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 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– (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 (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 | 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 . 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 . The cumulative effect of all four high structural similarity. As each kinase catalyzes the same phosphoryl-transfer reaction between the γ- 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 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. , 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- 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 the observation that nature seems to escape drug spectively) compared to wild type (KD = 26 ± 4 nM). It seemed treatment by amino acid changes in these dynamic hot spots that unlikely that such minor changes in overall affinity would result are responsible for the high affinity of imatinib to Abl, we set out in resistance against imatinib and opposes the resistance mech- to investigate the underlying molecular mechanism for their drug anism proposed in the literature. What are we missing? The resistance. Three imatinib resistance mutations were selected measured binding affinities are overall KD values comprising all (Fig. 1A) with the following criteria in mind: 1) they are major microscopic steps for drug binding (Fig. 2B). As shown previ- resistance mutations found in patients; 2) they are from the ously, the high specificity of imatinib toward its target Abl stems subset of the 15 dynamic hot spots; and 3) their mechanism is not from an induced-fit step after drug binding, and not from the understood, with either controversial or inconclusive prior lit- conformational-selection mechanism for aspartate–phenylalanine– erature studies (19, 20, 25, 26). F317L is located at the back of glycine-in/out, or subsequent binding (Fig. 2B) (35). The induced-fit the P loop, and the phenylalanine was suggested to be important step entails kinking of the P loop for extensive interactions between for van der Waals interactions and polar stacking with the aro- the protein and the drug. Since all three resistance mutations matic ring of imatinib (21, 24, 27–30). G250E is located in the P overlap with the residues important for this P-loop kinking (23), the loop, and its mechanism for resistance has not been well char- expectation is that these mutations alter the conformational dy- acterized (31, 32). Y253F, part of the P loop, forms a hydrogen namics after drug binding. bond with N322, which is thought to stabilize the kinked P-loop To test this hypothesis, stopped-flow experiments were per- conformation in the Abl drug-bound form (20, 23, 33–35). There formed to measure the kinetics of association and dissociation. In are additional conflicting reports for the Y253F mutant this experiment, Abl was rapidly mixed with varying concentrations

AB

Fig. 1. Drug-resistance mutants in Abl kinase overlap with residues important for induced-fit step in imatinib binding. (A) Wild-type Abl bound to imatinib shown in black [Protein Data Bank ID code 1OPJ (33)]. (A, Upper) Residues important for tight binding of imatinib (23) are highlighted as red spheres. Dark red residues are overlapping with resistance mutations that have been isolated from cancer patients. The three mutations investigated in this study are high- lighted with colored arrows and labeled. (A, Lower) Common kinase domain resistance mutations isolated from patients treated with imatinib are shown as brown spheres. Dark brown residues are mutations that overlap with the 15 residues important for imatinib binding. (B) Multiple sequence alignment of Src (weak imatinib binder; top rows), the ancestral protein (ANC-SA; middle rows) previously characterized to be an intermediate imatinib binder (23), and Abl (tight imatinib binder; bottom rows). Coloring of residues corresponds to A.

2of7 | www.pnas.org/cgi/doi/10.1073/pnas.1919221117 Hoemberger et al. Downloaded by guest on September 27, 2021 A

B

CD E

FG BIOCHEMISTRY

Fig. 2. Thermodynamics and kinetics of imatinib binding to wild-type (WT) and mutant forms of Abl showing imatinib resistance. (A) Macroscopic disso-

ciation constant (KD), measured by monitoring intrinsic tryptophan fluorescence at 5 °C. (B) Imatinib binding scheme to Abl comprising a conformational selection and an induced-fit step after drug binding. (C) Representative traces for stopped-flow fluorescence experiments at 5 °C illustrating double- exponential decay for imatinib binding (data in color, with double-exponential fit shown in black). a.u., arbitrary units. (D and E) Plot of observed rate obs obs constants for the fast binding step (kbind )(D) and the slow induced-fit step (kconf )(E) versus the concentration of imatinib. (F) Dissociation of imatinib obs from Abl wild-type, F317L, G250E, and Y253F. (G) Comparison between measured overall KD (KD ) from A and binding affinities calculated from microscopic kin rate constants (KD ); see also Table 2. (n = 3 experiments; mean ± SD.)

