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BCR-ABL1 Compound Mutations Combining Key Kinase Domain Positions Confer Clinical Resistance to Ponatinib in Ph Chromosome-Positive Leukemia
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.
- 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
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 and non-small cell lung cancer.
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, and Bradeen et al, 2006), nilotinib ( Weisberg et al., 2005 ), and dasatinib ( Shah et al., 2004 ), and the second-line therapy, bosutinib (Cortes et al, 2011 and 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, and 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. Anti-leukemic 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 and Cortes et al, 2013). After a hold due to safety concerns pertaining to vascular occlusion events, regulatory approval in the United States 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 a BCR-ABL1 allele harboring two or more mutations, 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 preclinical 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.
Key BCR-ABL1 Kinase Domain Positions Are Frequently Represented in Clinically Reported 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 1 A) 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 ( Figures 1 B and 1C). Each position has been implicated in resistance to one or more TKIs: imatinib (Bradeen et al, 2006 and Gorre et al, 2001), nilotinib (Bradeen et al, 2006, Ray et al, 2007, and Weisberg et al, 2005), dasatinib (Bradeen et al, 2006, Burgess et al, 2005, and Shah et al, 2004), bosutinib ( Redaelli et al., 2009 ), ponatinib ( O’Hare et al., 2009 ), and rebastinib (Chan et al, 2011 and 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 1 A). Clinical examples of T315I paired with all key positions except 299 and 317 have been reported ( Figure 1 B and Figure S1 A available online). Among 66 possible pairings of the 12 key positions, 30 (45%) have been reported to date ( Figures 1 B, 1C, and S1 B). Further variations at the specific substitution level also occur, for example T315I/F359C and T315I/F359V ( Figure 1 B) or E255K/F317L and E255V/F317I ( Figure 1 C).
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 Tyrosine Kinase Inhibitors Exhibit Differential Activity against BCR-ABL1 Compound Mutants
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, and Stagno et al, 2008). Except for I315M (see below), each single mutant was effectively inhibited by at least one TKI and exhibited a half-maximal inhibitory concentration (IC50) value below the average steady-state plasma TKI concentration reported for patients receiving the standard drug dose ( Figure 2 ). 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 2 B 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 2 D).
All non-T315I compound mutants analyzed were inhibited by one or more TKIs ( Figure 2 B). 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 2 B 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 2 E). 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 Tyrosine Kinase Inhibitor 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 and 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 (Suite 2012: GlideXP, version 5.8, Schrödinger, New York, NY, 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 3 A). Loss of Y253-F382 aromatic π-π stacking also pushed F382 into the ponatinib site ( Figures 3 B and S2 ), and disruption of the critical K271-E286 salt bridge in the inactive conformation repositioned residues L248, K271, E286, and R362 ( Figures 3 B 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 ( Figures 3 C and 3D). Thus, in vitro experimental results and computational modeling ( Figures 2 B, 2C, and 3 E) identify dasatinib as the only TKI that retains potency against Y253H/E255V at clinically relevant levels.
Conventional and Clonal Sequencing Establish Correlations between Baseline Mutations and 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 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 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: (1) T315I, (2) mutation other than T315I, or (3) no mutation. There were 31 patients that were in the chronic phase (CML-CP), 14 in the accelerated phase (CML-AP), and 19 in CML-BP or with Ph+ ALL. The cohort was heavily pretreated: 31 patients (48%) had been exposed to two TKIs and 29 (45%) to three or more TKIs. Prior TKI exposure, baseline mutation status, response, and outcome are summarized in Table 1 .
