood cells (SRBC) (Frezzato et al., 2014). Mesenchymal stromal cells (MSCs)

were isolated from iliac crest bone marrow (BM) aspirate of CLL patients under local anaesthesia and diluted 1:3 in Phosphate Buff-

ered Saline (PBS) (Euroclone; Milan, Italy) (Frezzato et al., 2014). For MSC culture, BMmononuclear cells (BMMCs) were isolated as

stated above and plated at a density of 1,000 cells/cm2 in DMEM (Euroclone) with 1,000 mg/mL glucose, L-glutamine, 10% heat-

inactivated FBS and 100 U/mL Penicillin, 100 mg/mL Streptomycin (Life Technologies; Paisley, UK). BMMC suspensions were incu-

bated at 37�C in humidified atmosphere containing 5% CO2 and allowed to attach for 7 days; at this time-point, the non-adherent

fraction was discarded and adherent cells were fed every week with fresh medium. These cells were maintained until confluence,

then they were removed by treatment with Accutase (Sigma-Aldrich; Milan, Italy), centrifuged and diluted 1:3 for subsequent expan-

sion in 25 cm2 flasks or cryopreserved for future use. 2x106 purified human primary B-CLL cells or healthy B cells were seeded onto a

confluent MSC layer and treated as indicated in the figure legends. After treatments, MSC cell death was then analysed after labeling

for 20 min with Annexin V-labelling at 37�C by fluorescent microscopy with a Leica DMI4000 microscope (Leica Microsystem, Wet-

zlar, Germany) in the case of MSC (Szabo et al, 2015) or by flow cytometry (FACS Canto II, BD BioSciences) in the case of CLL or

healthy B cells growing in suspension. (Leanza et al., 2013).

Cell Viability and Cell Death AssaysFor cell growth/viability (MTT) assays, adherent cells were seeded (0.005 to 0.01 x 106 cells/well) in standard 96-well plates and al-

lowed to grow in DMEM (200 mL) for 24 h to ensure attachment. The growthmediumwas then replaced in the dark with amedium that

contained the desired compound (from a mother solution in DMSO) at the final concentration. The final concentration of DMSO was

0.1% or lower in all cases (including controls). Non-toxic concentration of Cyclosporine H (CSH) (4 mM) was used as MDRi. After in-

cubation for 24 h, CellTiter 96� AQUEOUSOne solution (Promega, Italy) was added to each well as indicated by the supplier. Absor-

bance was measured at 490 nm to detect formazan formation using a Packard Spectra Count 96-well plate reader.

For cell death assays of non-adherent cells, such as Jurkat, B-CLL cells and B cells, a Becton Dickinson FACS Canto II flow cy-

tometer was used. The cells were incubated with the test substances for 24 hr, washed in HBSS, and resuspended at 33105 cells/mL

in DMEM without serum and Phenol Red, or in some experiments in HBSS. DMSO concentration was < 0.1% in all cases. A 200-mL

portion of each incubation sample was then placed in a test tube and Propidium Iodide (final concentration 1 mg/mL) and annexin-V-

FLUOS (Roche) (1 mL/sample) were added. Flow cytometry analysis was carried out after a further 20 min labelling period at 37�Cin the dark. Data were processed by quadrant statistics using BD VISTA software. Cell death of adherent cells was measured by


fluorescence microscopy. 0.023106 cells were seeded in a 24-well plate and treated for 24 h, as indicated, in 1 mL of DMEMwithout

Phenol Red and FBS. Following incubation, 1 mL/well of annexin-V-FLUOS (Roche) was added and cells were incubated for 20min in

the dark at 37�C. Cells were then analysed using a Leica DMI 4000 fluorescencemicroscope (LeicaMicrosystem,Wetzlar, Germany).