of imatinib, and quenching of intrinsic tryptophan fluorescence due an 11-fold dilution, the experiments were performed on a fluo- to drug binding was measured over time (Fig. 2C). All experiments rometer instead. The kinase was preincubated with twofold ex- were performed at 5 °C, since the initial binding of the drug to cess of drug at 5 °C for 30 min and rapidly diluted 30-fold into enzyme was too fast to be observed at 25 °C, as noted earlier (35). buffer. Dissociation of the complex, characterized by an increase Kinetic traces were recorded for 1 and 120 s to capture both the in intrinsic tryptophan fluorescence, was monitored over a time fast-binding and subsequent slower conformational changes. After of 1,600 s. The kinetics of this process was very similar for all correction for photobleaching, the short time traces were fit to a protein forms (Fig. 2F and Table 1) and can be assigned to the single-exponential function (SI Appendix,Figs.S2–S5, Insets)with krev of the induced-fit step. the observed rate constants for the mutants increasing linearly with The kinetics experiments revealed that F317L, G250E, and imatinib concentration corresponding to the second-order binding Y253F altered the conformational dynamics in the drug-bound step (Fig. 2D). The bimolecular rate constant (kon,Binding)obtained state and, in particular, the forward rate of the induced-fit step k from the slope and dissociation of imatinib (koff,Binding) deter- since the slow-off rate ( rev) obtained from the dilution experi- mined from the intercept (Fig. 2D) were comparable to wild type ment was the same. This qualitatively agrees with what was ob- −1 −1 −1 (kon,Binding = 1.0 ± 0.1 μM ·s , koff,Binding = 25 ± 4s ) The ob- served for the well-characterized gatekeeper mutant reported served rate constants for the second slow phase obtained from the earlier (23); however, the effect for these newly characterized longer time traces (SI Appendix,Figs.S2–S5) were nonlinearly mutants was much smaller. The qualitative agreement for F317L, dependent on imatinib concentration, with the plateau defined by G250E, and Y253F with the gatekeeper mutant suggests that the sum of forward and reverse rates of the induced-fit step (kfwd + changing the equilibrium of the induced-fit step might be a more krev;Fig.2E). general mechanism of how mutations alter binding of imatinib to The dilution experiments were performed for the enzyme/drug the kinase. Taken together, our kinetics data (Fig. 2 C–F) en- complex to monitor the dissociation and complete the kinetic abled a rigorous testing of the underlying binding model characterization of imatinib binding. Since imatinib binds tightly (Fig. 2B) by comparing the measured overall KD with the cal- to Abl and the setup of our stopped-flow machine only allows for culated macroscopic KD from the individual rate constants. The

