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MacDonagh, Lauren, Gray, Steven, Breen, Eamon, Cuffe, Sinead, Finn,Stephen, O’Byrne, Ken, & Barr, Martin(2018)BBI608 inhibits cancer stemness and reverses cisplatin resistance inNSCLC.Cancer Letters, 428, pp. 117-126.

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https://doi.org/10.1016/j.canlet.2018.04.008

Accepted Manuscript

BBI608 inhibits cancer stemness and reverses cisplatin resistance in NSCLC

Lauren MacDonagh, Steven G. Gray, Eamon Breen, Sinead Cuffe, Stephen P. Finn,Kenneth J. O'Byrne, Martin P. Barr

PII: S0304-3835(18)30269-6

DOI: 10.1016/j.canlet.2018.04.008

Reference: CAN 13848

To appear in: Cancer Letters

Received Date: 5 February 2018

Revised Date: 30 March 2018

Accepted Date: 5 April 2018

Please cite this article as: L. MacDonagh, S.G. Gray, E. Breen, S. Cuffe, S.P. Finn, K.J. O'Byrne, M.P.Barr, BBI608 inhibits cancer stemness and reverses cisplatin resistance in NSCLC, Cancer Letters(2018), doi: 10.1016/j.canlet.2018.04.008.

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Abstract

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths

worldwide. While partial or complete tumor regression can be achieved in patients,

particularly with cisplatin-based strategies, these initial responses are frequently short-

lived and are followed by tumor relapse and chemoresistance. Identifying the root of

cisplatin resistance in NSCLC and elucidating the mechanism(s) of tumor relapse, is of

critical importance in order to determine the point of therapeutic failure, which in

turn, will aid the discovery of novel therapeutics, new combination strategies and a

strategy to enhance the efficacy of current chemotherapeutics. It has been

hypothesized that cancer stem cells (CSCs) may be the initiating factor of resistance.

We have previously identified and characterized an aldehyde dehydrogenase 1 CSC

subpopulation in cisplatin resistant NSCLC. BBI608 is a small molecule STAT3 inhibitor

known to suppress cancer relapse, progression and metastasis. Here, we show that

BBI608 can inhibit stemness gene expression, deplete CSCs and overcome cisplatin

resistance in NSCLC.

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BBI608 inhibits cancer stemness and reverses cisplatin resistance in NSCLC

Lauren MacDonagh1, Steven G. Gray

1, Eamon Breen

2, Sinead Cuffe

1,3, Stephen

P. Finn1,4

, Kenneth J. O’Byrne1,5

and Martin P. Barr1.

1. Thoracic Oncology Research Group, School of Clinical Medicine, Trinity Translational

Medicine Institute, Trinity Centre for Health Sciences, St. James’s Hospital and Trinity

College Dublin, Ireland.

2. Flow Cytometry Facility, Trinity Translational Medicine Institute, Trinity Centre for

Health Sciences, St. James’s Hospital and Trinity College Dublin, Ireland.

3. Medical Oncology, St. James’s Hospital, Dublin, Ireland

4. Department of Histopathology, St. James’s Hospital and Trinity College Dublin,

Ireland.

5. Cancer & Ageing Research Program, Queensland University of Technology, Brisbane,

Australia.

Corresponding author: Dr Martin Barr, Thoracic Oncology Research Group, Trinity

Translational Medicine Institute, Trinity Centre for Health Sciences, St James’s Hospital,

Dublin 8, Ireland. Tel: 00-353-1-8963620; Email: mbarr@stjames.ie

Keywords: Non-small cell lung cancer, chemotherapy, cisplatin resistance, cancer stem cells,

aldehyde dehydrogenase, BBI608

Abbreviations: NSCLC: non-small cell lung cancer; ALDH: aldehyde dehydrogenase; CSC:

cancer stem cell; STAT3: signal transducer and activator of transcription 3

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Acknowledgments: The authors would like to thank the Thoracic Oncology Research Group,

St. James’s Hospital & Trinity College Dublin, for their scientific discussion and support.

Funding: This study was funded by Molecular Medicine Ireland (MMI) as part of the Clinical

& Translational Research Scholars Programme (CTRSP) under Cycle 5 of the Irish

Governments Programme for Research in Third Level Institutions (PRTLI) and co-funded

under the European Regional Development Fund (ERDF).

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1. Introduction

Lung cancer is the leading cause of cancer-related death worldwide, where non-small-

cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. NSCLC is

further subdivided into three histological subtypes; adenocarcinoma, squamous cell

carcinoma and large cell carcinoma [1, 2]. Despite the significant advances in personalized

medicine and the development of therapeutics specifically targeting driver mutations,

platinum-based chemotherapy remains the cornerstone of current NSCLC management

following its FDA-approval in 1978 [3]. Since then cisplatin has changed the face of solid

malignancy management and in particular the therapeutic management of lung cancer. To

date, platinum-based agents have been repeatedly described in the literature as the

cytotoxic foundation of many combination chemotherapeutic strategies in the treatment of

lung cancer. Studies have compared platinum- and non-platinum-based chemotherapy

strategies when treating advanced NSCLC and response rates were shown to be significantly

higher following platinum-based therapy [4, 5]. However, despite the accolades which

cisplatin has received, NSCLC 5-year survival rates remain dismal at <15% [6, 7]. When

treating NSCLC an initial response is observed following cisplatin exposure, however, in

many cases this initial response is not sustained and instead resistance begins to emerge,

thereby contributing to relapse and the poor 5-year survival [7]. Unfortunately, in current

clinical practice the development of cisplatin resistance has become a major clinical

challenge in the treatment and management of lung cancer patients. Alternative strategies

to overcome cisplatin resistance and identification of new and novel therapies are of vital

importance in order to enhance the therapeutic efficacy of currently available cytotoxic

drugs and to identify new drugs with more promising anti-cancer properties.

