bcr-abl1 compound mutations combining key kinase …

BCR-ABL1 Compound Mutations Combining KeyKinase Domain Positions Confer ClinicalResistance to Ponatinib in PhChromosome-Positive LeukemiaMatthew S. Zabriskie, University of UtahChristopher A. Eide, Oregon Health and Science UniversitySrinivas K. Tantravahi, University of UtahNadeem A. Vellore, University of UtahJohanna Estrada, University of UtahFranck E. Nicolini, Centre Hospitalier Lyon SudHanna Khoury, Emory UniversityRichard A. Larson, University of ChicagoMarina Konopleva, University of Texas MD Anderson Cancer CenterJorge E. Cortes, University of Texas MD Anderson Cancer Center

Only first 10 authors above; see publication for full author list.

Journal Title: Cancer CellVolume: Volume 26, Number 3Publisher: Elsevier (Cell Press): 12 month embargo | 2014-09-08, Pages428-442Type of Work: Article | Post-print: After Peer ReviewPublisher DOI: 10.1016/j.ccr.2014.07.006Permanent URL: https://pid.emory.edu/ark:/25593/tvhfw

Final published version: http://dx.doi.org/10.1016/j.ccr.2014.07.006

Copyright information:© 2014 Elsevier Inc.This is an Open Access work distributed under the terms of the CreativeCommons Attribution-NonCommercial-NoDerivatives 4.0 International License(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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BCR-ABL1 Compound Mutations Combining Key Kinase DomainPositions Confer Clinical Resistance to Ponatinib in PhChromosome-Positive Leukemia

Matthew S. Zabriskie1,§, Christopher A. Eide2,3,§, Srinivas K. Tantravahi1,4, Nadeem A.Vellore5, Johanna Estrada1, Franck E. Nicolini6, Hanna J. Khoury7, Richard A. Larson8,Marina Konopleva9, Jorge E. Cortes9, Hagop Kantarjian9, Elias J. Jabbour9, Steven M.Kornblau9, Jeffrey H. Lipton10, Delphine Rea11, Leif Stenke12, Gisela Barbany13, ThoralfLange14, Juan-Carlos Hernández-Boluda15, Gert J. Ossenkoppele16, Richard D. Press17,Charles Chuah18, Stuart L. Goldberg19, Meir Wetzler20, Francois-Xavier Mahon21, GabrielEtienne22, Michele Baccarani23, Simona Soverini23, Gianantonio Rosti23, PhilippeRousselot24, Ran Friedman25, Marie Deininger1, Kimberly R. Reynolds1, William L.Heaton1, Anna M. Eiring1, Anthony D. Pomicter1, Jamshid S. Khorashad1, Todd W.Kelley26, Riccardo Baron5, Brian J. Druker2,3, Michael W. Deininger1,4,*, and ThomasO'Hare1,4,*

1Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112 USA 2Division ofHematology and Medical Oncology, Oregon Health & Science University Knight Cancer Institute,Portland, OR 97239 USA 3Howard Hughes Medical Institute, Portland, OR 97239 USA 4Divisionof Hematology and Hematologic Malignancies, University of Utah, Salt Lake City, UT 84112 USA5Department of Medicinal Chemistry, College of Pharmacy, and The Henry Eyring Center forTheoretical Chemistry, University of Utah, Salt Lake City, UT 84112 USA 6HematologyDepartment 1F, Centre Hospitalier Lyon Sud, Pierre Bénite, & INSERM U1052, CRCL, Lyon69495, France 7Department of Hematology and Medical Oncology, Winship Cancer Institute ofEmory University, Atlanta, GA 30322 USA 8University of Chicago, Chicago, IL 60637 USA9Departments of Leukemia and Stem Cell Transplantation and Cellular Therapy, The University ofTexas MD Anderson Cancer Center, Houston, TX 77030 USA 10Allogeneic Blood and MarrowTransplantation Program, Department of Medical Oncology and Hematology, Princess Margaret

Correspondence: Thomas O'Hare, 2000 Circle of Hope, Room 4264, Salt Lake City, UT 84112 USA: Ph: 801-585-0256;[email protected]; Michael W. Deininger, 2000 Circle of Hope, Room 4280, Salt Lake City, UT 84112 USA; Ph:801-587-4640; [email protected].§Co-first author*Co-senior author.

OHSU and B.J.D. have a financial interest in MolecularMD.

Disclosure of Potential Conflicts of Interest: Technology used in this research has been licensed to MolecularMD. This potentialconflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and Integrity OversightCouncil. OHSU also has clinical trial contracts with Novartis and Bristol-Myers Squibb (BMS) to pay for patient costs, nurse and datamanager salaries, and institutional overhead. B.J.D. does not derive salary, nor does his lab receive funds, from these contracts.M.W.D. served on advisory boards and as a consultant for Bristol-Myers Squibb, ARIAD, and Novartis and receives research fundingfrom Bristol-Myers Squibb, Celgene, Novartis and Gilead. H.M.K. receives research funding from ARIAD. E.J.J. receivesconsultancy fees from ARIAD. S.S. is a consultant for BMS, Novartis and ARIAD.

Author Contributions: M.S.Z. and C.A.E. are co-first authors. M.S.Z. carried out all in vitro experiments, analyzed data, prepareddisplay items and assisted in writing the manuscript. C.A.E. analyzed data, organized and tabulated the sequencing data, prepareddisplay items and assisted in writing the manuscript. M.S.Z. and C.A.E. made conceptual contributions to the design of experiments.

NIH Public AccessAuthor ManuscriptCancer Cell. Author manuscript; available in PMC 2015 September 08.

Published in final edited form as:Cancer Cell. 2014 September 8; 26(3): 428–442. doi:10.1016/j.ccr.2014.07.006.

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Hospital, University of Toronto, Toronto, Ontario M5G 2M9, Canada 11Service des Maladies duSang, Hopital Saint-Louis, 75010 Paris, France 12Department of Hematology, Karolinska Institutetand University Hospital, 17176 Stockholm, Sweden 13Department of Molecular Medicine andSurgery, Karolinska Institutet, 17176 Stockholm, Sweden 14Hematology and Oncology, Universityof Leipzig, 04103 Leipzig, Germany 15Hematology and Medical Oncology Department, HospitalClínico Universitario, Valencia, Spain 16VU University Medical Center, Department ofHematology, Amsterdam 1081HV, Netherlands 17Department of Pathology and Knight CancerInstitute, Oregon Health & Science University, Portland, Oregon 97239 USA 18Department ofHematology, Singapore General Hospital, Program in Cancer and Stem Cell Biology, Duke-NUSGraduate Medical School, 169856 Singapore 19John Theurer Cancer Center at HackensackUniversity Medical Center, Hackensack, NJ 07601 USA 20Roswell Park Cancer Institute, Buffalo,NY 14263 USA 21Laboratoire d'Hematologie, Centre Hospitalier Universitaire de Bordeaux andLaboratoire Hematopoïese Leucemique et Cible Therapeutique, Inserm U1035, UniversiteBordeaux, 33076 Bordeaux, France 22Centre Regional de Lutte Contre le Cancer de Bordeaux etdu Sud-Ouest, Institut Bergonie, Departement d'Oncologie Medicale, Bordeaux, France23Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology “L. eA. Seràgnoli”, University of Bologna, Bologna 40138, Italy 24Service d'Hématologie etd'Oncologie, Université de Versailles, Paris, France 25Department of Chemistry and BiomedicalSciences and Centre for Biomaterials Chemistry, Linnaeus University, 391 82 Kalmar, Sweden26Department of Pathology, University of Utah, Salt Lake City, UT 84112 USA

Summary

Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-

ABL1 single mutants in Philadelphia chromosome-positive (Ph+) leukemia, including the

recalcitrant BCR-ABL1T315I mutant. However, emergence of compound mutations in a BCR-

ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1

compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib,

dasatinib, ponatinib, rebastinib and bosutinib. T315I-inclusive compound mutants confer high-

level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of

treatment outcomes in Ph+ leukemia patients. Structural explanations for compound mutation-

based resistance were obtained through molecular dynamics simulations. Our findings

demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance,

necessitating rational treatment selection to optimize clinical outcome.