Hoemberger et al. PNAS Latest Articles | 3of7 Downloaded by guest on September 27, 2021 Table 1. Rapid kinetics of imatinib binding to wild-type and reports on cooperativity for substrate binding. It was shown mutant forms of Abl previously that peptide and ATP exhibit negative cooperativity α > α < Binding Conformational change for wild-type Src and Abl [ 1 negative cooperativity; 1 positive cooperativity (44)]. = ± μ −1· −1 = ± −1 ATP WT kon 1.0 0.1 M s kfwd 1.5 0.1 s Compared to the wild-type affinity for ATP (KD = 69 ± = ± −1 = ± × −4 −1 koff 25 4s krev (17 5) 10 s 4 μM), only G250E showed a significant tighter binding to ATP = ± μ −1· −1 = ± −1 ATP ATP F317L kon 1.1 0.1 M s kfwd 1.3 0.1 s (KD = 47 ± 2 μM), while F317L (KD = 76 ± 1 μM) had = ± −1 = ± × −4 −1 ATP koff 25 3s krev (25 3) 10 s near identical, and Y253F (KD = 98 ± 5 μM) even weaker = ± μ −1· −1 = ± −1 G250E kon 1.3 0.1 M s kfwd 0.5 0.1 s binding to ATP (Fig. 4B and Table 3). For the peptide sub- − − − k = 25 ± 4s 1 k = (23 ± 3) × 10 4 s 1 Peptide off rev strate compared to wild type (KD = 1,109 ± 89 μM), both = ± μ −1· −1 = ± −1 Y253F kon 1.2 0.1 M s kfwd 0.9 0.1 s F317L and G250E, however, showed increased affinity, with − − − k = 25 ± 3s 1 k = (22 ± 3) × 10 4 s 1 Peptide off rev a ∼30% tighter binding for F317L (KD = 755 ± 11 μM) Results from fitting of rapid kinetics from stopped-flow experiments and, remarkably, almost threefold tighter affinity for G250E K Peptide = ± μ C (Fig. 2 and SI Appendix, Figs. S2–S5). Rate constants for the binding step ( D 438 43 M) (Fig. 4 ). In addition, kinase activity of wild type (WT) and mutants are shown in the second column: physical was increased for all resistance mutants compared to the wild k = ± −1 k = ± −1 binding (kon) and dissociation of imatinib (koff). Rate constants for slow con- type ( cat 19.0 0.5 s ): F317L ( cat 25.6 0.1 s ) and −1 formational change after imatinib binding are shown in the last column G250E (kcat = 24 ± 0.3 s ) were slightly more active, while (forward, k ; and reverse, k ). −1 fwd rev Y253F (kcat = 38 ± 2s ) was twice as fast (Fig. 4D). Finally, cooperativity between the different substrates was also substan- tially affected (Fig. 4E). K obs excellent agreement between the experimental ( D ) and cal- Markedly, these different changes of Abl’s properties taken K kin culated ( D ) overall dissociation constants for imatinib together fully recapitulate the observed increase in IC at cel- G 50 (Fig. 2 and Table 2) verifies the binding mechanism. lular concentrations of ATP for all resistance mutations (Fig. 4F). The quantitative analysis revealed the contribution of Effect of Resistance Mutation on IC50 Values for Imatinib Depends each parameter/step to the overall increase of IC50, illustrating Strongly on ATP Concentration. These relatively small changes of how single mutations in Abl can lead to imatinib resistance for the induced-fit step lad only to a twofold to threefold weaker patients by relatively small alterations to each individual overall affinity for imatinib. The small change in imatinib affinity parameter. for the resistance mutants alone is unlikely to be responsible for development of resistance. Why does drug affinity not define Discussion drug resistance? In patients, the effective outcome of imatinib Imatinib, and a number of other small-molecule drugs against treatment only depends on the drug’s ability to efficiently inhibit ’ protein kinases, has been very successful in the initial treatment the kinase s activity. In other words, what matters is how much of cancer. However, this original success has been diminished by drug is needed to reach wild-type level of activity for the mutated the occurrence of drug resistance in response to the treatment. proteins in the cellular context. Therefore, we next measured the In the IRIS (International Randomized Study of IFN and effect of imatinib on kinase activity at 25 °C, using conditions STI571) trial, ∼17% of patients exhibited imatinib resistance that are routinely used in the literature when reporting half- (45). Furthermore, Soverini et al. (12) showed that 12 to 63% of maximal inhibitory concentration (IC ) values (50 μM ATP; 50 imatinib-resistance patients across 11 studies exhibited mutations Fig. 3A). The increase in IC values for the mutant proteins 50 in the BCR-Abl kinase domain. While primary resistance (failure relative to wild-type were the same as the increases in K values D of initial treatment) is found in 21 to 48% of identified muta- for imatinib (Fig. 3A). Strikingly, when the IC experiments 50 tions, secondary resistance (failure of response after prolonged were repeated at cellular ATP concentration (5 mM), much treatment) makes up for a larger percentage, with 10 to 68% of larger effects were observed (Fig. 3B). The IC values were 6- to 50 imatinib-resistant cases (12, 13). To open the door for solving the 15-fold larger for the respective mutant proteins than wild type (Fig. 3C), potentially explaining why patients harboring any of problem of resistance to treatment, a first step is to understand these mutations are not responsive to imatinib treatment (34, the molecular mechanism by which mutations in the target protein 41–43). Since cancer cells have elevated intracellular ATP con- cause resistance to the drugs. This question has, of course, been asked in the literature due to the enormous importance and the centrations, we further tested whether the IC50 values changed at 10 mM ATP (SI Appendix, Fig. S7). The effect on IC50 values was obs very small, and the ratios between wild type and mutants were Table 2. Comparison between measured overall KD and the kin not affected. calculated macroscopic KD from kinetic scheme K kin,nM K obs,nM Quantitative Enzyme Kinetics Analysis of Resistance Mutations D D bind Reveals Underlying Mechanism for Resistance. One possible expla- WT KD = 25 ± 4 μM27± 926± 4 conf −4 nation for the bigger difference in drug affinity (IC50) at high Keq = (11 ± 3) × 10 bind ATP concentrations between wild type and mutants would be a F317L KD = 25 ± 4 μM42± 950± 10 conf −4 mechanism where resistance mutations tighten ATP binding, Keq = (19 ± 2) × 10 bind thus making it harder for imatinib to outcompete the nucleotide G250E KD = 25 ± 2 μM88± 26 78 ± 12 conf −4 at cellular concentrations. To determine if resistance mutations Keq = (46 ± 11) × 10 bind affect ATP binding affinity, we decided against a pure measure Y253F KD = 21 ± 3 μM53± 12 54 ± 4 conf −4 of ATP binding. In the cell, the kinase not only has to bind ATP, Keq = (26 ± 5) × 10