Cisplatin resistance is multifactorial in nature and the mechanisms involved in

inherent and acquired resistance are avenues of intense interest that may be targeted to

overcome this drug resistance phenotype. The cancer stem cell (CSC) hypothesis suggests

that a rare population of cells exists within tumors which display stem-like characteristics

such as increased expression of stemness-associated markers and the abilities of self-

renewal and differentiation, both of which are essential for tumor initiation, maintenance,

progression and metastasis [8, 9]. Cellular heterogeneity is a histological hallmark of many

solid tumors [10-12]. As such, the CSC hypothesis suggests that the heterogeneity observed

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in phenotypically diverse tumors may arise due to hierarchical cell dynamics produced as a

result of asymmetric division and differentiation of this rare CSC population [8, 9, 13, 14].

The ability of CSCs to asymmetrically divide enables these cells to simultaneously self-

perpetuate and generate differentiated progeny, thus giving rise to a heterogeneous tumor,

with a consistently maintained CSC population [15-17]. Furthermore, it has previously been

shown that the CSC population survives and thrives during periods of stress, thereby

resulting in the expansion of the highly resistant CSC subset during chemotherapeutic

exposure [18]. The existence of lung CSCs could explain why tumors display resistance to a

broad spectrum of chemotherapeutic agents that target and kill the bulk of the tumor but

induce the expansion and enrichment of the CSC subset [19-24].

Members of the aldehyde dehydrogenase (ALDH) family of cytosolic isoenzymes are

responsible for oxidising intracellular aldehydes, where they contribute to the oxidation of

retinol to retinoic acid in early stem cell differentiation [25]. Haematopoietic and neural

stem cells display high ALDH activity [26, 27]. Increased ALDH1 activity has been reported in

stem cell populations and more recently, it has been identified as one of the most promising

and universal CSC markers in a number of malignancies, including lung cancer [28-32].

Enhanced ALDH1 activity has been identified across a number of cisplatin resistant NSCLC

cell lines and tumor samples. Previous studies in our laboratory have identified ALDH1-

positive (ALDH1+ve) subpopulations in cisplatin resistant NSCLC sublines which are not

present in their corresponding sensitive counterparts [18]. These ALDH1+ve subpopulations

have been characterized and shown to exhibit numerous properties typical of CSCs,

including enhanced chemoresistance, the ability to asymmetrically divide and give rise to a

differentiated progeny while maintaining a niche CSC subpopulation, as well as the

increased gene expression of stemness markers (Nanog, Oct-4, Sox-2, Klf4 and cMyc) [18,

32-34]. Identification of this ALDH1+ve CSC subset within an in vitro model of cisplatin

resistant NSCLC has provided a foundation on which to further investigate the role of CSC

inhibition, manipulation and exploitation in cisplatin resistance [18]. ALDH1, therefore,

provides a promising and therapeutically viable CSC target, inhibition of which, in

combination with cytotoxic chemotherapeutics may result in the effective killing of this CSC

population in resistant tumors, thereby limiting relapse and tumor recurrence.

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Throughout the literature a number of other subpopulations have been identified and

characterized as CSC subsets in NSCLC, including CD117, CD44, CD133 and Hoechst-negative

Side Population [35-38]. Inhibition of ALDH1 and other CSC markers have shown promise in

the treatment of therapeutically resistant NSCLC [20, 22, 32, 34]. However, the multitude of

CSC markers in conjunction with the concepts of CSC hierarchy, tumor heterogeneity and

plasticity known drivers of chemoresistance, suggests that stem cell inhibition at a more

fundamental gene level may be a more practical and robust approach than superficially

targeting surface and enzymatic CSC markers, which are numerous and variable across solid

malignancies and histological subtypes [39-41]. We hypothesized that cancer stemness

inhibition at the point of gene transcription using the cancer stemness inhibitor, BBI608,

first reported in the literature by Li et al., could effectively deplete the CSC population

thereby reversing cisplatin resistance in NSCLC [42].

BBI608 is a small molecule inhibitor of signal transducer and activator of transcription

3 (STAT3), a critical mediator for the maintenance of cancer stemness, yet it is largely

dispensable in haematopoietic stem cells [42]. In this study, we show that BBI608 decreased

the ALDH1+ve CSC population while decreasing the mRNA expression of critical stemness

genes, resulting in the re-sensitization of chemoresistant lung cancer cells to the cytotoxic

effects of cisplatin. Exposure of the cisplatin resistant cell lines to BBI608 in combination

with cisplatin exacerbated the functional effects observed with BBI608 alone. These data

suggest that targeting CSCs in the context of cisplatin resistant NSCLC via stemness

inhibition using BBI608, in combination with cisplatin, may be an effective and novel

approach in the management of cisplatin resistant NSCLC patients.

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2. Materials and Methods

2.1 Drugs

Cisplatin was purchased from Sigma-Aldrich and dissolved in 0.15M NaCl. The cancer

stemness inhibitor, BBI608, was purchased from Abcam (Cambridge, UK) and dissolved in

dimethyl sulfoxide (DMSO).

2.2 Cell lines

The human large cell carcinoma cell line, NCI-H460 (hereafter referred to as H460)

and its resistant variant were kindly donated by Dr Dean Fennell, Centre for Cancer

Research and Cell Biology, Queen’s University Belfast [43]. The human adenocarcinoma cell

line, H1299, and its resistant subline were given as a gift from Dr Parviz Behnam-Motlagh,

Department of Medical Biosciences, Umeå University, Sweden. The SKMES-1 squamous cell

carcinoma cell line was purchased from the American Type Culture Collection (ATCC) (LGC

Promochem, UK). Cisplatin resistant (CisR) sublines were generated from each original

parental (PT) cell line by continuous exposure to cisplatin, as previously described [44].

Briefly, cells were treated with cisplatin (IC50) for 72hrs, after which time cisplatin-containing

media was removed and cells were allowed to recover for a further 72hrs. This development

period was carried out for 6 months, after which time IC50 concentrations were reassessed

and used as a maintenance dose for a further 6 months. H460 cells were grown in Roswell

Park Memorial Institute (RPMI-1640) media. H1299 and SKMES-1 cells were maintained in

Eagle’s Minimum Essential Medium (EMEM) supplemented with 2mM L-glutamine and 1×

non-essential amino acid (NEAA). For all cell lines, media was supplemented with 10% heat-

inactivated foetal bovine serum (FBS), penicillin (100U/ml) and streptomycin (100μg/ml)

(Lonza, UK). All cell lines were grown as monolayer cultures and maintained in a humidified

atmosphere of 5% CO2 at 37°C [44]. Cell lines were routinely tested for mycoplasma and

were previously tested and authenticated using the PowerPlex

16 HS System (Source

BioScience, UK).