|Total||T315I Mutation||Mutation Other than T315I||No Mutation||Total||T315I Mutation||Mutation Other than T315I||No Mutation||Total||T315I Mutation||Mutation Other than T315I||No Mutation|
|n = 31||n = 8||n = 9||n = 14||n = 14||n = 6||n = 3||n = 5||n = 19||n = 8||n = 5||n = 6|
|Prior TKI exposure, n (%)|
|1 TKI||3 (10)||2 (25)||0 (0)||1 (7)||0 (0)||0 (0)||0 (0)||0 (0)||1 (5)||1 (13)||0 (0)||0 (0)|
|2 TKIs||12 (39)||5 (63)||5 (56)||2 (14)||7 (50)||3 (50)||1 (33)||3 (60)||12 (63)||6 (75)||4 (80)||2 (33)|
|≥3 TKIs||16 (52)||1 (13)||4 (44)||11 (79)||7 (50)||3 (50)||2 (67)||2 (40)||6 (32)||1 (13)||1 (20)||4 (67)|
|Best hematologic response on ponatinib, n (%) a|
|<MaHR||1 (3)||0 (0)||0 (0)||1 (7)||0 (0)||0 (0)||0 (0)||0 (0)||3 (16)||0 (0)||1 (20)||2 (33)|
|MaHR||1 (3)||1 (13)||0 (0)||0 (0)||1 (7)||0 (0)||1 (33)||0 (0)||5 (26)||2 (25)||1 (20)||2 (33)|
|CHR||29 (94)||7 (89)||9 (100)||13 (93)||13 (93)||6 (100)||2 (67)||5 (100)||11 (58)||6 (75)||3 (60)||2 (33)|
|Best cytogenetic response on ponatinib, n (%) b|
|<pCyR||12 (39)||2 (25)||5 (56)||5 (36)||6 (43)||2 (33)||1 (33)||3 (60)||8 (42)||1 (13)||2 (40)||5 (83)|
|pCyR||4 (13)||1 (13)||0 (0)||3 (21)||3 (21)||1 (17)||1 (33)||1 (20)||5 (26)||4 (50)||1 (20)||0 (0)|
|CCyR||15 (48)||5 (63)||4 (44)||6 (43)||5 (36)||3 (50)||1 (33)||1 (20)||6 (32)||3 (38)||2 (40)||1 (17)|
|Best molecular response on ponatinib, n (%) c|
|<MMR||20 (65)||4 (50)||6 (67)||10 (71)||10 (71)||4 (67)||2 (67)||4 (80)||18 (95)||8 (100)||4 (80)||6 (100)|
|≥MMR||11 (35)||4 (50)||3 (33)||4 (29)||4 (29)||2 (33)||1 (33)||1 (2)||1 (5)||0 (0)||1 (20)||0 (0)|
|Median duration of ponatinib treatment, months (range)||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 (0.5–16.3)||5.6 (1.3–19.5)||2.9 (2.0–16.3)|
|Remain on ponatinib therapy, n (%)||14 (45)||5 (63)||4 (44)||5 (36)||6 (43)||2 (33)||2 (67)||2 (40)||0 (0)||0 (0)||0 (0)||0 (0)|
|Discontinued, n (%)||17 (55)||3 (38)||5 (56)||9 (64)||8 (57)||4 (67)||1 (33)||3 (60)||19 (100)||8 (100)||5 (100)||6 (100)|
|Outcome follow-up, n (%)|
|Alive at last follow-up||28 (90)||6 (75)||8 (89)||14 (100)||8 (57)||3 (50)||2 (67)||3 (60)||7 (37)||3 (38)||1 (20)||3 (50)|
|Deceased at last follow-up||3 (10)||2 (25)||1 (11)||0 (0)||6 (43)||3 (50)||1 (33)||2 (40)||12 (63)||5 (63)||4 (80)||3 (50)|
|BCR-ABL1 cloning and sequencing, n (%)|
|Baseline samples analyzed||31 (100)||8 (100)||9 (100)||14 (100)||13 (93)||6 (100)||2 (67)||5 (100)||19 (100)||8 (100)||5 (100)||6 (100)|
|Longitudinal samples analyzed||7 (23)||2 (25)||4 (44)||1 (7)||4 (29)||1 (17)||2 (67)||1 (20)||3 (16)||1 (13)||1 (20)||1 (17)|
|End of treatment samples analyzed||4 (13)||0 (0)||1 (11)||3 (21)||5 (36)||3 (50)||0 (0)||2 (40)||9 (47)||4 (50)||2 (40)||3 (50)|
|Compound mutations emergent/persistent in failure||0 (0)||0 (0)||0 (0)||0 (0)||1 (7)||1 (17)||0 (0)||0 (0)||6 (32)||3 (38)||3 (60)||0 (0)|
a MaHR, Major hematologic response; CHR, complete hematologic response.
b pCyR, partial cytogenetic response; CCyR, complete cytogenetic response.
c MMR, major molecular response.