but also the second substrate, its target protein. Therefore, a full kin Comparison of the overall KD calculated from kinetic data (KD ) with the steady-state analysis of enzyme activity was performed to determine obs K Peptide K ATP experimentally measured macroscopic KD (KD ). Kinetic data are used to the binding of both substrates ( D and D ), their coopera- bind calculate individual equilibrium constants for binding (KD, ) and the tivity (α), and the catalytic turnover (kcat). To extract these parameters, conf kin induced-fit step (Keq ). The overall, kinetic KD is calculated according activity was measured at varying ATP/peptide concentrations, and kin ¼ bindp conf= þ conf to KD KD Keq 1 Keq and can be compared to the macroscopic, obs curves were globally fit to the equation for a sequential random experimentally determined value, KD (Fig. 2A) to validate the binding Bi–Bi mechanism (SI Appendix,Eq.1and Fig. 4A). The α parameter scheme. WT, wild type.

4of7 | www.pnas.org/cgi/doi/10.1073/pnas.1919221117 Hoemberger et al. Downloaded by guest on September 27, 2021 A

C

B BIOCHEMISTRY

Fig. 3. Kinase inhibition by imatinib (IC50) for resistance mutants exposes puzzle. (A and B)IC50 curves were measured at low (50 μM; A) and cellular (5 mM; B) ATP concentrations using 2 mM peptide substrate at 25 °C. (A, Right and B, Right)IC50 values are plotted showing the effect of ATP concentration on IC50.(C) Comparison of changes in KD (Fig. 2) with changes in IC50 for the imatinib-resistant mutants of Abl relative (rel.) to wild type at low and high ATP con- centrations. Errors are SD of triplicate experiments.

