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Cumulative Mechanism of Several Major Imatinib-Resistant Mutations in Abl Kinase

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Cumulative Mechanism of Several Major Imatinib-Resistant Mutations in Abl Kinase Cumulative mechanism of several major imatinib-resistant mutations in Abl kinase Marc Hoembergera,b,1, Warintra Pitsawonga,b, and Dorothee Kerna,b,2 aDepartment of Biochemistry, Brandeis University, Waltham, MA 02454; and bHHMI, Brandeis University, Waltham, MA 02454 Edited by Brian J. Druker, Oregon Health and Science University, Portland, OR, and approved July 6, 2020 (received for review November 4, 2019) Despite the outstanding success of the cancer drug imatinib, one ability to self-renew (14, 15). Alternatively, BCR-Abl–dependent obstacle in prolonged treatment is the emergence of resistance mechanisms involve either BCR-Abl amplification, mutations in mutations within the kinase domain of its target, Abl. We noticed the Abl’s regulatory domains that exert their resistance via al- that many patient-resistance mutations occur in the dynamic hot lostery (16), or mutations in the Abl kinase domain, which are spots recently identified to be responsible for imatinib’s high se- thought to modulate the kinase–drug interaction (17, 18). The lectivity toward Abl. In this study, we provide an experimental development of mutations in the Abl kinase domain is the most analysis of the mechanism underlying drug resistance for three commonly reported mechanism for resistance toward imatinib major resistance mutations (G250E, Y253F, and F317L). Our data treatment. For many kinase-domain mutations, the cause for settle controversies, revealing unexpected resistance mechanisms. resistance has been assumed to be a direct interference with drug The mutations alter the energy landscape of Abl in complex ways: binding (19–21). While this hypothesis is compelling, no quan- increased kinase activity, altered affinity, and cooperativity for the titative analysis has been performed, and the exact rationale for substrates, and, surprisingly, only a modestly decreased imatinib imatinib resistance remains obscure. Food and Drug Administration- affinity. Only under cellular adenosine triphosphate (ATP) concen- approved second- and third-generation inhibitors are effective trations, these changes cumulate in an order of magnitude in- against several of the imatinib-resistant mutants, but bear the same crease in imatinib’s half-maximal inhibitory concentration (IC50). problem of becoming ineffective due to new resistance mutations. A These results highlight the importance of characterizing energy promising allosteric inhibitor Abl001 that binds to the myristoylation landscapes of targets and its changes by drug binding and by re- site is in clinical trial (22). sistance mutations developed by patients. Here, we experimentally investigated three mutations that are BIOCHEMISTRY among the most commonly found resistance mutations in pa- Abl kinase | tyrosine kinase | cancer research | imatinib resistance tients treated with imatinib. We found that, contrary to previous hypotheses for these mutations, where resistance was suggested – inases are key enzymes in many crucial cellular-signaling to originate simply by disrupting the kinase drug interaction, it is Kprocesses and, when aberrant, often lead to the develop- a combination of multiple effects that lead to resistance. Sur- ment of cancer (1–3). Thus, it is not surprising that they have prisingly, single mutations result only in small changes in drug ’ been major targets for modern drug design. However, a major affinity, but simultaneously alter Abl s turnover rate, affinities of obstacle in designing orthosteric drugs for protein kinases is their ATP, and its target substrate. The cumulative effect of all four high structural similarity. As each kinase catalyzes the same phosphoryl-transfer reaction between the γ-phosphate of aden- Significance osine triphosphate (ATP) and its protein substrate (usually a tyrosine, serine, or threonine residue), designed orthosteric One obstacle for the prolonged success of the wonder drug drugs are subject to off-target effects. Yet, highly selective, imatinib in leukemia has been the emergence of resistance orthosteric drugs are possible, as shown by the success story of mutations within the Abl kinase domain. Here, we elucidate the anticancer drug imatinib, a potent inhibitor of Abl kinase. the molecular mechanism for resistance of three major muta- Imatinib binds with 3,000-fold higher affinity to its target Abl tions in patients treated with imatinib. Unpredictably, the kinase relative to its closest homolog, with a high degree of se- single-site resistance mutations act via a cumulative effect of quence (54%) and structural similarity, Src kinase (4). This high an only modest decrease in drug affinity, combined with an