See also Tables S3–S5 .
Patients with a T315I Baseline Mutation
T315I was detected at baseline in 22/64 patients (34.4%), including eight CML-CP, six CML-AP, five CML-BP, and three Ph+ ALL patients ( Table S3 ). There were three patients that 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 17/64 patients (26.6%), representing all non-T315I key positions except 244 and 311: nine CML-CP, three CML-AP, two CML-BP, and three 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, five CML-AP, four CML-BP, and two 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 ( Tables 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 4 A). Molecular dynamics simulations traced the reduced affinity of ponatinib toward E255V/T315I compared to T315I alone to reorientation of the P loop and C-helix necessary to accommodate the hydrophobic V255 side chain ( Figure 4 B). 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 4 C 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 5 A 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.
Last, 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 5 C). 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 5 D) and were insensitive to all other TKIs tested except rebastinib (IC50: 322.9 nM) ( Figures 2 B and 2C). Both ponatinib and rebastinib were effective only at clinically unachievable concentrations ( Figure 2 B). Among these three examples, the T315I-inclusive EOT mutation was also detectable at baseline in only one case (E255V/T315I), suggesting the mutation was acquired on therapy or was below the detection limit of cloning and sequencing in the other two cases.
There were two additional patients in our study (#17 and #18; Tables S3 and S6 ) that 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 6 A; 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 and 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 6 B) exceeded all tested single and compound mutants except E255V/T315I (IC50: 659.5 nM). Molecular dynamics simulations demonstrated direct encroachment of the methionine residue on the ponatinib site ( Figures 6 C and 6D) and that adjustments at positions 269, 290, 317, 359, and 381 also disfavor ponatinib binding ( Figures 6 D, 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 Tyrosine Kinase Inhibitor 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 2 C). 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 7 A; 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 7 A).
In contrast, three patients with non-T315I compound mutations at baseline achieved durable responses on ponatinib. Patient #34 ( Figure 7 B; Tables S4 and S9 ) presented with CML-AP and was treated with imatinib and dasatinib prior to starting ponatinib. There were two mutations (F317I and F359V) identified in the baseline sample that were confirmed as a predominant F317I/F359V compound mutation by cloning and sequencing ( Figure 7 B). 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.
There were two additional patients (#23 and #27; Tables S4 , S9 ) with non-T315I compound mutations predominant at baseline that 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 7 C). 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 7 C), 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 7 D) 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 ( Figures 1 B 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.
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 and 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 United States, 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 preempt 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 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 and 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 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 nonrandom 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, nontolerated effects on kinase function ( Corbin et al., 2002 ) or fitness of the mutated clone (Griswold et al, 2006, Shah et al, 2007, and 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 and 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 and 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 et al., 2014 ). In contrast, our comprehensive, direct experimental comparison of compound mutants and findings in cell-based resistance analyses identify Y253H/T315I as 3.5-fold more ponatinib resistant than T315I/F359V ( O’Hare et al., 2009 ).
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 acute myeloid leukemia ( Smith et al., 2013 ) and nonsmall cell lung cancer (Awad et al, 2013 and 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.
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 (0–10,240 nM) for 72 hr. Proliferation was assessed by methanethiosulfonate-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 Ficoll-separation. 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 (<2 ×107 cells), the RNA isolation described below was done with an aliquot of MNCs. See also Supplemental Experimental Procedures .
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 and Shah et al, 2007). 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 (Protein Data Bank [PDB] entry 2GQG ; Tokarski et al., 2006 ) and inactive (PDB entry 2HYY ; Manley et al., 2005 ) conformations of ABL1 kinase were created. The Nanoscale Molecular Dynamics 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 .
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 .
M.S.Z. and C.A.E. are co-first authors. M.S.Z. carried out all in vitro experiments, analyzed data, prepared display items, and assisted in writing the manuscript. C.A.E. analyzed data, organized and tabulated the sequencing data, prepared display items, and assisted in writing the manuscript. M.S.Z. and C.A.E. made conceptual contributions to the design of experiments.