2.3 Aldefluor assay

The Aldefluor assay (Stem Cell Technologies, Canada) was used to identify and isolate

cell populations with ALDH1 enzymatic activity. The assay was carried out according to

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manufacturer’s instructions. Briefly, cells (5 × 105) were suspended in Aldefluor assay buffer

containing activated Aldefluor reagent, BODIPY-aminoacetaldehyde (BAAA) for 45 mins. The

Aldefluor reagent is a fluorescent non-toxic ALDH1 substrate that freely diffuses into intact

viable cells. In the presence of ALDH1, BAAA is converted to BOPIDY-aminoacetate (BAA),

which is retained within the cells expressing ALDH1. A specific ALDH1 inhibitor, DEAB, was

used to inhibit the BAAA-BAA conversion and acts as an internal negative control for

background fluorescence. The brightly fluorescent ALDH1+ve cells were detected using the

green fluorescence channel (520-540nm). ALDH1 activity was measured using a CyAnTM

ADP

flow cytometer (Dako, USA). The ALDH1+ve cell subset from each cisplatin resistant NSCLC

subline acted as the in vitro model of CSCs following previous characterization and

confirmation in our laboratory [18].

2.4 Reverse transcriptase polymerase chain reaction (RT-PCR)

Total RNA was extracted using TRI Reagent (Molecular Research Center, OH, USA)

according to manufacturer’s instructions. cDNA was generated using the SuperScript III

reverse transcriptase kit (Invitrogen) and Oligo dT(20) primers (Eurofins MWG Operon,

Ebersberg, Germany) according to the manufacturer’s instructions. Gene expression analysis

(mRNA) of stem cell-associated and CSC markers was carried out using the primer

sequences (Table I). Template cDNA was initially denatured at 95°C for 5mins, followed by

35-40 amplification cycles consisting of denaturation at 95°C for 1min, primer-specific

annealing for 1min and extension at 72°C for 1min. Cycles were followed by an elongation

step of 72°C for 10mins. PCR products were resolved on 2% agarose gels containing

ethidium bromide. Images were acquired using the Fusion FX imaging system (Vilber

Lourmat, Germany). Product quantification was performed using ImageJ densitometry

software. Gene expression was normalized to endogenous β-actin controls and was

expressed as fold-change.

2.5 Cell proliferation

Cell proliferation was measured using a Cell Proliferation BrdU ELISA (Roche

Diagnostics Ltd., UK), according to manufacturer’s instructions. Briefly, cells (H460, H1299

and SKMES-1) were seeded at 2.5 × 103cells/well in a 96-well plate. Following overnight

incubation, cells were treated for 72hrs with cisplatin (0-100μM) alone or in combination

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with BBI608 (1 μM) [45]. Absorbance was recorded at 450 nm and sensitivity to BBI608 and

cisplatin was calculated based as a percentage of cell proliferation relative to untreated

controls, which were set at 100%.

2.6 Clonogenic survival

Cells in the exponential growth phase were harvested by trypsinization. Cell

suspensions were adjusted to optimal cell numbers in complete medium and seeded in 6-

well plates. Cell seeding densities were optimized to ensure at least 50 viable colonies were

visible at the end of the clonogenic incubation period (Supplementary file). A positive colony

is defined to consist of at least 50 cells as previously described [46]. Cells were treated with

increasing concentrations of cisplatin (0-10μM) for 72hrs, alone or in combination with

BBI608 (1μM), after which time, culture media was removed and replaced with fresh

treatment-free media and re-incubated for a further 10 days. Colonies were fixed and

stained with 25% (v/v) methanol, 0.05% (w/v) crystal violet for 30 mins. Residual stain was

removed by rinsing wells gently with tap water. Colonies were counted using the

ColCountTM

colony counter (Oxford Optronix Ltd, Oxford, UK). Plating efficiencies (PE) were

calculated using the formula: PE = Number of colonies/Number of cells seeded. The

percentage surviving fraction (SF) was calculated using the formula: SF = (PE treated

colonies/PE untreated) ×100.

2.7 Apoptosis

Apoptosis was measured by flow cytometry using dual Annexin V and propidium

iodide (PI) staining to identify early and late apoptotic cells from live and necrotic cells [47].

Briefly, cells (1 × 105) were seeded in 6-well plates and treated with increasing

concentrations of cisplatin (0-100μM) alone, or in combination with BBI608 (1μM) in cell

culture media for 48hrs. Untreated control cells were treated with media only. Following

treatments, supernatants and adherent cells were collected, washed and stained with

Annexin V-FITC (IQ Products, The Netherlands) and 1µg/ml PI (Invitrogen). Apoptotic cells

were measured using a CyAnTM

ADP flow cytometer (Dako, USA). Forward scatter versus

side scatter plots were used to eliminate debris, while forward scatter versus pulse width

plots isolated single cells for analysis.

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2.8 Statistical Analysis

Analysis between groups was tested using analysis of variance (ANOVA). Statistical

comparison of two means was carried out using an unpaired two-tailed Student’s t-test.

Significance was defined as p≤0.05. Data are graphically represented as mean ± standard

error of the mean (SEM). All data was analysed using GraphPad InStatTM

(version 5)

statistical software.

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3. Results

3.1 BBI608 depletes ALDH1+ve CSC populations

Following characterization and confirmation of cisplatin resistance in the CisR NSCLC

sublines relative to their PT counterparts, H460, H1299 and SKMES-1 NSCLC cell lines were

investigated for the presence of a CSC population [18, 44]. As shown in Figure 1, a greater

proportion of ALDH1+ve cells were observed across the CisR sublines relative to their

corresponding PT controls. Isolation and characterization of ALDH1+ve subpopulations was

previously carried out in our laboratory, confirming the stem-like nature of the ALDH1+ve

cells across a number of parameters when compared to the ALDH1-negative (ALDH1-ve)

bulk cell populations [18].