obvious nature of this question. Surprisingly, literature reports have words, subtle changes in the energy landscape of Abl by single been highly controversial, conflicting, or puzzling. A number of the point mutations, when added together, lead to resistance kinase domain resistance mutations have not even been experi- in patients. mentally investigated, but mechanisms have been assumed from What are the implications of our findings in respect to pub- static structural data, such as crystal structures and molecular- lished results on this question? Y253F is located at the P loop of dynamics simulation-based studies (13, 17, 24, 27, 33, 46, 47). We the kinase and forms a hydrogen bond with N322. This led to the started this project of characterizing the molecular mechanism of hypothesis in the literature that substitution of the tyrosine by the three major resistance mutations with an interesting observation phenylalanine would disrupt a hydrogen bond, thus removing that many resistance mutations overlap in position with the dynamic crucial interaction between kinase and drug and impair binding hot spots essential for the induced-fit step of imatinib binding (23). of the drug (21, 30, 48), leading to resistance. In addition to this From this finding, we reasoned that the induced fit will be structural interpretation, there have been conflicting reports of hampered by the mutations, and, hence, the drug affinities will activities and transformation efficiencies for Y253F. Allen and be reduced. Wiedemann (49) reported an increased transformation potency While this hypothesis qualitatively held true, we were sur- for Y253F, as well as an increased total phosphotyrosine content prised to find that this effect was much too small to explain drug in cell lysates from cells expressing full-length BCR-Abl–Y253F. resistance for any of the mutants. Nonetheless, the results rein- However, they were unable to see a higher activity in in vivo force that 1) these mutations do not alter the physical binding assays when comparing autophosphorylation of full-length c-Abl step of imatinib, but 2) alter the induced-fit step. This supports with full-length c-Abl–Y253F (49). In a later paper, Rou- the hypothesis that changes of these 15 dynamic hot spots act miantsev et al. (38) were also unable to show increased phos- cumulatively in causing high affinity of imatinib to Abl via this phorylation, neither for autophosphorylation nor for in vitro altered induced-fit step. assays using synthetic peptides as substrates. To solve this apparent puzzle of drug resistance, we analyzed The authors concluded that for increased catalytic activity of the effect of these three resistance mutations on altering the Y253F, expression of BCR-Abl–Y253F in cells was necessary free-energy landscape of the kinase and its effect on catalysis, as (38). Corbin et al. (39) investigated Y253F in the context of a well as on the competition of the drug with ATP. Only at cellular kinase domain-only construct, but could not detect an increase in ATP concentrations does the combination of a 1) small alter- activity for Y253F; however, they observed an increase in binding ation in the induced-fit step of imatinib binding with 2) an in- affinity to ATP. Studies by Griswold et al. (36), also utilizing a crease in enzymatic activity and 3) tighter binding to their kinase domain-only construct, showed increased transformation substrate peptide combined with substrate cooperativity yield an potency, as well as increased catalyticactivityincellular-outgrowth order-of-magnitude increased IC50 value for imatinib. In other assays and substrate-phosphorylation assays; however, their

Hoemberger et al. PNAS Latest Articles | 5of7 Downloaded by guest on September 27, 2021 A

BC DE F

Fig. 4. Resistance mutations cumulatively change the energy landscape of Abl kinase. Kinase activity for wild-type and resistant mutant forms of Abl at varying peptide and ATP concentrations (symbols, raw data; dashed line, fit to general velocity equation for a sequential random Bi–Bi reaction [see also SI

Appendix, Eq. 1]). (B–E) The KD for ATP (B), KD for peptide substrate (C), kinase turnover rate (kcat)(D), and cooperativity between substrates (α)(E) are altered by these individual mutations. (F) Fold change in IC50 of mutant forms relative (rel.) to wild type (WT) at 5 mM ATP, dissected into the individual contributions ATP Peptide from KD imatinib, kcat, Km , and Km . Note that the latter two also contain α. Errors shown in A are SD of mean for triplicate experiment; errors in B–D are uncertainties of the global fit for each enzyme form. Errors in E are propagated errors from A–D. The Welch t test was used to determine significance of the differences, and bars are marked. *P < 0.05; **P < 0.01.