Introduction

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 (Druker et al., 2006) have

dramatically improved the prognosis of chronic myeloid leukemia (CML) and, to a lesser

extent, Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL).

However, TKI resistance occurs in 20-30% of CML patients (O'Hare et al., 2012) and is

commonly attributable to point mutations in the BCR-ABL1 kinase domain. The TKIs

approved for first-line therapy, imatinib (Apperley, 2007; Azam et al., 2003; Bradeen et al.,

2006), nilotinib (Weisberg et al., 2005), and dasatinib (Shah et al., 2004), and the second-

Zabriskie et al. Page 2

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line therapy, bosutinib (Cortes et al., 2011; Redaelli et al., 2009), demonstrate overlapping

resistance profiles, with the BCR-ABL1T315I mutant a shared vulnerability (O'Hare et al.,

2012). Additionally, some patients fail therapy despite inhibition of BCR-ABL1, implicating

activation of alternative, BCR-ABL1 kinase-independent resistance mechanisms (Dai et al.,

2004; Donato et al., 2003; Hochhaus et al., 2002).

Ponatinib (O'Hare et al., 2009) is a high-affinity, pan-BCR-ABL1 TKI with the unique

property of inhibiting BCR-ABL1T315I. Antileukemic activity has been observed in clinical

trials of ponatinib, including patients with BCR-ABL1T315I, although responses in patients

with blastic phase CML (CML-BP) or Ph+ ALL are typically transient (Cortes et al., 2012;

Cortes et al., 2013). After a hold due to safety concerns pertaining to vascular occlusion

events, regulatory approval in the U.S. was reinstated for patients with refractory Ph+

leukemia harboring BCR-ABL1T315I or for whom no other TKI is indicated (Senior, 2014).

A risk of sequential TKI treatment is the selection of BCR-ABL1 compound mutants,

defined as harboring ≥2 mutations in the same BCR-ABL1 allele, that have the potential to

confer resistance to multiple TKIs (Shah et al., 2007). Vulnerability of ponatinib to certain

two-component compound mutations was demonstrated in pre-clinical studies (O'Hare et al.,

2009), suggesting they may emerge as a clinical problem in patients treated with ponatinib.

Importantly, ultra-deep sequencing of serial samples from Ph+ leukemia patients who had

received sequential TKI treatment showed that the majority (76%) of BCR-ABL1 compound

mutations were two-component mutations, as compared to 21% triple and 3% quadruple

mutations (Soverini et al., 2013). Progress in the development of a next generation

sequencing approach spanning the BCR-ABL1 kinase domain in a single read was recently

reported (Kastner et al., 2014).

The ability of available TKIs to address resistance due to clinically reported BCR-ABL1

compound mutants has yet to be investigated. In this study, we inventoried clinically

reported BCR-ABL1 compound mutations and established in vitro TKI sensitivity profiles

of BCR-ABL1 compound mutants against a panel of clinically available TKIs.

Results

Key BCR-ABL1 Kinase Domain Positions are Frequently Represented in ClinicallyReported Compound Mutants

Over 100 BCR-ABL1 kinase domain point mutations have been linked with clinical

imatinib resistance (Apperley, 2007), and resistance profiles for newer BCR-ABL1 TKIs are

mainly comprised of subsets of these mutations. In the current study, all uses of the term

‘compound mutation’ refer to two-component compound mutations unless otherwise stated.

Thorough inventory of clinical BCR-ABL1 compound mutations associated with TKI

resistance reported in the published literature identified a limited list of 12 kinase domain

positions (Figure 1A) comprising the majority of compound mutations, which we refer to as

key positions. All clinically reported compound mutations (100%) in Figure 1 include a key

position, and the majority (65%) involve two (Figure 1B and 1C). Each position has been

implicated in resistance to one or more TKIs: imatinib (Bradeen et al., 2006; Gorre et al.,

2001), nilotinib (Bradeen et al., 2006; Ray et al., 2007; Weisberg et al., 2005), dasatinib



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(Bradeen et al., 2006; Burgess et al., 2005; Shah et al., 2004), bosutinib (Redaelli et al.,

2009), ponatinib (O'Hare et al., 2009) and rebastinib (Chan et al., 2011; Eide et al., 2011).

The key residues in native BCR-ABL1 are: M244, G250, Q252, Y253, E255, V299, F311,

T315, F317, M351, F359, and H396 (Figure 1A). Clinical examples of T315I paired with all

key positions except 299 and 317 have been reported (Figures 1B and S1A). Among 66

possible pairings of the 12 key positions, 30 (46%) have been reported to date (Figures 1B,

1C and S1B). Further variations at the specific substitution level also occur, for example

T315I/F359C and T315I/F359V (Figure 1B) and E255K/F317L and E255V/F317I (Figure

1C).

Relevance for these key positions in TKI resistance is further supported by baseline

conventional sequencing traces of 439 patients entering the phase 2 Ponatinib Ph+ ALL and

CML Evaluation (PACE) trial (Cortes et al., 2013). Enrollment required: 1) resistance to or

unacceptable toxicity from nilotinib or dasatinib or 2) a documented baseline T315I

mutation. Mutations occurring in more than 1 patient were confined to 16 positions,

including 11/12 key positions (all except Q252). In total, 95.4% (270/283) of the mutations

observed in >1 patient among baseline PACE specimens occurred at key positions. For

PACE end of treatment (EOT) specimens, 93.8% (15/16) of two-component compound

mutations inferred from conventional sequencing involved two key positions (Table S1).

Clinically Available TKIs Exhibit Differential Activity Against BCR-ABL1 CompoundMutants

Proliferation assays comparing six TKIs were performed with Ba/F3 cells expressing native

BCR-ABL1, BCR-ABL1 single mutants at each of the 12 key positions, and clinically

reported BCR-ABL1 compound mutants (Khorashad et al., 2008; Kim et al., 2010; Shah et

al., 2007; Stagno et al., 2008). Except for I315M (see below), each single mutant was

effectively inhibited by at least one TKI and exhibited an IC50 value below the average

steady-state plasma TKI concentration reported for patients receiving the standard drug dose

(Figure 2A). The six TKIs displayed partially overlapping resistance profiles for BCR-

ABL1 single mutants, with T315I inhibited only by ponatinib and rebastinib.

T315I-inclusive compound mutants were insensitive to all TKIs except ponatinib and

rebastinib, which exhibited only marginal efficacy in most cases (Figures 2B and 2C). The

most resistant mutant, E255V/T315I (IC50: 659.5 nM), exhibited 11.9- and 22.7-fold higher

ponatinib resistance than E255V (IC50: 55.6 nM) or T315I (IC50: 29.1 nM; Table S2),

respectively. The IC50 for E255V/T315I is >6.5-fold the average steady-state plasma

concentration (101 nM) for patients receiving ponatinib at the PACE starting dose (45 mg/

day; (Cortes et al., 2012)). The Q252H/T315I, T315I/M351T, T315I/F359V and T315I/

H396R mutants exhibited marginal ponatinib sensitivity (IC50: 84.8-114.3 nM) and high-

level rebastinib resistance (IC50: 464.6-955.3 nM). M244V/T315I was the only T315I-

inclusive compound mutant in the panel predicted to be sensitive to ponatinib and rebastinib

at clinically achievable levels (Figure 2; Table S2). In vitro sensitivity of T315I-inclusive

mutants was correlated to the degree of BCR-ABL1 Y393 phosphorylation (a marker of

kinase activity) by immunoblot analysis (Figure 2D).