To differentiate the percentage of apoptotic B- and T-cells from the same B-CLL patient or from healthy subjects, the following

antibodies were used: Annexin V-alexa 568 (Roche), and anti-CCR7 fluorescein isothiocyanate (FITC; CCR7-FITC: FAB197F)

(R&D System, Minneapolis, MN, USA), anti-CD3 phycoerythrin-cyanin 7 (PE-Cy7;CD3-PEcy7: 557851) and anti-CD19 allophyco-

cyanin (APC; CD19-APC: 555415) (Becton Dickinson, Franklin Lakes, NJ, USA) (Frezzato et al., 2014). The percentage of CD3+/

CCR7- TEM cells was 39.4±8% and of CD3+/CCR7+ cells was 60.6±8% in the 3 examined B-CLL patients (Figure 6D). The per-

centages of CD3+CCR7- and of CD3+CCR7+ cells in PBMCs from 3 subjects were 38±9% and 62±10%, respectively

(Figure S6F).

Western BlotFollowing lysis of cells, the pelleted membranes were resuspended in TES buffer and separated by SDS-PAGE in a 10% polyacryl-

amide gel containing 6 M Urea. Protein concentration was determined using the BCA method in a 96 well plate (200 mL total volume

for each well) incubating at 37�C in the dark for 30 min. Absorbance at 540 nm was measured by a Packard Spectra Count 96 well

plate reader. After separation by electrophoresis, gels were blotted overnight at 4�C onto Polyvinylidene fluoride (PVDF) membranes.

After blocking with a 10% solution of defattedmilk, themembranes were incubated with the following primary antibodies overnight at

4�C: anti-Kv1.3 (1:200, rabbit polyclonal, Alomone Labs APC-101); anti-GAPDH (1:1000, mouse monoclonal, Millipore MAB374). Af-

ter washing, themembraneswere developed using corresponding anti-mouse or anti-rabbit secondary antibodies (Calbiochem). The

antibody signal was detected with enhanced chemiluminescence substrate (SuperSignal West Pico Chemiluminescent Substrate,

Thermo Scientific).

Determination of Immune Cell SubpopulationsAt the end of in vivo experiments, 16 days after tumor cell injection and regular treatment with PAP-1 derivatives or cisplatin, as

described above, blood, thymus, spleen and iLNs were collected. Total cell number was determined in blood using a Burker cham-

ber. The other organs were mechanically dissociated to separate cells and also in this case total cells number was counted.

Following, 1x106 cells for each condition were labeled using the antibodies as indicated in Figure 5E for 15 min at 4�C in the dark.

Labeled cells were then analyzed by flow cytometry.

For the determination of immune cell number directly in the tumor (Figure S5D), after 10 days from tumor cells injection, mice

were injected i.p. with PAP-1 and its derivatives and then sacrificed. Each tumor was removed from the flank, and meshed in

1 ml cold PBS in a 12 well plate. After filtering and washing with PBS, cells were counted and 1 x 106 cells were used for each

FACS staining. Samples were blocked with True Stain Fc (1:100 in PBS) at 4�C first, and then the desired antibody mix was added

(prepared 2x, in PBS) with the same conditions. After washing, samples were analyzed by flow cytometry. Biotinylated antibodies

were further incubated with streptavidin-conjugated antibodies. FoxP3 staining was performed, according to the manufacturers

protocol. Samples were measured with a FACSCalibur (BD) instrument and analyzed using a BD CellQuest Pro software. Results

are shown as the x-fold change of the different populations in relation to the mean value of the untreated. The antibodies used are

listed in the following table.