measured activities are extremely low and only measured at Considering the findings in this study with previously pub- 100 μM ATP. Griswold et al. (36) concluded that differences lished data from Yun et al. (40) for EGFR suggests that the to published results might stem from insensitive assays using cumulative mechanism for resistance is a more broadly applica- Western blotting of substrate stained with pY antibodies. Our ble one than thought. Our results expose the necessity for careful data not only resolve the controversy about the underlying quantitative analysis of the enzymatic properties, along with the mechanism of resistance mutations by Y253F, but they dem- drug-binding mechanism of target proteins to understand their onstrate the need to characterize all changes to the enzyme respective resistance causing mutant forms. Enzymatic kits are that contribute to the overall resistance of patients’ mutant commonly used in research for probing catalytic activity of ki- proteins to imatinib. The increase in activity or the changes in nases and other targets and the effect of inhibitors. However, it is drug affinity alone are not sufficient for the observed resis- crucial for the user of such kits to keep in mind that many of tance in patients. these assays rely on low ATP or substrate concentrations and Similarly, the proposed simple mechanism of disruption of van could lead to misinterpretation. der Waals interactions with the aromatic ring of imatinib by the F317L mutation (21, 24, 27–30) is not the sole mechanism by Materials and Methods which this mutation causes drug resistance. Finally, we describe Expanded methods are available within SI Appendix. In brief, different the mechanism underlying resistance for one of the most com- constructs of Abl kinase were expressed and purified in Escherichia coli as monly isolated resistance mutations (G250E) that had not been described (23, 35). Rapid kinetics of imatinib binding to Abl kinase were quantitatively studied before. We are able to show that G250E measured via stopped-flow fluorescence, similar to that reported in ref. 23. has fundamental effects on the energy landscape of the kinase Kinase activity was measured by using a peptide substrate in a coupled and shows the strongest effect on both drug and ATP affinity. enzymatic assay that detects adenosine diphosphate production through

Table 3. Steady-state kinetic parameters for wild-type and mutant forms of Abl ATP Peptide −1 ATP Peptide Construct KD , μM KD , μM kcat,s α Calc. Km @ 2 mM peptide, mM Calc. Km @ 2 mM ATP,mM

WT 69 ± 2 1,107 ± 57 19.0 ± 0.5 1.8 ± 0.2 97 ± 8 1,975 ± 236 F317L 76 ± 1755± 11 25.6 ± 0.1 1.48 ± 0.02 100 ± 13 1,343 ± 176 G250E 47 ± 2438± 43 24.0 ± 0.3 2.2 ± 0.3 96 ± 13 952 ± 157 Y253F 98 ± 5 1,164 ± 50 38 ± 2 1.4 ± 0.3 104 ± 14 1,617 ± 344

Measured and derived steady-state parameters. Shown are fits of activity assays with varying concentrations of both peptide substrate (0.25 to 2 mM) and ATP (25 μM to 2 mM) shown in Fig. 4 and SI Appendix, Fig. S6. Data are fitted to the general velocity equation for a sequential random Bi–Bi reaction (see also ATP Peptide SI Appendix, Eq. 1). This yields catalytic activity (kcat), binding affinity of ATP (KD ), and binding affinity of peptide substrate (KD ), as well as proportionality factor α. α quantifies the degree that binding of substrate one either increases (a < 1) or decreases (a > 1) affinity of the other substrate ATP Peptide to the enzyme. These parameters can then be used to calculate the Km as well as Km at fixed concentrations of the other substrate (see also SI Appendix, Eqs. 2 and 3). Calc., calculated.