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All non-T315I compound mutants analyzed were inhibited by one or more TKIs (Figure

2B). For some compound mutants (e.g.Y253H/F317L), several TKI options exist. For

others, a single TKI stands out as the leading choice, most notably dasatinib for Y253H/

E255V (Figures 2B and 2C; Table S2). Superiority of dasatinib compared to ponatinib

(which demonstrate similar low nanomolar IC50s against native BCR-ABL1) against this

particular compound mutant was further confirmed by immunoblot analysis, which revealed

substantial residual pBCR-ABL1 signal for ponatinib compared to dasatinib at 100 nM

(Figure 2E). All other non-T315I compound mutants were effectively suppressed by

ponatinib at concentrations below the steady-state plasma concentrations of 45 mg/day (101

nM), and 30 mg/day (84 nM) doses. In addition to Y253H/E255V, only E255V/V299L

(IC50: 42.8 nM) and F317L/F359V (IC50: 53.2 nM) were not inhibited at the steady-state

plasma concentration for the 15 mg/day (35 nM) dose. It is conceivable that ponatinib doses

lower than 15 mg/day may not be able to prevent the emergence of additional compound

mutants. In summary, non-T315I BCR-ABL1 compound mutants exhibited a spectrum of

TKI sensitivities, suggesting in vitro resistance profiles may serve as a guide for clinical TKI

selection.

Computational Modeling of Y253H/E255V Rationalizes Differential TKI Sensitivity

Ponatinib binds to the ABL1 kinase domain in the DFG-out mode, recognizing an inactive

conformation of the kinase (O'Hare et al., 2009; Zhou et al., 2011). The binding site of

ponatinib is centered on the adenine pocket of the enzyme and extends from the phosphate-

binding loop (P-loop) to the C-helix region. By contrast, dasatinib binding is accompanied

by fewer conformational constraints and is less dependent on direct P-loop and C-helix

interactions (Tokarski et al., 2006). Since ponatinib compared favorably with dasatinib

against all non-T315I compound mutants except Y253H/E255V, we investigated structural

features that account for the striking difference in the case of this compound mutant

(ponatinib IC50: 203.5 nM; dasatinib IC50: 18.1 nM; Table S2). Molecular dynamics

simulations were carried out for a protracted interval (100 ns), and docking simulations were

performed using the GlideXP method (Schrödinger, 2012) on a collection of 50 Y253H/

E255V conformations extracted at regular intervals. Introduction of Y253H and E255V

noticeably shifted the P-loop, impinging on the ponatinib binding site (Figure 3A). Loss of

Y253-F382 aromatic π-π stacking also pushed F382 into the ponatinib site (Figures 3B and

S2) and disruption of the critical K271-E286 salt bridge in the inactive conformation

repositioned residues L248, K271, E286, and R362 (Figure 3B and S2). In contrast,

modeling predicted that dasatinib forms a new hydrogen bond with H253 in the Y253H/

E255V mutant and that realignments relative to native BCR-ABL1 do not obstruct dasatinib

binding (Figure 3C and 3D). Thus, in vitro experimental results and computational modeling

(Figures 2B, 2C and 3E) identify dasatinib as the only TKI that retains potency against

Y253H/E255V at clinically relevant levels.

Conventional and Clonal Sequencing Establish Correlations Between Baseline Mutationsand Response to Ponatinib

To understand the role of compound mutations for ponatinib response and resistance, we

received and analyzed 100 specimens from 64 patients treated on the PACE trial (n=50) or

ponatinib expanded access program (n=14), using both conventional Sanger sequencing and



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clonal sequencing of an average of 85 individual BCR-ABL1 kinase domain amplicons per

specimen. Clinical specimens originated from patients enrolled at centers participating in the

PACE trial that elected to participate in an investigator-initiated companion protocol. The

cloning and sequencing approach is an order of magnitude more sensitive and conclusively

differentiates compound from polyclonal mutations, allowing greater insight into the role of

compound mutations in TKI resistance. Pre-ponatinib baseline samples were evaluated for

all patients; for 30 patients, longitudinal and/or EOT specimens were also analyzed. Patients

were grouped according to baseline mutation status assessed by conventional sequencing: (i)

T315I, (ii) mutation other than T315I, or (iii) no mutation. Thirty-one patients were in the

chronic phase (CML-CP), 14 in the accelerated phase (CML-AP) and 19 in the blastic phase

of CML (CML-BP) or with Ph+ ALL. The cohort was heavily pretreated: 31 patients (48%)

had been exposed to 2 TKIs and 29 (45%) to 3 or more TKIs. Prior TKI exposure, baseline

mutation status, response and outcome are summarized in Table 1.

Patients with a T315I baseline mutation—T315I was detected at baseline in 22/64

patients (34.4%), including 8 CML-CP, 6 CML-AP, 5 CML-BP and 3 Ph+ ALL patients

(Table S3). Three patients carried a second baseline mutation: K285E (#2), F317L (#10) or

H396R (#18).

Patients with a mutation other than T315I at baseline—Non-T315I baseline

mutations were found in 17/64patients (26.6%), representing all non-T315I key positions

except 244 and 311: 9 CML-CP, 3 CML-AP, 2 CML-BP and 3 Ph+ ALL (Table S4). Most

baseline samples (11/17; 64.7%) harbored a mutation at a single position; six had mutations

at two positions: F317L; E450G (#23), F317L; E459K (#27), E255V; F317L (#32), F317I;

F359V (#34), Y253H; E255V (#35) and G250E; F317L (#39).

Patients with no baseline BCR-ABL1 mutation—Lack of a baseline mutation was

observed in 25/64 patients (39.1%): 14 CML-CP, 5 CML-AP, 4 CML-BP and 2 Ph+ ALL

(Table S5). No patient lacking a baseline mutation who discontinued ponatinib harbored a

compound mutation at EOT, suggesting a degree of BCR-ABL1-independent resistance

prior to initiating ponatinib therapy as well as at ponatinib failure. In the following, we

evaluated outcomes on ponatinib therapy for patients carrying a baseline T315I or non-

T315I mutation.

T315I-Inclusive Compound Mutations are Associated with Ponatinib Failure

Our in vitro profiling of T315-inclusive compound mutants predicts that most pairings with

a second key position will confer moderate- to high-level ponatinib resistance (Figure 2).

Accordingly, we observed three patients who discontinued ponatinib due to marked

expansion of a T315I-inclusive compound mutation. Patient #38 (Table S4 and S6)

presented with Ph+ ALL previously refractory to imatinib and dasatinib. Cloning and

sequencing (n=84 clones) confirmed a predominant E255V mutation (85% of clones),

including as an E255V/T315I compound mutation (17% of clones). Transient response to

ponatinib was followed by rapid hematologic relapse and a detection of a dominant E255V/

T315I compound mutation (69% of clones; Figure 4A). Molecular dynamics simulations

traced the reduced affinity of ponatinib toward E255V/T315I compared to T315I alone to re-



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orientation of the P-loop and C-helix necessary to accommodate the hydrophobic V255 side

chain (Figure 4B). These adjustments force the L248 and I315 side chains into the ponatinib

site, repositioning residues M290, F359, and D381, and reducing the distance between F382

and I315, which narrows the channel into which ponatinib normally binds (Figures 4C and

S2).