Treatment of the CisR sublines with BBI608 (1μM) significantly decreased the

presence of the ALDH1+ve CSC population in each CisR subline to levels comparable to

those observed in the corresponding parental (PT) cells. BBI608 significantly reduced the

ALDH1+ve population of the large cell carcinoma H460 CisR subline from 2.81% to 0.03%

(p<0.01). Similar effects were observed upon treatment of the H1299 and SKMES-1 CisR

sublines with BBI608, which resulted in a significant decrease in ALDH1+ve cell subsets in

both resistant sublines by, 9.66% (p<0.001) and 14.04% (p<0.001), respectively. These data

indicate the potential of BBI608 to deplete the ALDH1+ve CSC populations present in

cisplatin resistant NSCLC cells.

3.2 BBI608 down-regulates the expression of stemness-associated genes

To determine whether BBI608 influenced the stemness potential of the CisR sublines

at a transcriptional level, mRNA expression of a number of embryonic stem cell markers

(Nanog, Oct-4, Sox-2, Klf4 and cMyc) and CSC-associated genes (CD133 and ALDH1) were

examined by end-point reverse transcriptase-PCR (Figure. 2).

Exposure of the CisR sublines to BBI608 altered their stemness gene expression

profile. Treatment of the H460 CisR subline with BBI608 significantly decreased expression

of a number of stemness factors, including Nanog (5.26-fold, p<0.05), Oct-4 (3.70-fold,

p<0.05), Sox-2 (1.79-fold, p<0.05) and cMyc (12.5-fold, p<0.001), in addition to the CSC-

specific markers CD133 (4.35-fold, p<0.05) and ALDH1 (13.33-fold, p<0.05). In the H1299

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CisR subline, BBI608 showed a less remarkable effect on the expression of stemness-

associated genes, where Oct-4 and cMyc were significantly down-regulated, 1.3-fold

(p<0.05) and 1.54-fold (p<0.01), respectively. Interestingly, BBI608 significantly increased

Sox-2 mRNA expression in H1299 CisR cells relative to untreated controls (p<0.001), in

contrast to that observed for H460 and SKMES-1 CisR sublines. In both H460 and H1299 CisR

sublines, no significant effects were observed on Klf4 gene expression in response to

BBI608. Treatment of the cisplatin resistant squamous cell carcinoma, SKMES-1 subline with

BBI608 yielded similar results, where BBI608 induced a significant down-regulation of Nanog

(10.31-fold, p<0.01), Oct-4 (1.33-fold, p<0.01), Sox-2 (7.14-fold, p<0.01) and ALDH1 (9.09-

fold, p<0.001). In contrast, however, BBI608 significantly increased Klf-4 gene expression in

this subline (p<0.01).

Taken together, these data are largely in agreement with that reported in the current

literature suggesting that BBI608 depletes CSCs and down-regulates stemness and CSC-

associated genes at the transcriptional level [42, 48].

3.3 BBI608 re-sensitizes chemoresistant NSCLC cells to cisplatin and inhibits cell

proliferation

To assess the effect of BBI608 on the proliferative capacity of cisplatin resistant NSCLC

sublines and its ability to restore cisplatin sensitivity, CisR sublines were treated with

increasing concentrations of cisplatin, alone and in combination with BBI608 (1μM). The

effects on cell proliferation and cisplatin IC50 concentrations were determined relative to

untreated cells. IC50 concentrations (Table II) were used as a confirmatory measure of

cisplatin resistance of the CisR sublines relative to their PT sensitive counterparts.

BBI608 significantly decreased the proliferative capacity across all CisR sublines

(Figure. 3) as an independent treatment relative to untreated cells. BBI608 was introduced

in combination with cisplatin in a dose-escalation study and assessed relative to cisplatin

alone. BBI608 in combination with cisplatin significantly inhibited cell proliferation across all

resistant NSCLC sublines representing each histological subtype of this cancer type. These

data were reflected in IC50 concentrations (Table II), in which the combination treatment of

the CisR sublines with BBI608 and cisplatin reduced the cisplatin IC50 concentrations to less

than those observed in their cisplatin-only treated cisplatin sensitive counterparts. While

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BBI608 alone significantly reduced cell proliferation of all CisR sublines, cisplatin (10-100μM)

in combination with BBI608 significantly enhanced this anti-proliferative effect in the highly

cisplatin resistant and aggressive H1299 CisR subline relative to BBI608 alone.

Collaboratively, these data support our hypothesis that BBI608 warrants further

investigation as a novel agent in the re-sensitization of NSCLC cells to the cytotoxic effects of

cisplatin. Importantly, these significantly decreased changes in cisplatin IC50 concentrations,

when used in combination with BBI608, may be indicative for this combination treatment as

an effective clinical approach for frontline therapy in lung cancer. In doing so, such a

treatment strategy could potentially delay or indeed prevent the development of platinum

resistance in these patients.

3.4 BBI608 decreases the presence of a robustly resistant subpopulation of NSCLC cells by

reducing clonogenic survival

To determine the presence of a robustly resistant population within the cisplatin

resistant NSCLC sublines that are capable of survival and expansion following exposure to

cisplatin, cells were treated with either cisplatin alone or in combination with BBI608 (1μM).

The clonogenic survival ability of H460, H1299 and SKMES-1 CisR sublines were assessed

relative to untreated cells (Figure.4).

Treatment of the CisR sublines with BBI608 alone significantly depleted the surviving

cell fraction relative to untreated cells. Similarly, when resistant cell lines were treated with

BBI608 in combination with increasing concentrations of cisplatin (0.1-10μM), the surviving

fraction was significantly reduced relative to untreated cells across each CisR subline.

Moreover, combination treatments decreased clonogenic survival to a significantly greater

extent than BBI608 alone across CisR sublines. Together, these data highlight the potential

benefit of combining BBI608 with current chemotherapy drugs such as cisplatin to decrease

NSCLC survival and as a means of overcoming cisplatin resistance.