Name and Dilution Clone Company

TruStain fcX (anti-mouse CD16/32) 1:100 Clone 93 Biolegend

CD3-PE 1:500 Clone 17A2 BD Bioscience

CD4-Fitc 1:1000 Clone GK1.5 eBioscience

CD8a-APC 1: 500 Clone 53-6.7 eBioscience

CD19-PE 1: 1000 Clone eBio1D3 eBioscience

MHCII I-Ab APC 1: 500 Clone AF6-120.1 eBioscience

CD25 Biotin 1: 1000 Clone 7D4 BD Biosciences

FoxP3-PE (Staining Set) 1: 400 Clone FJK-16s eBioscience

F4/80 Alexa Fluor488 1: 500 Clone BM8 eBioscience

CD11b Biotin 1: 2000 Clone M1/70 eBioscience

CD11c Fitc 1: 1000 Clone N418 eBioscience

CD204 Alexa Fluor 647 1:5 Clone 2F8 Bio-Rad

Ly-6G (Gr-1) APC 1:750 Clone RB6-8C5 eBioscience

PE-Streptavidin 1: 2000 BD Biosciences

APC-Streptavidin 1: 2000 BD Biosciences


Oxygen Consumption Assay and Activity of Respiratory Chain ComplexesRespiration was measured by using an XF24 Extracellular Flux Analyzer (Seahorse, Bioscience), which measures the oxygen con-

sumption rate (OCR) (Manago et al., 2015a, 2015b). Adherent B16F10 cells were seeded at 153103 cells/well in 200 mL of their culture

medium and incubated for 24h at 37�C in humidified atmosphere with 5% CO2. The medium was then replaced with 670 mL/well of

high-glucose DMEMwithout serum and supplemented with 1mM sodium pyruvate and 4mML-glutamine. The oxygen consumption

rate (OCR) wasmeasured with an extracellular flux analyzer (Seahorse) at preset time intervals upon the preprogrammed additions of

the following compounds: oligomycin to 1 mg/mL, FCCP to 300 nM, Antimycin A to 1 mM final concentrations. All chemicals were

added in 70 mL of DMEM. Amassive loss of cells because of death and detachment was excluded by direct microscopic observation

of the cells at the end of each experiment (not shown).

The activity of mitochondrial respiratory chain complexes and ATP synthase was assayed in vitro using rat liver mitochondriamem-

brane fractions as described below (Manago et al., 2015a).

Complex I activity: To assay NADH-CoQ oxidoreductase (complex I) activity, rat liver mitochondria (RLM) membrane fractions

(50 mg prot./mL) were incubated with 10 mM alamethicin, 3 mg/mL bovine serum albumin (BSA), 10 mM Tris–HCl (pH 8.0),

2.5 mM NaN3 and 65 mM coenzyme Q1 (CoQ1). In order to start the reaction, 100 mM NADH was added. Changes in absorbance

(340 nm) were monitored at 37�C using an Agilent Technologies Cary 100 UV–Vis spectrophotometer. After 6 min, 2 mM rotenone

was added to assess the rotenone- (and thus complex I-) independent activity to be subtracted. Complex III activity: To assay

CoQ cytochrome c oxidoreductase (complex III) activity, RLM (10 mg prot./mL) were added to a cuvette containing 50mMpotassium

phosphate buffer, pH 7.5, 10 mM alamethicin, 3 mg/mL BSA, 2.5 mM NaN3, 2 mM rotenone, 0,025% TWEEN, and 75 mM oxidized

cytochrome c. The reaction was started adding 75 mM of reduced decylubiquinol; changes in absorbance were monitored at

550 nm, 37�C. After 6 min, 2 mg/mL antimycin was added for assessment of complex III-independent activity. ATP-synthase activity:

Mitochondrial F0F1 ATPase activity was measured by coupling the production of ADP to the oxidation of NADH via the pyruvate

kinase and lactate dehydrogenase reaction (coupled assay). RLM (20 mg prot./mL) were added to a reaction mixture (pH 7.6) con-

taining 250 mM sucrose, 10 mM Tris–HCl, 200 mM EGTA–Tris, 1 mM NaH2PO4, 6 mM MgCl2, 2 mM rotenone, 10 mM alamethicin,

3 mg/mL BSA, 1 mM phosphoenol- pyruvate (PEP), 0.1 mM NADH, pyruvate kinase (PK; 20 units/mL), lactate dehydrogenase

(LDH; 50 units/mL). Absorbance was measured at 340 nm, 25�C. The addition of 500 mM ATP started the reaction; after 6 min,

1 mg/mL oligomycin A was added to evaluate F0F1-ATPase-independent ATP hydrolysis. Activities were evaluated from the changes

in the slope of the absorbance vs. time plot; data are expressed as % of the control (i.e., the activity without any addition of PAP-1

derivatives).