A second patient (#36, CML-BP; Table S4) had a baseline F359C mutation and later

experienced disease progression attributable to a T315I/F359C mutation that was not

detectable in the baseline clonal sequencing profile (Figures 5A and 5B; Table S7). This

compound mutant was recovered in Ba/F3 BCR-ABLT315I cell-based resistance screens for

ponatinib (O'Hare et al., 2009) and rebastinib (Eide et al., 2011), in line with our in vitro

profiles implicating mutant pairing of these two positions in moderate and high-level

resistance to these TKIs, respectively.

Lastly, a CML-AP patient (#12; Tables S3 and S7) with a baseline T315I mutation treated

with ponatinib (45 mg/day) experienced disease progression with a T315I/E453K mutation

(90% of clones) not detected at baseline by conventional sequencing or cloning and

sequencing (Figure 5C). The E453K mutation has been reported in imatinib resistance

(Soverini et al., 2013) but not compound mutation-based resistance. Ba/F3 BCR-

ABL1T315I/E453K cells showed a substantially higher level of ponatinib resistance (IC50:

93.4 nM) relative to those expressing the T315I mutant (Figure 5D) and were insensitive to

all other TKIs tested except rebastinib (IC50: 322.9 nM) (Figure 2B and 2C). Both ponatinib

and rebastinib were effective only at clinically unachievable concentrations (Figure 2B).

Among these three examples, the T315I-inclusive EOT mutation was also detectable at

baseline in only one case (E255V/T315I) and only by clonal sequencing, suggesting the

treatment failure-causing mutation was acquired on therapy or was below the detection limit

of cloning and sequencing in the other two cases.

Two additional patients in our study (#17 and #18; Tables S3 and S6) had ponatinib resistant

EOT mutations that already predominated at baseline (Y253H/T315I and T315I/H396R,

respectively; Figure S3). Altogether, these findings suggest that T315I-inclusive compound

mutations significantly impair ponatinib binding and typically lead to clinical resistance and

relapse.

The I315M Mutation Emanates from T315I and Confers High-Level Ponatinib Resistance

Nearly every instance of BCR-ABL1 kinase domain mutation-based ponatinib failure was

attributable to a compound mutation pairing two key positions. However, in the case of a

Ph+ ALL patient (#22; Figure 6A; Tables S3 and S8) with a baseline T315I mutation who

achieved a complete cytogenetic response (CCyR) on ponatinib (45 mg/day) but progressed

after 7 months, longitudinal and EOT cloning and sequencing revealed a change of I315 to

methionine (I315M) through a single nucleotide change (ATT to ATG). We previously

recovered the ponatinib resistant I315M mutant in Ba/F3 BCR-ABL1T315I cell-based

resistance screens (Eide et al., 2011; O'Hare et al., 2009), and in vitro profiling of Ba/F3

BCR-ABL1I315M cells confirmed pan-TKI resistance. The level of ponatinib resistance

conferred by the I315M mutation (IC50: 577.5 nM; Figure 6B) exceeded all tested single and

compound mutants except E255V/T315I (IC50: 659.5 nM). Molecular dynamics simulations



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demonstrated direct encroachment of the methionine residue on the ponatinib site (Figure

6C and 6D) and that adjustments at positions 269, 290, 317, 359 and 381 also disfavor

ponatinib binding (Figure 6D; Figure S2) and disrupt the hydrophobic spine architecture

(Azam et al., 2008). These findings illustrate that I315M as a single point mutation can lead

to ponatinib treatment failure.

Non-T315I Compound Mutations Impart Differential Levels of TKI Resistance

In vitro evaluation of non-T315I compound mutants showed varying levels of TKI

sensitivity across the panel, with 7/8 mutants demonstrating sensitivity to ponatinib (Figure

2C). Among patients for whom EOT samples were available, only one demonstrated clear

evidence of a non-T315I compound mutation at failure (#37, Ph+ ALL; Figure 7A; Tables

S4 and S9). Following treatment with imatinib and dasatinib, this patient exhibited a

baseline F317I mutation. The patient experienced disease progression and discontinued

ponatinib (45 mg/day), with EOT sequencing revealing an E255V/F317I mutation (100% of

clones; Figure 7A).

In contrast, three patients with non-T315I compound mutations at baseline achieved durable

responses on ponatinib. Patient #34 (Figure 7B; Tables S4 and S9) presented with CML-AP

and was treated with imatinib and dasatinib prior to starting ponatinib. Two mutations

(F317I and F359V) identified in the baseline sample were confirmed as a predominant

F317I/F359V compound mutation by cloning and sequencing (Figure 7B). The patient

achieved rapid complete hematologic response (CHR) and a major molecular response

(MMR) within 7 months on ponatinib, at which time the majority (87%) of sequenced

clones remained F317I/F359V. At last follow-up of 16 months, the patient continued to

maintain MMR.

Two additional patients (#23 and #27; Tables S4 and S9) with non-T315I compound

mutations predominant at baseline showed marked reduction in the abundance of the

compound-mutant clones on ponatinib therapy. After failing four successive TKIs, Patient

#23 exhibited an F317L/E450G baseline compound mutation (83% of clones; Figure 7C).

The patient achieved a CHR and stable disease on ponatinib for over 2 years, discontinuing

for undisclosed reasons. EOT sequencing showed no F317L/E450G (Figure 7C), suggesting

this mutant is resistant to previous TKIs but sensitive to ponatinib. Similarly, CML-CP

Patient #27 (Tables S4 and S9) exhibited an F317L/E459K baseline mutation (98% of

clones; Figure 7D) following failure of imatinib and dasatinib. After a year on ponatinib and

achievement of CHR but neither CCyR nor MMR, F317L/E459K was reduced to a minor

component (6% of clones), suggesting sensitivity to ponatinib. Compound mutants pairing

E459K with M244V, G250E, V299L (Kim et al., 2009) and T315I have been reported

(Figure 1B and 1C); the current study reports F317L/E459K as a confirmed clinical

compound mutation. In summary, T315I-inclusive compound mutations almost uniformly

confer high-level resistance to all clinically available TKIs including ponatinib, while a

fraction of non-T315I compound mutants remain sensitive to one or more TKIs.



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Discussion

Drug resistant compound mutations within the BCR-ABL1 kinase domain are an emerging

clinical problem for patients receiving sequential TKI therapy. As we predicted for ponatinib

and rebastinib, some of these mutations confer resistance that is several-fold higher than that

of either contributing mutation in isolation (Eide et al., 2011; O'Hare et al., 2009). We

investigated the role of BCR-ABL1 compound mutations in TKI resistance, focusing on

ponatinib due to its unique effectiveness against the T315I single mutant and clinical

availability.

In the U.S., ponatinib is approved for patients with refractory CML or Ph+ ALL harboring a

T315I mutation or for whom no other TKI is indicated, based on results of the PACE trial

demonstrating significant activity at a median follow-up of 15 months (Cortes et al., 2013).

Despite the impressive efficacy of ponatinib, our findings indicate that compound mutations

are an important route of therapy escape, and it is conceivable that dose reduction from 45

mg to 30 mg/day, as recommended for patients with a good response to ponatinib, may

increase the emergence of drug resistant compound mutants.