Oregon Health & Science University (OHSU) and B.J.D. have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and Integrity Oversight Council. OHSU also has clinical trial contracts with Novartis and Bristol-Myers Squibb (BMS) to pay for patient costs, nurse and data manager salaries, and institutional overhead. B.J.D. does not derive salary, nor does his laboratory receive funds, from these contracts. M.W.D. served on advisory boards and as a consultant for BMS, ARIAD, and Novartis and receives research funding from BMS, Celgene, Novartis, and Gilead. H.M.K. receives research funding from ARIAD. E.J.J. receives consultancy fees from ARIAD. S.S. is a consultant for BMS, Novartis, and ARIAD.
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 nonfinancial support and permission to perform an investigator-initiated companion study (P.I.: M.D.) to study mechanisms of resistance to ponatinib in patients 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 in conjunction with grant P30 CA042014 awarded to the Huntsman Cancer Institute (T.O.). T.O. is supported by the NIH/NCI (R01 CA178397). S.K.T is a recipient of 2013 Research Training Award for Fellows from the American Society of Hematology. A.M.E. is currently a Fellow of the Leukemia & Lymphoma Society (5090-12). This work was 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 & Lymphoma Society (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 at the XSEDE supercomputers (award TG-CHE120086), and the Director’s Discretionary Program (Epigenetics), which used resources of the Argonne Leadership Computing Facility, supported by the Office of Science of the U.S. Department of Energy (DE-AC02-06CH11357).
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1 Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
2 Division of Hematology and Medical Oncology, Oregon Health & Science University Knight Cancer Institute, Portland, OR 97239, USA
3 Howard Hughes Medical Institute, Portland, OR 97239, USA
4 Division of Hematology and Hematologic Malignancies, University of Utah, Salt Lake City, UT 84112, USA
5 Department of Medicinal Chemistry, College of Pharmacy and The Henry Eyring Center for Theoretical Chemistry, University of Utah, Salt Lake City, UT 84112, USA
6 Hematology Department 1F, Centre Hospitalier Lyon Sud, Pierre Bénite, INSERM U1052, CRCL, Lyon 69495, France
7 Department of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA
8 University of Chicago, Chicago, IL 60637, USA
9 Departments of Leukemia and Stem Cell Transplantation and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
10 Department of Medical Oncology and Hematology, Allogeneic Blood and Marrow Transplantation Program, Princess Margaret Hospital, University of Toronto, Toronto ON M5G 2M9, Canada
11 Service des Maladies du Sang, Hospital Saint-Louis, 75010 Paris, France
12 Department of Hematology, Karolinska Institutet and University Hospital, 17176 Stockholm, Sweden
13 Department of Molecular Medicine and Surgery, Karolinska Institutet, 17176 Stockholm, Sweden
14 Hematology and Oncology, University of Leipzig, 04103 Leipzig, Germany
15 Hematology and Medical Oncology Department, Hospital Clínico Universitario, 46010 Valencia, Spain
16 Department of Hematology, VU University Medical Center, Amsterdam 1081HV, the Netherlands
17 Department of Pathology and Knight Cancer Institute, Oregon Health & Science University, Portland, OR 97239, USA
18 Department of Hematology, Singapore General Hospital, Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, 169856 Singapore, Singapore
19 John Theurer Cancer Center at Hackensack University Medical Center, Hackensack, NJ 07601, USA
20 Roswell Park Cancer Institute, Buffalo, NY 14263, USA
21 Laboratoire d’Hematologie, Centre Hospitalier Universitaire de Bordeaux and Laboratoire Hematopoïese Leucemique et Cible Therapeutique, Inserm U1035, Universite Bordeaux, 33076 Bordeaux, France
22 Departement d’Oncologie Medicale, Centre Regional de Lutte Contre le Cancer de Bordeaux et du Sud-Ouest, Institut Bergonie, 33076 Bordeaux, France
23 Department of Experimental, Diagnostic, and Specialty Medicine, Institute of Hematology “L. e A. Seràgnoli,” University of Bologna, 40138 Bologna, Italy
24 Service d’Hématologie et d’Oncologie, Université de Versailles, 75010 Paris, France
25 Department of Chemistry and Biomedical Sciences and Centre for Biomaterials Chemistry, Linnaeus University, 391 82 Kalmar, Sweden
26 Department of Pathology, University of Utah, Salt Lake City, UT 84112, USA
∗ Corresponding author
∗∗ Corresponding author
27 Co-first author
28 Co-senior author
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