Mitochondrial Morphology, ROS Production and Membrane PotentialTo measure mitochondrial membrane potential and ROS levels, B cells either from CLL patients or from healthy subjects were incu-

bated for 20 min at 37�. After incubation, the compounds indicated in the figure legend were added and the decrease in TMRM fluo-

rescence or the increase in MitoSOX fluorescence, respectively, was measured by flow cytometry. The data reported are the median

values of the fluorescence intensity distributions (5,000 cells were counted). B16F10 cells (50,000 cells/well) were seeded on cover-

slips in a 12-well plate in 1 mL of their culture medium. After 24 h, cells were incubated with 20 nM TMRM or 1mMMitoSOX in HBSS

(Thermo Fisher Scientific) for 20 min at 37�C in the dark. After incubation compounds were added as indicated in the figure and the

decrease in TMRM fluorescence or the increase in MitoSOX fluorescence was followed at the indicated time points by fluorescence

microscopy using a Leica DMI 6000 fluorescencemicroscope equipped with confocal setup (LeicaMicrosystem,Wetzlar, Germany).

Nigericin has been reported to cause inner mitochondrial membrane hyperpolarization, without causing an acute effect on the cyto-

solic pH or on the plasma membrane potential (e.g. Akhmedov et al., 2010; Zhang et al., 2001).

To observe mitochondrial morphology in B16F10 cells as well as in human primary fibroblasts, mitochondria were stained with

200 nM Mitotracker green for 20 min at 37�C. Transmission electron microscopy was performed as described in Carraretto et al.,

2013. Briefly, samples were fixed overnight in a 2.5% v/v glutaraldehyde solution in 100 mM sodium cacodylate, pH 7.2, at 4�C.Following washing, postfixation was performed in a 1% OsO4 solution in 100 mM sodium cacodylate, pH 7.2, at 4�C. Sectionswere contrasted with a saturated uranyl acetate solution in 100% ethanol for 15 min, followed by incubation in a 1% w/v lead citrate

solution in 100% ethanol for 7 min. Finally, the samples were observed with a Tecnai G2 Spirit transmission electron microscope (Fei

electron microscopes) operating at 100 kV.

In Vivo Experiments and ImmunohistochemistryThe animal experiments and care complied with the institutional guidelines of institutional authorities (see above in ‘‘animal studies’’

section). For in vivo experiments, B16F10 or Colo357 cells were grown to sub-confluency in a medium as specified above. The cells

were detached with cell dissociation solution (Becton Dickinson, Heidelberg, Germany), washed twice in PBS and subcutaneously

injected into the right flank of C57BL/6Jmice in the case of themelanomamodel (Leanza et al., 2012), or injected into the pancreas of

SCID mice in the case of PDAC (Zaccagnino et al., 2016). For the melanoma model, the treatment with the derivatives PCARBTP,

PAPTP or PAP-1 at the indicated dosages was initiated at post-injection day 5 and repeated at days 7, 9 and 11. For experiments

with NAC,micewere pretreated with intraperitoneal injection of 0.7 mg/gbwNAC 1 hr before each injection of PAP-1 or its derivatives.

Tumors were removed 16 days after initiation and the tumor volumes were measured. Volume was determined as the product of

length, width and height. For the PDAC model, the orthotopic injection was performed as previously described (Tepel et al.,

2006). In detail, humanmetastatic pancreas adenocarcinoma cells Colo357 were detached with Accutase solution (Healthcare, Little


Chalfont, UK), resuspended at the concentration of 106 cells/mL in 25 mL of Matrigel (BD-Biosciences, Heidelberg, Germany) and

stored on ice. After median laparotomy, 25mL of cell suspension were injected into the tail of the pancreas. The animals were

randomly designated to the treatment procedure. The therapy was initiated ten days after tumor inoculation and spanned