Our in vitro studies predict that a 30 mg/day dose would maintain efficacy against 7/8 non-

T315I compound mutants tested in our panel. At a daily dose of 15 mg, ponatinib is

predicted to pre-empt outgrowth of 5/8 non-T315I compound mutants in our panel, with the

Y253H/E255V, E255V/V299L and F317L/F359V mutants remaining potentially

problematic. In contrast, therapeutic utility is less promising with respect to T315I-inclusive

compound mutants, where 9/10 in our panel showed little or no sensitivity to ponatinib or

any of the other TKIs tested. We provide examples of clinical ponatinib failure attributable

to E255V/T315I, T315I/F359C, Y253H/T315I, T315I/H396R, and T315I/E453K. Given the

unique efficacy of ponatinib against the T315I single mutant and its current revised U.S.

clinical indication, a significant fraction of future patients treated with ponatinib will be

expected to harbor a T315I mutation at baseline. More sensitive, routine screening of

baseline samples from these patients may be warranted to determine whether problematic

T315I-inclusive compound mutants are present.

Detection of two mutations by conventional sequencing does not provide sufficient

information to identify the best treatment option since this may represent two clones, each

with a single mutation. In contrast, cloning and sequencing readily discerns compound from

polyclonal mutations. For example, while Patients #10, #26, and #35 each had two baseline

mutations by conventional sequencing, cloning and sequencing demonstrated mutual

exclusivity of these mutations at the clonal level. Verification that Y253H and E255V exist

in different clones as opposed to as a highly ponatinib resistant Y253H/E255V compound

mutant (Patient #35) is of importance for clinical decision-making.

In our study, no patient beginning ponatinib with native BCR-ABL1 by conventional

sequencing exhibited a causative compound mutation at EOT, similar to reports on BCR-

ABL1 single mutants (Khorashad et al., 2008; Soverini et al., 2009). These results suggest

that patients who failed multiple TKIs without a BCR-ABL1 mutation are unlikely to

experience ponatinib failure due to emergence of a resistance-conferring compound



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mutation. Effective therapy for these patients may require a synthetic lethality approach

involving blockade of a second pathway in addition to BCR-ABL1. Additionally, consistent

with inferred compound mutational status data reported in the PACE trial (Cortes et al.,

2013), we found detection of compound mutations at EOT to be more frequent among

CML-BP and Ph+ ALL patients than those with CML-CP, suggesting an increased risk of

compound mutation-based ponatinib resistance in advanced disease. Although we identified

a number of resistance-conferring compound mutants at EOT, longer follow-up and a larger

number of specimens will be required to make definitive prognostic use of baseline

sequencing profiles of patients beginning a new TKI.

Among the 12 key positions identified, there appear to be pairing constraints for generation

of TKI resistant compound mutants. For example, the current snapshot of reported

compound mutations includes position 315 in compound with 9/11 other key positions,

whereas position 252 has only been reported in compound with positions 255 and 315. We

also observed that among the 66 possible pairwise combinations of the 12 key residues, the

spectrum and frequency of pairings reported to date appear to represent a non-random

distribution (χ2=42.39; dF=3; p<0.0001). While additional resistant pairings will

undoubtedly be observed in the future, findings to date may suggest that only a limited

number of compound mutation possibilities avoid deleterious, non-tolerated effects on

kinase function (Corbin et al., 2002) or fitness of the mutated clone (Griswold et al., 2006;

Shah et al., 2007; Skaggs et al., 2006) and are consistent with our observation that the

number of missense mutations tolerated by the kinase appears to be limited.

The broad potency of ponatinib against BCR-ABL1 point mutants can be traced to the

extensive network of contacts that stabilize its binding to the kinase domain. However,

certain pairings of mutations, each of which is susceptible to a given TKI in isolation, confer

increased resistance when present as a compound mutation. Molecular dynamics-guided

modeling performed for Y253H/E255V, E255V/T315I and I315M reveals commonalities

that could aid in designing TKIs to treat compound mutants. For instance, several compound

mutations involving the P-loop result in significant distortion of this region, suggesting it

may prove advantageous to minimize direct TKI/P-loop interactions. Also intriguing is our

characterization of an I315M point mutation in clinical resistance to ponatinib due to direct

encroachment of the mutant side chain on drug binding (Patient #22). Notably, ponatinib is a

poor inhibitor of kinases in which methionine is the native ‘gatekeeper’ position analogous

to BCR-ABL1 position 315. For example, the insulin receptor is ∼500 fold less sensitive to

ponatinib than ABL1T315I (O'Hare et al., 2009). By contrast, the T315A mutant is uniquely

resistant to dasatinib and inhibited by each of the other five TKIs including ponatinib

(Burgess et al., 2005; Shah et al., 2004). These findings argue that efforts to develop future

BCR-ABL1 TKIs should also consider the capacity to accommodate multiple different

specific substitutions at the gatekeeper position.

Computational methods have been applied to BCR-ABL1 single and compound mutants to

predict TKI binding, including ponatinib (Gibbons et al., 2014; Tanneeru and Guruprasad,

2013). The use of different computational methods and the inherent limitations of modeling

mandate experimental validation of predictions. For example, one computational approach

identified T315I/F359V as ∼4-fold more resistant to ponatinib than Y253H/T315I (Gibbons



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et al., 2014). In contrast, our comprehensive, direct experimental comparison of compound

mutants and findings in cell-based resistance analyses (O'Hare et al., 2009) identify Y253H/

T315I as 3.5-fold more ponatinib resistant than T315I/F359V.

It is impossible to predict exactly how many patients diagnosed with CML or Ph+ ALL will

develop resistance due to BCR-ABL1 compound mutations. Fortunately, most CML-CP

patients treated with TKIs upfront achieve and maintain profound responses and are at a low

risk for acquiring compound mutations. In contrast, TKI failure remains common in CML-

BP and Ph+ ALL, and the incidence of compound mutations may increase with the number

of successive TKI therapies (Shah et al., 2007). Although patients with compound mutations

represent only a minority of Ph+ leukemias, they currently lack a targeted therapy option and

their prognosis is poor (Cortes et al., 2013), highlighting the need to identify therapeutic

strategies that minimize mutational escape in Ph+ leukemia. In addition, our findings are

relevant to other cancers in which compound mutations are a predicted mechanism of

therapy escape, including AML (Smith et al., 2013) and NSCLC (Awad et al., 2013; Davare

et al., 2013). Development of therapeutic strategies to control and target compound

mutation-based resistance in Ph+ leukemia will also provide a blueprint for similar discovery

in other cancers.

Experimental Procedures

Inhibitors

Inhibitors were prepared as 10.0 mM stock solutions in phosphate-buffered saline (imatinib)

or DMSO and stored at -20 °C. Serial dilutions of stock solutions were carried out before

each experiment.

Cellular Proliferation Assays

Ba/F3 BCR-ABL1-expressing cells were plated in 96-well plates (2×103 cells/well) and

incubated in 2-fold escalating concentrations of dasatinib, ponatinib (0-768 nM), imatinib,

nilotinib, rebastinib, or bosutinib (0-10240 nM) for 72 h. Proliferation was assessed by

methanethiosulfonate (MTS)-based viability assay (CellTiter 96 AQueous One; Promega).

IC50 values are reported as the mean of three independent experiments performed in

quadruplicate. See also: Supplemental Experimental Procedures.