3.5 BBI608 circumvents therapeutic resistance and induces apoptotic cell death

In order to assess the potential of BBI608 to induce lung cancer cell death, apoptosis

was measured by Annexin V/PI staining and flow cytometry following treatment with

BBI608 (1μM) alone and in combination with increasing concentrations of cisplatin (0-

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100μM). Apoptotic cell death was assessed relative to untreated controls and cisplatin-only

treated cells (Fig. 5).

Treatment of resistant sublines with BBI608 as a lone therapeutic agent significantly

induced apoptotic cell death across all NSCLC subtype cell line representatives, albeit to

varying degrees of cell death. The greatest induction of BBI608-associated cell death was

observed in the H460 CisR subline, with 99.27±0.03% (p<0.001) cells undergoing apoptosis

relative to untreated controls, indicating particular sensitivity of the cisplatin resistant large

cell carcinoma subline to the cytotoxic effects of BBI608. BBI608 alone significantly induced

apoptotic cell death in H1299 and SKMES-1 CisR sublines, 42.05±4.84% (p<0.001) and

52.14±1.47 (p<0.001), respectively. When investigated in combination with increasing

concentrations of cisplatin (1-100μM), significant increases in apoptosis were observed in

each CisR subline relative to cisplatin alone.

BBI608 significantly induced apoptosis when used as a single therapeutic agent.

Further statistical comparisons were applied to determine the pro-apoptotic effect of

combining BBI608 with cisplatin relative to BBI608 alone. This cisplatin-BBI608 combination

significantly increased the percentage of apoptotic cells in H1299 CisR cells at

concentrations of cisplatin ranging from 40-100μM relative to BBI608 alone. A similar effect

was observed at all concentrations of cisplatin in the SKMES-1 CisR subline. Cumulatively,

these data indicate that BBI608 may have therapeutic potential as either a monotherapy or

as part of a combination strategy and further supports our hypothesis that BBI608 may aid

in the circumvention of cisplatin resistance in NSCLC cells.

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4. Discussion

Lung cancer is the most common cancer worldwide and the greatest contributor to

cancer-related death [2, 49]. The current efficacy of lung cancer treatment is hampered by

the development of resistance that renders current therapeutics ineffective. The cancer

stem cell population has been suggested as the root cause of resistance; however numerous

CSC subpopulations have been identified across multiple cancer types. This variability in the

presentation and identification of CSCs means that CSC population identifiers or cell surface

markers may not be the most viable or robust therapeutic target. With this in mind, a broad

spectrum CSC inhibitor, such as BBI608 may hold the greatest promise as an inhibitor of this

robustly resistant subpopulation.

This study investigated the effects of the small molecule stemness/STAT3 inhibitor,

BBI608, on the expression of critical genes involved in the maintenance of cancer stem cells.

In the seminal publication by Li et al, BBI608 was initially introduced and investigated as a

stemness inhibitor in the context of tumor relapse in a xenograft model of pancreatic

cancer, where its effects were compared to the chemotherapeutic agent, gemcitabine [42].

Gemcitabine inhibited the growth of PaCa-2 xenograft tumors, however, following cessation

of gemcitabine treatment, animals soon relapsed and further tumor growth was recorded.

Similarly, BBI608 significantly inhibited tumor growth compared to untreated controls.

However, unlike gemcitabine following cessation of BBI608 treatment, no relapse of tumor

regrowth was observed for the duration of the study [42]. Li et al. utilised an intra-splenic

nude mouse model system (ISMS) to evaluate the anti-metastatic potential of BBI608. In

this model, colon cancer cells (HT29) were injected into the splenic capsule of nude mice,

where these cancer cells have the ability to spontaneously metastasise to the liver. Using

this model, BBI608 was found to effectively inhibit metastases to the liver and spleen in the

ISMS model [42]. Treatment of the CSC Side Population and non-CSC cells with doxorubicin

showed resistance within the CSC population, while treatment with BBI608 resulted in cell

death in both CSC and non-CSC cell populations, with increased sensitivity observed within

the CSC population. BBI608 inhibited expression of a number of stemness genes, including

Nanog, Oct-4, Sox-2, Klf-4 and cMyc, in addition to the CSC marker, ALDH1 [42]. Krüppel-like

factor 4 (Klf4) is a transcription factor with opposing roles in different human cancers. Its

overexpression in several cancers is correlated with a poor prognosis. The expression and

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role of Klf4 in lung cancer however, remains unclear. It is a bifunctional transcription factor

that can either activate or repress transcription using different mechanisms, depending on

the target gene. Therefore, depending on the cell type or cell context, Klf4 may act either as

a tumor suppressor gene or as an oncogene. In our study, Klf4 mRNA remained unchanged

in H460 and H1299 CisR sublines but was significantly increased the squamous cell SKMES-1

CisR cell line in response to BBI608. Klf4 has been reported to function as a tumor

suppressor gene in lung cancer. Its expression was downregulated in 21 of 25 primary lung

cancers and ectopic expression of Klf4 suppressed lung cancer cell proliferation and

clonogenic formation in vitro. Moreover, transfection of lung cancer cells with the Klf4 gene

also suppressed tumor growth in vivo [50]. Our findings that BBI608 induced Klf4 gene

expression in SKMES-1 cisplatin resistant cells may be a consequence, at least in part, of the

molecular mechanism underlying the tumor-suppressive function of this gene in squamous

cell lung cancer. Further studies however are warranted to further explore this possibility, as

only few studies have investigated the role and differences in expression of Klf4 among the

different histological subgroups of lung cancer.

BBI608 was investigated in relation to cancer progression by Zhang et al [48] in

prostate cancer. BBI608 inhibited cell proliferation, colony formation and migration, while

increasing the sensitivity of prostate cancer cells to the cytotoxic effects of docetaxel.