20 days. For the therapy, the inhibitors were administered at the indicated dose intraperitoneally. The control group was treated

with a solution containing DMSO and physiological saline buffer. All animals were examined daily for general signs of distress and

complications. Thirty days after cell inoculation, the animals were sacrificed and tumor weight was determined after blood removal

(Zaccagnino et al., 2016). Tissues obtained in in vivo melanoma experiments were included and the Hematoxylin/Eosin staining and

TUNEL assay were performed following the following protocols (Leanza et al., 2012). Untreatedmice or those treated with PAP-1 and

its derivatives were sacrificed and immediately perfused at low pressure via the right heart with 0.9% NaCl for 2 min followed by

4% paraformaldehyde for 10 min. Organs, including the brain, heart, liver, kidney and spleen, were then removed and further fixed

in 4% paraformaldehyde for 36 h. Tissue was serially dehydrated and embedded in paraffin for sectioning at a thickness of 7 mm. The

sections were then dewaxed, rehydrated and incubated in 0.1M citrate buffer (pH 6.0) at 350W for 4min in amicrowave. The samples

were immediately cooled in PBS and incubatedwith TMR- or FITC-coupled dUTP in the presence of terminal deoxynucleotidyl-trans-

ferase (Roche Diagnostics) for 30 min at 37�C. They were then placed in 70�C PBS for 10 min and subsequently cooled. These sec-

tions were stained for 2 min with hematoxylin and washed with water prior to being mounted in Mowiol and evaluated using a Leica

TCS-SP2 microscope (Leica Microsystem, Wetzlar, Germany). Haematoxylin and eosin stainings were also performed with tissue

prepared as described above.

For TUNEL assay of tumor tissues, mice were injected with B16F10 cells and tumor was allowed to grow for 11 days. Mice were

either left untreated or treated with a single dose of PAPTP or PCARBTP (5 nmol/gbw and 10 nmol/gbw, respectively). After removal

of the tumor (24 hr following treatment with the compounds), analysis was performed as on healthy tissues.

Pharmacokinetic AnalysisTissue distribution of various compounds was assayed in C57BL/6J mice. PAP-1 and its derivatives at the reported dosages (see

legend to Figure 5A) were injected into control mice or into mice 11 days following injection of B16F10 cells (for tumor samples)

and 2, 4 and 8 h after the treatment the various tissues were collected, 100mgwere weighed, PBS (1 vol) was added, and themixture

containing tissue cut into small pieces was homogenized using an electric pestle. The samples were vortexed (2 min) and then sta-

bilized and extracted adding 0.43 M acetic acid, 100 mM 5-MOP as internal reference, acetone (v/v/v: 0.1, 0.1, 10), vortexed (2 min),

sonicated (2 min), and centrifuged (12,000 g, 7 min, 4�C); the supernatant was collected, concentrated, and finally analyzed via

HPLC-UV according to previously established protocols for other compounds (Azzolini et al., 2014).

ElectrophysiologyIC50 values were determined in whole-cell patch clamp experiments for Kv1.1, Kv1.3 and Kv1.5 expressed in CHO cells by Chantest

Ltd, UK and for Kv1.3 in Jurkat cells in our laboratory. In the case of CHO cells whole-cell peak current values following pulse appli-

cation to +50 mV from a holding potential of -50 mV were measured and used for determination of the concentration at which half of

the currentmeasured under control conditions was obtained. To test in Jurkat cells whether PAP-1 derivatives block the closed Kv1.3

channel or a post-activation state, we first applied a 400-ms depolarizing pulse to +70 mV to elicit a control Kv1.3 current and then

perfused the indicated concentrations of PAPTP or PCARBTP into the bath while the channel was closed. After a 3-min interval in

order to allow diffusion of PAP-1 derivatives to the cell interior, a set of depolarizing pulses to +70 mV were applied every 45 s

and the inhibitory efficiency was determined at steady–state block (Schmitz et al., 2005). IC50 values were determined by curve fitting

using the Origin Program set. Whole-cell currents were acquired with a EPC-7 amplifier (List, Darmstadt, Germany; sampling rate,