Isolation of Primary Ph+ Leukemia Cells from Blood or Bone Marrow

All patients were consented in accordance with the Declaration of Helsinki and the Belmont

Report, and University of Utah Institutional Review Board approved all studies with human

specimens. Mononuclear cells (MNCs) were isolated from primary patient peripheral blood

or bone marrow specimens by Ficollseparation. CD34+ cells were enriched by magnetic

column separation using a CD34 human microbead kit and the POSSELDS program

(AutoMACS; Miltenyi). Purity of the CD34+ fraction was determined to be >90% by

fluorescence-activated cell sorting. If MNC yield was limiting (<2e7 cells), the RNA

isolation described below was done with an aliquot of MNCs. See also: Supplemental

Experimental Procedures.



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Conventional and Clonal Sequencing of the BCR-ABL1 Kinase Domain

RNA obtained from primary Ph+ leukemia cell lysates (QIAGEN RNeasy Mini Kit) served

as template for cDNA synthesis (BioRad iScript cDNA Synthesis Kit) as recommended by

the manufacturer. Amplification of the BCR-ABL1 kinase domain was done by two-step

PCR to exclude amplification of normal ABL1 (Khorashad et al., 2006). PCR products were

electrophoresed on a 2% agarose gel to confirm amplification, purified (QIAquick PCR

Purification Kit; QIAGEN) and subjected to 1) conventional Sanger sequencing in both

directions using BigDye terminator chemistry on an ABI3730 instrument (Khorashad et al.,

2006), and 2) cloning and sequencing of amplified fragments introduced into E. coli TOP10

cells (TOPO cloning system; Invitrogen) (Khorashad et al., 2013). For cloning and

sequencing, individual bacterial colonies (average: 85/specimen; range: 23-100), each

carrying a recombinant plasmid with a single BCR-ABL1 kinase domain amplicon inserted,

were subjected to BCR-ABL1 kinase domain amplification and Sanger sequenced in both

directions (Beckman Coulter Genomics). DNA sequence analysis was done with Mutation

Surveyor software (SoftGenetics) (O'Hare et al., 2009).

Immunoblot Analysis of BCR-ABL1 Tyrosine Phosphorylation

Ba/F3 cells expressing native or compound mutant BCR-ABL1 were cultured for 4 hr in

standard medium alone or with escalating concentrations of TKI, followed by boiling for 10

min in SDS-PAGE loading buffer. Lysates were separated on 4-15% Tris-glycine gels,

transferred, and immunoblotted with antibodies for the BCR N-terminus (3902; Cell

Signaling) and phospho-ABL1 (Y393 [1a numbering]; Cell Signaling).

Molecular Dynamics Simulations

Mutant conformations of the ABL1 kinase were prepared using standard methods to

generate ABL1Y253H/E255V, ABL1E255V/T315I, and ABL1I315M. For each mutant, both the

active (PDB entry 2GQG (Tokarski et al., 2006)) and inactive (PDB entry 2HYY (Manley et

al., 2005)) conformations of ABL1 kinase were created. The NAMD simulation package

was used for molecular dynamics simulation, and the Amber ff12SB force field was

employed for standard protein parameters. See also: Supplemental Experimental Procedures.

Docking Simulations

The Schrödinger suite of programs (Suite 2012: Maestro, version 9.3) was used for docking

studies. In the final 50 ns of the simulation, 50 conformations were extracted as docking

receptors. Selected conformations were prepared using Protein Preparation Wizard. Ligands

(ponatinib and dasatinib) were prepared (Suite 2012: LigPrep, version 2.5) and initial

docking simulation was performed using the GlideXP module (version 5.7) of the

Schrödinger program. To enhance binding conformations and allow receptor flexibility,

docked conformations were subjected to induced fit simulations. Docking scores were

computed using the GlideXP module. See also: Supplemental Experimental Procedures.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.



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Acknowledgments

The authors acknowledge the mentorship of Professor John M. Goldman (1938-2013), Imperial College London,and dedicate this paper to his legacy. We thank ARIAD Pharmaceuticals for non-financial support and permissionto perform an investigator-initiated companion study (P.I.: M.D.) to study mechanisms of resistance to ponatinib inpatients treated on the PACE trial. We thank Qian Yu, David J. Anderson and Ira L. Kraft for technical assistance.H.J.K. thanks the Georgia Cancer Coalition for a tissue bank-supporting grant. We acknowledge support inconjunction with grant P30 CA042014 awarded to the Huntsman Cancer Institute (T.O.). T.O. is supported by theNIH/NCI (R01 CA178397). S.K.T is a recipient of 2013 Research Training Award for Fellows from the AmericanSociety of Hematology. A.M.E. is currently a Fellow of the Leukemia & Lymphoma Society (5090-12). This workwas supported by Howard Hughes Medical Institute and NIH/NCI MERIT award R37CA065823 (B.J.D.). M.W.D.is supported by the NIH (HL082978-01, 5 P01 CA049639-23 and R01 CA178397), was a Leukemia & LymphomaSociety (LLS) Scholar in Clinical Research (7036-01), and is an investigator on LLS SCOR7005-11. R.B.acknowledges funding from the University of Utah Department of Medicinal Chemistry, a computing allocation atthe XSEDE supercomputers (award TG-CHE120086) and the Director's Discretionary Program (Epigenetics),which used resources of ALCF, supported by the Office of Science of the U.S. Department of Energy (DE-AC02-06CH11357).

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Zhou T, Commodore L, Huang WS, Wang Y, Thomas M, Keats J, Xu Q, Rivera VM, ShakespeareWC, Clackson T, et al. Structural mechanism of the Pan-BCR-ABL inhibitor ponatinib(AP24534): lessons for overcoming kinase inhibitor resistance. Chem Biol Drug Des. 2011; 77:1–11. [PubMed: 21118377]



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Highlights

BCR-ABL1 compound mutations can lead to clinical failures of ponatinib and other

TKIs

Nearly all non-T315I compound mutants are sensitive to at least one approved TKI

T315I-inclusive compound mutants confer resistance to all TKIs, including

ponatinib

Structural modeling provides a basis for design of TKIs targeting compound mutants



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Significance

In patients with Ph+ leukemia, control of TKI resistance due to BCR-ABL1 single

mutants is now achievable, but the ability of clinically available TKIs to target BCR-

ABL1 compound mutants has yet to be thoroughly investigated. Results from this study

reveal that BCR-ABL1 compound mutations impart various levels of TKI resistance,

underscoring the need for definitive sequencing screens to distinguish these from

polyclonal mutations and suggesting that optimal therapy selection for patients harboring

compound mutations will improve disease control in Ph+ leukemia. These findings may

also apply to other malignancies in which compound mutations are a predicted route of

therapy escape, such as acute myeloid leukemia (AML) and non-small-cell lung cancer

(NSCLC).



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Figure 1. Clinically Reported BCR-ABL1 Compound Mutations are Centered on 12 KeyPositions(A) Crystal structure of the ABL1 kinase domain in complex with imatinib (PDB entry

2HYY). Twelve key positions accounting for most clinical BCR-ABL1 TKI resistance,

including compound mutation-based resistance, are highlighted (orange; T315 is in red). The

phosphate-binding (yellow) and activation loops (green) are indicated. (B, C) Key resistance

positions (orange) in clinically reported T315I-inclusive (B) and non-T315I BCR-ABL1

compound mutants (C). Substitutions at non-key positions are in gray. 1, Shah et al., 2007;

2, Khorashad et al., 2008; 3, Stagno, et al., 2008; 4, Kim et al., 2010; 5, Kim, D.W. et al.,

Blood 2010 abstract 3443; 6, Khorashad et al., 2013; 7, Smith, C.C., et al., Blood 2011,

abstract 3752; 8, Soverini, et al. 2013; 9, Cortes, et al. 2013; 10, Hochhaus et al., 2013; *,

the current study. See also Figure S1 and Table S1.