BBI608 decreased the presence of CD44+/CD133

+ CSCs while inhibiting stemness gene

expression and decreased the ability of the stemness-high cancer cell subpopulation to grow

spheres on ultra-low attachment plates. In a xenograft model of prostate cancer using the

cell lines, PC-3 and 22RV1, BBI608 inhibited tumor growth relative to untreated or docetaxel

alone [48]. Following these initial preclinical studies of BBI608 in the literature, phase II and

III clinical trials followed, many of which are ongoing across a number of advanced

malignancies, and in combination with numerous chemotherapeutic agents (Table III) [42,

48]. BBI608 is currently being investigated in relation to gastric cancer and gastric cancer

CSCs in the phase III BRIGHTER trial, following on from the reported phase I and II results

[51, 52]. Alternate STAT3 inhibitors have shown similar effects to BBI608, PMMB-187

inhibited constitutive/inducible STAT3 activation, transcriptional activity, nuclear

translocation and downstream target gene expression in STAT3-dependent breast cancer

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cells MDA-MB-231, while dramatically suppressing MDA-MB-231 xenograft tumor growth

[53].

Many stem-like cells commonly overexpress markers such as Nanog, Oct-4, Sox-2, Klf4

and cMyc, where these genes play important roles in the regulation of self-renewal and

tumorigenicity in CSC populations of several cancer types. However, a growing body of CSC

research has also highlighted a number of CSC-specific markers that have been shown to

display a stem-like phenotype, and include ALDH1 and CD133 [54, 55], the expression of

which has been reported to be altered in a number of solid tumors in response to therapy.

In a study of oral squamous cell carcinomas (OSCC), CSC-like markers expressed in cisplatin

resistant oral carcinomas such as Nanog and Oct-4 became expanded during the acquisition

of cisplatin resistance in OSCC. It was postulated based on these findings that

overexpression of these stemness markers may promote cisplatin resistance in OSCCs that

subsequently recur [56]. A recent study from our laboratory suggests that ALDH1+ve cells

not only correlate with acquired cisplatin resistance but out-survive their ALDH1-ve

counterparts during cisplatin therapy. We reported that relative to ALDH1-ve

subpopulations isolated from chemoresistant NSCLC cell lines, ALDH1+ve subsets had

significantly increased proliferative and survival abilities when challenged with cisplatin

chemotherapy. In addition to this observed increase in cisplatin resistance, an up-regulation

of stemness genes and CSC makers such as ALDH1 and CD133 was shown [18]. ALDH1

overexpression is associated with poor prognosis in NSCLC patients and high ALDH1

expression is significantly associated with a more aggressive and advanced pathological

grade and stage [33]. Furthermore, increased ALDH1 expression has been associated with

increased metastasis in multiple cancers, including inflammatory breast cancer [57], [58].

While studies examining a direct link between ALDH1 expression and response to anti-

cancer therapies are more limited, some have examined cohorts of patients undergoing

neoadjuvant chemotherapy, chemoradiotherapy or radiotherapy followed by complete

surgical resection. In one study, the 5-year overall survival rate of patients with CD133-

positive or ALDH1-positive tumors was significantly worse than that of patients with both

CD133-negative and ALDH1-negative expression (44.9% vs. 90.0%, respectively;

p=0.042)[59]. The expression of these CSC markers following chemoradiotherapy (CRT)

correlated significantly with a poor prognosis in NSCLC patients. A multivariate analysis also

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identified expression of ALDH1 in NSCLC patients as a significant independent prognostic

factor for disease-free survival [60]. The authors of this study reported that the 5-year

disease-free survival rate for patients with high cancer cell expression of ALDH1 was

significantly lower than those with low ALDH1 levels (47.3% vs. 21.5%, respectively;

p=0.023). These data clearly indicate that CSC-related marker positivity may be assessed

prior to chemotherapy-based interventions and could have prognostic value for patients

with NSCLC who are treated with neoadjuvant therapy.

This is the first study to investigate BBI608 in the context of cisplatin resistant NSCLC

and the ability of BBI608 to deplete ALDH1+ve CSCs, thereby re-sensitizing NSCLC cell lines

to the cytotoxic effects of cisplatin. Treatment of cisplatin resistant NSCLC cell lines with

BBI608 significantly decreased the ALDH1+ve CSC subpopulations across all NSCLC subtypes,

a previously unreported effect of this novel small molecule inhibitor. When used in

combination with cisplatin, BBI608 significantly reduced proliferation and survival while

simultaneously increasing apoptosis in the cisplatin resistant cell lines compared to cisplatin

alone. In addition to the examination of the functional effects of BBI608, end-point PCR

gene expression analysis revealed a significant down-regulation of the mRNA expression of

embryonic stem cell markers and CSC-specific markers following BBI608 treatment. These

results support the potential use of BBI608 as an efficacious anti-cancer therapeutic agent

[42, 48]. While BBI608 is a first-in-class cancer stemness inhibitor and clinical trials indicate

that it is well-tolerated, it has been reported to display predominantly mild adverse effects

[61]. It has demonstrated potent anti-tumor and anti-metastatic effects with no significant

pharmacokinetic interactions when used as part of these combination therapeutic regimens.

BBI608 (napabucasin) has to date shown variable efficacy in a number of different

cancer types, both as a monotherapy and in combination with conventional

chemotherapeutic agents. However, early-phase trials have demonstrated the anti-tumor

efficacy of BBI608 in patients when treated with BBI608 in combination with standard

chemotherapy agents, with preclinical data suggesting that it can synergize with agents such

as paclitaxel to overcome drug resistance [62]. It has more recently been reported to

potentially play a role in malignancies where there is an urgent and unmet need for

effective therapeutics, such as advanced pancreatic adenocarcinoma [63]. In a double-blind

randomized phase III trial of napabucasin versus placebo in refractory advanced colorectal

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cancer, patients were randomly assigned (1:1) to receive placebo or napabucasin through a

web-based system with a permuted block method, after stratification by ECOG performance

status, KRAS status, previous VEGF inhibitor treatment, and time from diagnosis of

metastatic disease. Napabucasin (480 mg) or matching placebo was taken orally every 12 h.