specificity of imatinib for Abl has made it an effective treatment increase in enzyme activity and altered substrate affinity/ for chronic myelogenous leukemia (CML), a pathological con- cooperativity. Strikingly, this combination indeed leads to at dition that is caused by a fusion product of the gene for Abl least an order-of-magnitude higher IC50 values for imatinib, but (located on chromosome 9) with the BCR gene on chromosome only under cellular ATP concentrations. Our findings settle a 22, thereby forming a shortened chromosome termed the Phil- longstanding controversy, and concepts found here are likely adelphia chromosome. As a consequence of the formation of this to play a role in drug resistance in other targets. Philadelphia chromosome, parts of the regulatory N terminus of Abl are replaced with BCR, which then leads to the expression of Author contributions: M.H. and D.K. designed research; M.H. and W.P. performed re- a constitutively active BCR-Abl (5–9). Since imatinib is one of search; M.H. and W.P. analyzed data; and M.H. and D.K. wrote the paper. the few drugs on the market that is highly selective with mini- Competing interest statement: D.K. is co-founder of Relay Therapeutics and MOMA Ther- apeutics. D.K. is an inventor on pending patents applied for by Brandeis University that mizing side effects (10), it has been the frontline treatment for describes compositions and methods for modulating kinase activity (US20180334510A1 most CML patients and has proven to be very effective. Imatinib and US20190038582A1) and on pending patents of a biophysical platform for drug de- is also highly effective against the kinase c-kit in gastrointestinal velopment based on energy landscape (PCT/US2016/15171). stromal cancer (11). This article is a PNAS Direct Submission. However, one problem BCR-Abl–positive patients have faced Published under the PNAS license. over last decade has been the emergence of resistance to imatinib 1Present address: Bioassays & High-Throughput Screens, Biotherapeutic and Medical Sci- (12, 13). Both BCR-Abl–independent and BCR-Abl–dependent ences, Biogen, Cambridge, MA 02142. mechanisms are possible ways patients can acquire resistance to- 2To whom correspondence may be addressed. Email: [email protected]. ward drug treatment. Examples of BCR-Abl–independent mecha- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ nisms include mutations in transporters that mediate drug influx or doi:10.1073/pnas.1919221117/-/DCSupplemental. persistence of drug-resistant cancer stem cells that retain their www.pnas.org/cgi/doi/10.1073/pnas.1919221117 PNAS Latest Articles | 1of7 Downloaded by guest on September 27, 2021 effects reflects a substantially changed energy landscape and indicating a possible effect on catalytic activity; both an increase explains the resistance to imatinib by these mutations. (36, 37) and no effect (38, 39) on catalytic activity was reported. Results Thermodynamics and Kinetics of Imatinib Binding to Resistance Resistance Mutations Overlap with Dynamic Hot Spots Essential for Mutants. While there have been reports that epidermal growth Imatinib Selectivity. Previously, we had studied the origin of factor receptor (EGFR) resistance mutations can lead to an in- imatinib specificity using ancestral sequence reconstruction. By crease in kinase activity or altered ATP affinity (40), the bulk of studying the evolutionary trajectory of Abl from its closest ho- the literature on resistance mutations observed for Abl come to molog, Src, we identified 15 key residues that are responsible for the conclusion that resistance mutations in Abl’s kinase domain the tight affinity and high selectivity of imatinib to Abl (23). mainly work by affecting binding of the drug to the kinase. To These 15 residues are distributed across the N-terminal lobe of test this assumption, we first measured the affinity of imatinib to the kinase (Fig. 1A) and were suggested to alter the flexibility of the three resistance mutants of Abl via intrinsic tryptophan the P loop, resulting in the observed conformational changes fluorescence at 5 °C (Fig. 2A; see SI Appendix, Fig. S1 for data at after imatinib binding that are crucial for high affinity and se- 25 °C). Since formation of the BCR-Abl fusion product fully lectivity (23, 24). Interestingly, when we now compared the lo- removes the autoinhibition by the N terminus of Abl, we decided cation of these 15 residues with known resistance mutations to use the Abl kinase domain only constructs as a suitable mimic found in cancer patients that were treated with imatinib, many of of the fusion product. To our surprise, none of these mutations the resistance mutations overlapped with the 15 dynamic hot decreased binding by more than threefold (KD = 50 ± 10 nM, spots in Abl (Fig. 1). 78 ± 12 nM, and 54 ± 4 nM for F317, G250E, and Y253F, re- Intrigued by
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