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Figure 2. Single and Compound BCR-ABL1 Mutants Exhibit Differential TKI Sensitivity inBa/F3 Cells(A, B) Cell proliferation IC50 values of TKIs against BCR-ABL1 single mutants (A), T315I-

inclusive (B, left) and non-T315I (B, right) compound mutants. Mean IC50 values are

plotted. Blue lines denote average steady-state plasma concentration of each TKI in patients

taking the standard daily dose: imatinib (400 mg; 3377 nM) (Peng et al., 2004; Gozgit et al.,

Blood 2013, abstract 3992), nilotinib (400 mg twice daily; 2754 nM) (Kantarjian et al.,

2006; Gozgit et al., Blood 2013, abstract 3992), dasatinib (100 mg; 27 nM) (Gozgit et al.,

Blood 2013, abstract 3992; Talpaz et al., 2006), ponatinib (15, 30, and 45 mg/day doses are

represented; 35, 84, and 101 nM respectively) (Cortes et al., 2012; Gozgit et al., Blood 2013,

abstract 3992), rebastinib (150 mg twice daily; 350 nM) (Chan et al., 2011; Eide et al., 2011)

and bosutinib (500 mg; 287 nM) Gozgit et al., Blood 2013, abstract 3992). (C) Heat map of

TKI IC50s for single and compound mutants. I315M (see Figure 6) and T315I/E453K (see

Figure 5) are included for comparison. A color gradient from green (sensitive) to yellow

(moderately resistant) to red (highly resistant) denotes the IC50 sensitivity to each TKI:

imatinib (green: <1000 nM; yellow: 1000-4000 nM; red: >4000 nM); nilotinib (green: <200

nM; yellow: 200-1000 nM; red: >1000 nM); dasatinib (green: <25 nM; yellow: 25-150 nM;

red: >150 nM); ponatinib (green: <25 nM; yellow: 25-150 nM; red: >150 nM); rebastinib



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(green: <100 nM; yellow: 100-1000 nM; red: >1000 nM); bosutinib (green: <150 nM;

yellow: 150-1000 nM; red: >1000 nM). (D, E) BCR-ABL1 tyrosine phosphorylation (Y393)

immunoblot analysis of T315I-inclusive (D) and non-T315I (E) compound mutants. See also

Table S2.



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Figure 3. Molecular Dynamics Simulations of ABL1Y253H/E255V Provide a StructuralExplanation for the Ineffectiveness of Ponatinib Compared to Dasatinib(A) Structural alignment of native ABL1 kinase (PDB entry 3IK3 bound to ponatinib;

orange) with the ABL1Y253H/E255V mutant kinase (blue). Ponatinib is shown in wireframe.

Positions within 4 Å of ponatinib were designated for structural alignment using only the Cα

atoms. Native positions Y253 and E255 (green) and mutated positions H253 and V255 (red)

are shown as spheres. The region of the backbone (P-loop, C-helix and DFG loop) that

showed major conformational change is highlighted in either orange (native) or blue

(Y253H/E255V). (B) Positions of selected residues in the ponatinib site of native ABL1

(orange) and ABL1Y253H/E255V (cyan). (C) Dasatinib was docked to a representative model

of the ABL1Y253H/E255V mutant structure using MD simulation. Key residues involved in

binding of dasatinib are shown (cyan). The Y253H substitution establishes a new hydrogen

bond with dasatinib, in addition to the existing T315-dasatinib hydrogen bond (red dashed

lines). The chemical structure of dasatinib is shown using a space-filling wireframe

representation. (D) Computational docking of dasatinib (black) and ponatinib (gray) into the

mutant ABL1Y253H/E255V kinase domain (Glide XP; Schrödinger, 2012). (E) Fold-change in

cellular IC50 for BCR-ABL1Y253H/E255V relative to native BCR-ABL1 for dasatinib (black)

and ponatinib (gray). Error bars represent +/- SEM. See also Figure S2.



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Figure 4. BCR-ABL1E255V/T315I Exhibits Severely Compromised TKI Binding and is Detectedat Clinical Relapse on Ponatinib(A) Summary of BCR-ABL1 cloning and sequencing for Patient #38 (Ph+ ALL) at baseline

(upper) and EOT (lower) by cloning and sequencing individual amplicons. Conventional

sequencing results and prior TKIs are shown. Light red bars represent mutations detected at

key positions; dark red fractions of bars indicate additional mutations at other positions. For

bars designated “None”, light and dark red fractions represent native BCR-ABL1 and all

mutations not occurring at key positions, respectively. In this patient, a dominant, pan-TKI

resistant E255V/T315I mutant was detected at the time of ponatinib failure. (B) Molecular

dynamics comparison of E255V/T315I (blue; V255 in red) and T315I mutants (orange;

E255 in green). (C) Structural realignments in E255V/T315I (cyan) compared to the T315I

mutant (orange). See also Figure S3 and Table S6.



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Figure 5. Clinical Emergence of T315I-Inclusive Compound Mutants Can Lead to Relapse(A) Summary of BCR-ABL1 cloning and sequencing of individual clones for Patient #36

(CML-BP) at baseline (upper) and EOT (lower). Conventional sequencing results and prior

TKIs are shown. Bar graphs are as in Figure 4. (B) Fold-change in ponatinib Ba/F3 cellular

IC50 values for BCR-ABL1F359V and BCR-ABL1T315I/F359V compared to native BCR-

ABL1. (C) Summary of BCR-ABL1 cloning and sequencing for Patient #12 (CML-AP).

Samples were analyzed as in (A) at baseline (upper), while on ponatinib (middle), and at

EOT (lower). (D) Fold-change in ponatinib Ba/F3 cellular IC50 values for BCR-ABL1T315I

and BCR-ABL1T315I/E453K compared to native BCR-ABL1. All error bars represent +/-

SEM. See also Table S7.



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Figure 6. Acquired I315M Point Mutation-Based Ponatinib Failure in a Patient HarboringT315I at Onset of Therapy(A) Summary of BCR-ABL1 cloning and sequencing for Patient #22 (Ph+ ALL) at baseline

(upper), longitudinally on ponatinib therapy (middle), and at EOT (lower) by cloning and

sequencing individual clones. Conventional sequencing results and prior TKIs are shown.

Bar graphs are as in Figure 4. (B) Fold-change in ponatinib Ba/F3 cellular IC50 values for

BCR-ABL1I315M and BCR-ABL1T315I compared to native BCR-ABL1. Error bars represent

+/- SEM. (C) Ponatinib (in wireframe) in complex with ABL1T315I (orange); ABL1I315M

(blue) is superimposed. Structural alignment of the T315I and I315M mutants was

performed as in Figure 3. (D) M315 penetrates deeply into the ponatinib site, and structural

adjustments at positions 269, 290, 293, 317, 359 and 381 also disfavor ponatinib binding.

See also Figure S4 and Table S8.



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Figure 7. Certain Non-T315I Compound Mutants Lead to Clinical Relapse, While Others AreSusceptible to Ponatinib(A) Summary of BCR-ABL1 cloning and sequencing for Patient #37 (Ph+ ALL) at baseline

(upper) and EOT (lower) by cloning and sequencing individual amplicons. Conventional

sequencing results and prior TKIs are shown. Bar graphs are as in Figure 4. (B) Summary of

BCR-ABL1 cloning and sequencing for Patient #34 (CML-AP) at baseline (upper) and while

responding to ponatinib (lower). (C) Summary of BCR-ABL1 cloning and sequencing for

Patient #23 (CML-CP) at baseline (upper) and EOT (lower). (D) Summary of BCR-ABL1



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cloning and sequencing for Patient #27 (CML-CP) at baseline (upper) and while responding

to ponatinib (lower). See also Table S9.