5 kHz; filter, 1 kHz), digitalized through a Axon DigiData 1322A (Molecular Devices), and stored on a computer. Currents were

compensated for pipette and membrane capacitance and for series resistance; leak currents were not subtracted. The bath solution

was composed, in mM, of 170 NaCl, 1 CaCl2, MgCl2, 10 HEPES, pH 7.4 with NaOH; the intracellular solution contained 134 KCl,

10 EGTA, 1 CaCl2, MgCl2, 10 HEPES, 10 glucose, pH 7.4 with KOH. Patch-clamp recordings were analyzed using the Clampfit

8.1 software from the pClamp suite (Molecular Devices).

ElectrocardiographyA cohort of twelve six months-old C57Bl/6J male mice was chosen for electrocardiography experiments and divided in four groups:

each group was randomly assigned to a compound testing or to the negative control (i.e. injection with PBS plus DMSO). Electro-

cardiography (ECG) was performed under inhalation anesthesia: each mouse was anesthetized by a mixture of isoflurane (Tanaka

and Nishikawa, 1999) and oxygen, delivered by inhalation with a proper mask. A slight increase in RR is a well-known effect of

isoflurane (Tanaka and Nishikawa, 1999). Once anesthesia state was raised and verified, by absence of the paw withdrawal reflex,

two small needles were inserted subcutaneously in the mouse anterior paws (explorative electrodes) and one in the posterior left

one (reference electrode). ECG was performed by a PowerLab 8/35 (ADInstruments), and acquired by LabChart 7 Pro software

(ADInstruments) at a frequency of 10 kHz, with no filters. ECG was performed under control conditions for 5 min, and afterwards

the chosen compounds was injected intraperitoneally; the recording was continued for 30 min after injection. PAPTP, PAP-1, and

PCARBTP were diluted in PBS and administered at the following doses: 5 nmol/gbw, 20 nmol/gbw, and 10 nmol/gbw, respectively.

Recordings were analyzed with LabChart 7 Pro software (ADInstruments), averaging every 20 heartbeats and analyzing singularly

every average for identifying P, Q, R, S, and T waves. RR and PR intervals, as well as QRS duration and corrected QT interval (QTc)


weremeasured in control and in the presence of the compound/vehicle for eachmousewithin each group; the values were then aver-

aged for each group. Statistically significant difference between each couple (control and compound/vehicle) was assessed by

paired t-test. Statistically significant difference between the four groups was then assessed for each parameter by means of two

ways ANOVA analysis.

Mitochondrial DNA (mtDNA) QuantificationWe determined mtDNA copy numbers with the TaqMan probe system and Applied Biosystem 7500 realtime PCR as described

in (Franzolin et al., 2015). Genomic DNA was extracted by Puregene Core kit B (Qiagen) from human fibroblasts maintained in

culture for 6 days in the continuous presence of 0.1 mM PAPTP. Mitochondrial rRNA 12S TaqMan probe 6FAM-50-TGCCAGCC

ACCGCG-30-MGB (Applied Biosystems) and primers rRNA 12S forward (50-CCACGGGAAACAGCAGTGATT-30) and reverse (50-CTATTGACTTGGGTTAATCGTGTGA-30) were used to quantify mtDNA. For nuclear DNA, we used RNase P primers and probe VIC mix

(Applied Biosystems). To quantify mtDNA and nuclear DNA we used calibration curves generated by serial dilution of a mixture of

plasmids carrying the two PCR amplicons. Each DNA sample was analyzed in triplicate.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistically significant difference between each couple (control and compound/vehicle) was assessed by paired t-test. Statistically

significant difference between the four groups was then assessed for each parameter by means of two-way ANOVA analysis. Box

plots were obtained using the Origin6.1 Program Set.


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