All patients received best supportive care. The primary endpoint was overall survival

assessed in an intention-to-treat analysis. This was the final analysis of this particular trial

(NCT01830621). Although there was no difference in overall survival between groups in the

overall unselected population, STAT3 was highlighted as an important target for the

treatment of colorectal cancer with elevated pSTAT3 expression [64]. An additional study

which was conducted by the Canadian Cancer Trials Group (CCTG, former National Cancer

Institute of Canada Clinical Trials Group), assessed the efficacy and safety of BBI608 with

Best Supportive Care (BSC) compared with placebo and BSC in patients with advanced

colorectal cancer [65]. The primary objective of this study was overall survival (OS) with

secondary objectives being progression free survival (PFS), disease control rate (DCR),

safety, quality of life, health economics, pharmacokinetics, and correlative biomarkers.

Biomarker analyses included nuclear pSTAT3 assessed by immunohistochemistry. In

unselected patients, there was no significant difference observed in OS, PFS, or DCR

between BBI608 and placebo treatment in the intent-to-treat analysis. Although there were

no safety concerns serious enough to warrant terminating the trial, grade 3 GI adverse

events were significantly more frequent in napabucasin-treated patients (17%) compared

with placebo controls. While the study was designed to recruit 650 patients, this was

ultimately terminated following completion of the first interim analysis of the initial 96

patients. The DCR met protocol-defined criteria for termination of the trial. Further

enrolment of new patients and drug delivery to existing patients was ceased.

Given the promising preclinical results of BBI608 in combination therapies [45],

several clinical trials have assessed combinations in cancer patients. The phase III

CanStem111P combination trial investigated BBI608 in the combination setting in patients

with metastatic pancreatic ductal adenocarcinoma. This trial evaluated the efficacy of

BBI608 plus weekly BBI608-paclitaxel (125mg/m2) immediately followed by gemcitabine

(1000mg/m2) compared with weekly BBI608-paclitaxel (125mg/m2) followed by immediate

gemcitabine (1000mg/m2) which was administered on days 1, 8, and 15 of every 28-day

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cycle. This trial (NCT0299373) is currently recruiting. More recently, a phase Ib/II clinical trial

(BBI608-201CIT) has been initiated that will administer BBI608 in combination with different

immune checkpoint inhibitors such as ipilimumab, nivolumab, and pembrolizumab in adult

patients with advanced cancers (NCT02467361), in addition to a trial combining BBI606 with

pembrolizumab (NCT02851004) in metastatic colorectal cancer. Our data, together with

those highlighted in the current literature, support the use of BBI608 as a potential re-

sensitizing agent in the setting of platinum resistance through its ability to deplete putative

ALDH1-positive cancer stem cell subpopulations in NSCLC tumors and warrants further

investigation in vivo.

Conflict of interest Statement: There are no conflicts of interest.

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[65] D.J. Jonker, L.M. Nott, T. Yoshino, The NCI CTG and AGITG CO.23 trial: a phase III randomized

study of BBI608 plus best supportive case (BSC) versus placebo (PBO) plus BSC in patients (Pts) with

pretreated advanced colorectal carcinoma (CRC) [abstract no. TPS3660]. J Clin Oncol, 32 (2014),

(Suppl) 3660.

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Target Sequence (5’-3’)

Nanog Forward: TTGGAGCCTAATCAGCGAGGT

Reverse: GCCTCCCAATCCCAAACAATA

Oct-4 Forward: ATTCAGCCAAACGACCATCT

Reverse: GTTTTCTTACTAGTCACGTGCGG

Sox-2 Forward: GGAGCTTTGCACGAAGTTTG

Reverse: GGAAAGTTGGGATCGAACAA

Klf4 Forward: CACACTTGTGATTACGCGGG

Reverse: CCCGTGTGTTTACGGTAGTGC

cMyc Forward: AGGTTTGCTGTGGCCTCCAG

Reverse: CCTCGGATTCTCTGCTCTCCTC

CD133 Forward: GAGAAAGTGGCATCGTGCAA

Reverse: CACGTCCTCCGAATCCATTC

ALDH1 Forward: GCCATAACAATCTCCTCTGCT

Reverse: CATGGAAACCGTACTCTCCC

β-actin Forward: TGTTTGAGACCTTCAACACCC

Reverse: AGCACTGTGTTGGCGTACAG

Table I. Primer sequences for RT-PCR gene expression studies

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Cell Line

Cisplatin IC50 (μM)

Cisplatin Cisplatin + BBI608

H460 PT 3.96 0.41

CisR 17.29 0.40

H1299 PT 6.82 0.42

CisR 60.79 1.94

SKMES-1 PT 4.69 0.40

CisR 13.42 0.40

Table II. Comparison of IC50 cisplatin concentrations across cisplatin sensitive and

resistant NSCLC cell lines treated with cisplatin alone and in combination with BBI608.

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Study NCT Identifier

A Study of BBI608 in Adult Patients with Advanced, Refractory

Hematologic Malignancies

NCT02352558

A Study of BBI608 Administered With Paclitaxel in Adult Patients

with Advanced Malignancies

NCT01325441

A Study of BBI608 in Adult Patients with Advanced Colorectal

Cancer

NCT01776307

A Study of BBI608 in Combination with Standard

Chemotherapies in Adult Patients with Advanced

Gastrointestinal Cancer

NCT02024607

A Study of BBI608 in Combination with Standard

Chemotherapies in Adult Patients with Pancreatic Cancer

NCT02231723

A Study of BBI608 Administered in Combination with Immune

Checkpoint Inhibitors in Adult Patients with Advanced Cancers

NCT02467361

A Study of BBI608 in Combination with Sorafenib, or BBI503 in

Combination with Sorafenib in Adult Patients with

Hepatocellular Carcinoma

NCT02279719

A Study of BBI608 in Combination with Temozolomide in Adult

Patients with Recurrent or Progressed Glioblastoma

NCT02315534

A Study of BBI608 and BBI503 Administered in Combination to

Adult Patients with Advanced Solid Tumors

NCT02432326

A Study of BBI608 Plus weekly Paclitaxel to Treat Gastric and

Gastro-Esophageal Junction Cancer (BRIGHTER)

NCT02178956

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A Study of BBI608 Administrated with Sorafenib in Adult