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Tab

le 1

Pat

ient

Cha

ract

eris

tics

: P

rior

TK

I E

xpos

ure,

Bas

elin

e M

utat

ion

Stat

us, R

espo

nse

and

Out

com

e

See

also

Tab

les

S3-S

5.

CM

L-C

PC

ML

-AP

CM

L-B

P/P

h+ A

LL

Tot

aln=

31

T31

5Im

utat

ion

n=8

Mut

atio

not

her

than

T31

5In=

9

No

mut

atio

nn=

14T

otal

n=14

T31

5Im

utat

ion

n=6

Mut

atio

not

her

than

T31

5In=

3

No

mut

atio

nn=

5T

otal

n=19

T31

5Im

utat

ion

n=8

Mut

atio

not

her

than

T31

5In=

5

No

mut

atio

nn=

6

Prio

r T

KI

expo

sure

– n

(%

)

1

TK

I3

(10)

2 (2

5)0

(0)

1 (7

)0

(0)

0 (0

)0

(0)

0 (0

)1

(5)

1 (1

3)0

(0)

0 (0

)

2

TK

Is12

(39

)5

(63)

5 (5

6)2

(14)

7 (5

0)3

(50)

1 (3

3)3

(60)

12 (

63)

6 (7

5)4

(80)

2 (3

3)

≥3

TK

Is16

(52

)1

(13)

4 (4

4)11

(79

)7

(50)

3 (5

0)2

(67)

2 (4

0)6

(32)

1 (1

3)1

(20)

4 (6

7)

Bes

t hem

atol

ogic

res

pons

e on

pon

atin

ib –

n (

%)a

<

MaH

R1

(3)

0 (0

)0

(0)

1 (7

)0

(0)

0 (0

)0

(0)

0 (0

)3

(16)

0 (0

)1

(20)

2 (3

3)

M

aHR

1 (3

)1

(13)

0 (0

)0

(0)

1 (7

)0

(0)

1 (3

3)0

(0)

5 (2

6)2

(25)

1 (2

0)2

(33)

C

HR

29 (

94)

7 (8

9)9

(100

)13

(93

)13

(93

)6

(100

)2

(67)

5 (1

00)

11 (

58)

6 (7

5)3

(60)

2 (3

3)

Bes

t cyt

ogen

etic

res

pons

e on

pon

atin

ib –

n (

%)b

<

pCyR

12 (

39)

2 (2

5)5

(56)

5 (3

6)6

(43)

2 (3

3)1

(33)

3 (6

0)8

(42)

1 (1

3)2

(40)

5 (8

3)

pC

yR4

(13)

1 (1

3)0

(0)

3 (2

1)3

(21)

1 (1

7)1

(33)

1 (2

0)5

(26)

4 (5

0)1

(20)

0 (0

)

C

CyR

15 (

48)

5 (6

3)4

(44)

6 (4

3)5

(36)

3 (5

0)1

(33)

1 (2

0)6

(32)

3 (3

8)2

(40)

1 (1

7)

Bes

t mol

ecul

ar r

espo

nse

on p

onat

inib

– n

(%

)c

<

MM

R20

(65

)4

(50)

6 (6

7)10

(71

)10

(71

)4

(67)

2 (6

7)4

(80)

18 (

95)

8 (1

00)

4 (8

0)6

(100

)

≥M

MR

11 (

35)

4 (5

0)3

(33)

4 (2

9)4

(29)

2 (3

3)1

(33)

1 (2

)1

(5)

0 (0

)1

(20)

0 (0

)

Tre

atm

ent f

ollo

w-u

p

M

edia

n du

ratio

n of

pon

atin

ib tr

eatm

ent,

mos

. (ra

nge)

13.6

(2.

8-34

.2)

11.1

(2.

8-32

.4)

15.1

(6.

1-30

.4)

13.6

(3.

3-34

.2)

17.1

(3.

6-30

.8)

19.3

(5.

5-30

.8)

17.6

16.

6-27

.6)

13.8

(3.

6-29

.7)

3.7

(0.4

-19.

5)3.

6 (0

.5-1

6.3)

5.6

(1.3

-19.

5)2.

9 (2

.0-1

6.3)

R

emai

n on

pon

atin

ib th

erap

y –

n (%

)14

(45

)5

(63)

4 (4

4)5

(36)

6 (4

3)2

(33)

2 (6

7)2

(40)

0 (0

)0

(0)

0 (0

)0

(0)

D

isco

ntin

ued

– n

(%)

17 (

55)

3 (3

8)5

(56)

9 (6

4)8

(57)

4 (6

7)1

(33)

3 (6

0)19

(10

0)8

(100

)5

(100

)6

(100

)

Out

com

e fo

llow

-up

– n

(%)

A

live

at la

st f

ollo

w-u

p28

(90

)6

(75)

8 (8

9)14

(10

0)8

(57)

3 (5

0)2

(67)

3 (6

0)7

(37)

3 (3

8)1

(20)

3 (5

0)

D

ecea

sed

at la

st f

ollo

w-u

p3

(10)

2 (2

5)1

(11)

0 (0

)6

(43)

3 (5

0)1

(33)

2 (4

0)12

(63

)5

(63)

4 (8

0)3

(50)


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CM

L-C

PC

ML

-AP

CM

L-B

P/P

h+ A

LL

Tot

aln=

31

T31

5Im

utat

ion

n=8

Mut

atio

not

her

than

T31

5In=

9

No

mut

atio

nn=

14T

otal

n=14

T31

5Im

utat

ion

n=6

Mut

atio

not

her

than

T31

5In=

3

No

mut

atio

nn=

5T

otal

n=19

T31

5Im

utat

ion

n=8

Mut

atio

not

her

than

T31

5In=

5

No

mut

atio

nn=

6

BC

R-A

BL

1 cl

onin

g an

d se

quen

cing

– n

(%

)

B

asel

ine

sam

ple

anal

yzed

31 (

100)

8 (1

00)

9 (1

00)

14 (

100)

13 (

93)

6 (1

00)

2 (6

7)5

(100

)19

(10

0)8

(100

)5

(100

)6

(100

)

L

ongi

tudi

nal s

ampl

e(s)

ana

lyze

d7

(23)

2 (2

5)4

(44)

1 (7

)4

(29)

1 (1

7)2

(67)

1 (2

0)3

(16)

1 (1

3)1

(20)

1 (1

7)

E

nd o

f tr

eatm

ent (

EO

T)

sam

ple

anal

yzed

4 (1

3)0

(0)

1 (1

1)3

(21)

5 (3

6)3

(50)

0 (0

)2

(40)

9 (4

7)4

(50)

2 (4

0)3

(50)

C

ompo

und

mut

atio

n em

erge

nt/p

ersi

sten

t in

failu

re0

(0)

0 (0

)0

(0)

0 (0

)1

(7)

1 (1

7)0

(0)

0 (0

)6

(32)

3 (3

8)3

(60)

0 (0

)

a Maj

or h

emat

olog

ic r

espo

nse:

MaH

R, c

ompl

ete

hem

atol

ogic

res

pons

e: C

HR

.

b Part

ial c

ytog

enet

ic r

espo

nse:

pC

yR, c

ompl

ete

cyto

gene

tic r

espo

nse:

CC

yR.

c Maj

or m

olec

ular

res

pons

e: M

MR

.


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