Patients with Advanced Hepatocellular Carcinoma

NCT02358395

A Study of BBI608 in Combination with Pemetrexed and

Cisplatin in Adult Patients with Malignant Pleural Mesothelioma

NCT02347917

A Study of BBI608 in Adult Patients with Advanced Malignancies NCT01775423

Special Combination of BBI608 and Pembrolizumab NCT02851004

BBI608 and Best Supportive Care vs Placebo and Best

Supportive Care in Patients With Pre-treated Advanced

Colorectal Carcinoma

NCT01830621

A Study of BBI608 Administered with FOLFRI + Bevacizumab in

Adult Patients with Metastatic Colorectal Cancer

NCT02641873

A Study of Napabucasin (BBI-608) Plus weekly Paclitaxel Versus

weekly Paclitaxel Alone in Patients with Advanced, Previously

Treated, Non-Squamous Non-Small Cell Lung Cancer

NCT02826161

A Study of Napabucasin (BBI-608) in Combination with FOLFRI in

Adult Patients with Previously Treated Metastatic Colorectal

Cancer

NCT02753127

A Study of Napabucasin Plus Nab-Paclitaxel with Gemcitabine in

Adult Patients with Metastatic Pancreatic Adenocarcinoma

NCT02993731

Table III. BBI608 trials listed as active, recruiting or completed (www.clinicaltrials.gov)

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Figure Legends

Figure 1. BBI608 decreases ALDH1 activity in cisplatin resistant NSCLC cells. Following

validation of a resistance phenotype in an isogenic panel of cisplatin resistant (CisR) H460

(large cell carcinoma), H1299 (adenocarcinoma) and SKMES-1 (squamous cell carcinoma)

sublines relative to their parental (PT) controls, the presence of a putative cancer stem cell

subpopulation with ALDH1 activity (ALDH1+ve) was examined. The presence of ALDH1+ve

cells was measured by flow cytometry using the Aldefluor assay in the PT and CisR cell lines.

Cells were incubated with the Aldefluor ALDH1 substrate, which in the presence of ALDH1 is

retained within the cell. ALDH1 activity was determined relative to an internal negative

control, in which cells were incubated with the Aldefluor ALDH1 substrate and specific

ALDH1 inhibitor, DEAB. The DEAB inhibited control was used to determine background

fluorescence, from which gates were set. Upon identification of distinct ALDH1+ve

subpopulations across the CisR sublines examined, cells were treated with BBI608 (1μM) for

72hrs. Representative flow plots are shown. Data are representative of three independent

experiments and are presented as Mean ± SEM (NS; not statistically significant, **p<0.01,

***p<0.001).

Figure 2. BBI608 down-regulates stemness gene expression. Cisplatin resistant (CisR)

sublines were treated with BBI608 (1μM) for 72hrs and gene expression of embryonic stem

cell factors (Nanog, Oct-4, Sox-2, Klf4 and cMyc) and CSC-associated genes (ALDH1 and

CD133) were measured relative to untreated cells, by end-point PCR. β-actin was used as a

endogenous control to which data were normalized. Data are representative of three

independent experiments and are presented as Mean ± SEM (*p<0.05, **p<0.01,

***p<0.001).

Figure 3. BBI608 reduces proliferation and enhances the anti-proliferative effect of cisplatin

across cisplatin resistant NSCLC cells. NSCLC cisplatin resistant sublines were treated with

increasing concentrations of cisplatin (0-100μM) in the presence or absence of BBI608

(1μM) for 72hrs. Proliferation was measured by BrdU an assessed as a percentage of

untreated controls, which were set at 100%. The anti-proliferative effects following

platinum-based combination treatments were determined relative to cisplatin-only treated

CisR cells (*p<0.05, **p<0.01, ***p<0.001) and relative to BBI608 treatment alone

(##p<0.01, ###p<0.001). Data are representative of three independent experiments and are

presented as Mean ± SEM.

Figure 4. BBI608 decreases clonogenic survival. Cisplatin resistant lung cancer sublines were

treated with increasing concentrations of cisplatin (0-100μM) in the presence or absence of

BBI608 (1μM) for 72hrs and clonogenic survival was assessed using the clonogenic survival

assay. Clonogenic survival was determined relative to untreated cells, which were set as

100%. The effect of BBI608 alone was determined relative to untreated cells and

combination treatments with cisplatin were determined relative to cisplatin-only treated

cells (*p<0.05, **p<0.01, ***p<0.001). The effects of BBI608 in combination with cisplatin

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were also assessed relative to BBI608 alone (#p<0.05, ##p<0.01). Data are representative of

three independent experiments and are presented as Mean ± SEM.

Figure 5. BBI608 circumvents therapeutic resistance and induces apoptotic cell death.

Cisplatin resistant sublines were treated with BBI608 (1μM) alone or in combination with

increasing concentrations of cisplatin (0-100μM) for 48hrs. Apoptosis was measured by flow

cytometry using Annexin V/PI dual-staining. Gates were set using untreated control samples

for each cell line. The pro-apoptotic effect of BBI608 alone was determined relative to

untreated cells, while the effect of the BBI608-cisplatin combination strategy was

determined relative to cisplatin-only treated cells (*p<0.05, **p<0.01, ***p<0.001). The

effect of BBI608 in combination with cisplatin was also assessed relative to BBI608 alone

(#p<0.05, ##p<0.01, ###p<0.001). Data are representative of three independent

experiments and are presented as Mean ± SEM.

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Highlights

• BBI608 depleted an ALDH1-positive cancer stem cell population in a model of

cisplatin resistant NSCLC

• BBI608 altered stemness gene expression

• BBI608 decreased the proliferative capacity and clonogenic survival ability of

cisplatin resistant lung cancer cells, an effect that was significantly enhanced in

combination with cisplatin

• BBI608 re-sensitized chemoresistant lung cancer cells to the cytotoxic effects of

cisplatin chemotherapy and significantly induced cell apoptosis

• The use of BBI608 as a novel small molecule inhibitor in the treatment of cisplatin

resistant NSCLC warrants further investigation