6of7 | www.pnas.org/cgi/doi/10.1073/pnas.1919221117 Hoemberger et al. Downloaded by guest on September 27, 2021 coupling with pyruvate kinase and lactate dehydrogenase, allowing for Data Availability. All data and scripts are available in the paper and monitoring of oxidation of NADH to NAD+. All activity assays were per- SI Appendix. formed in 50 mM Tris·HCl (pH 8.0), 500 mM NaCl, 1 mM Tris(2-carboxyethyl) phosphine, 5% dimethyl sulfoxide, 20 mM MgCl2, and 0.3 mg/mL bovine ACKNOWLEDGMENTS. We thank Renee Otten for helping with the lmfit serum albumin. Data fitting was performed with the Python package package and fitting of steady-state data. This work was supported by lmfit (50). the HHMI.

1. F. G. Giancotti, Deregulation of cell signaling in cancer. FEBS Lett. 588, 2558–2570 27. E. P. Reddy, A. K. Aggarwal, The ins and outs of BCR-Abl inhibition. Genes Cancer 3, (2014). 447–454 (2012). 2. E. K. Greuber, P. Smith-Pearson, J. Wang, A. M. Pendergast, Role of ABL family kinases 28. M. Azam et al., Activity of dual SRC-ABL inhibitors highlights the role of BCR/ABL in cancer: From leukaemia to solid tumours. Nat. Rev. Cancer 13, 559–571 (2013). kinase dynamics in drug resistance. Proc. Natl. Acad. Sci. U.S.A. 103, 9244–9249 (2006). 3. E. Gocek, A. N. Moulas, G. P. Studzinski, Non-receptor protein tyrosine kinases sig- 29. D. Bixby, M. Talpaz, Mechanisms of resistance to inhibitors in chronic naling pathways in normal and cancer cells. Crit. Rev. Clin. Lab. Sci. 51, 125–137 (2014). myeloid leukemia and recent therapeutic strategies to overcome resistance. Hema- 4. M. A. Seeliger et al., c-Src binds to the cancer drug imatinib with an inactive Abl/c-Kit tology (Am. Soc. Hematol. Educ. Program) 2009, 461–476 (2009). conformation and a distributed thermodynamic penalty. Structure 15, 299–311 30. C. B. Gambacorti-Passerini et al., Molecular mechanisms of resistance to imatinib in (2007). Philadelphia-chromosome-positive leukaemias. Lancet Oncol. 4,75–85 (2003). 5. O. Hantschel, Allosteric BCR-ABL inhibitors in Philadelphia chromosome-positive 31. S. Lovera et al., The different flexibility of c-Src and c-Abl kinases regulates the ac- acute lymphoblastic leukemia: Novel opportunities for drug combinations to over- cessibility of a druggable inactive conformation. J. Am. Chem. Soc. 134, 2496–2499 come resistance. Haematologica 97, 157–159 (2012). (2012). 6. A. Sirvent, C. Benistant, S. Roche, Cytoplasmic signalling by the c-Abl tyrosine kinase in 32. P. S. Georgoulia, G. Todde, S. Bjelic, R. Friedman, The catalytic activity of Abl1 single normal and cancer cells. Biol. Cell 100, 617–631 (2008). and compound mutations: Implications for the mechanism of drug resistance muta- – 7. S. Balabanov, M. Braig, T. H. Brümmendorf, Current aspects in resistance against ty- tions in chronic myeloid leukaemia. Biochim. Biophys. Acta Gen. Subj. 1863, 732 741 rosine kinase inhibitors in chronic myelogenous leukemia. Drug Discov. Today. (2019). Technol. 11,89–99 (2014). 33. D. L. Gibbons et al., Molecular dynamics reveal BCR-ABL1 polymutants as a unique 8. G. K. Lambert, A.-K. Duhme-Klair, T. Morgan, M. K. Ramjee, The background, dis- mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy. Proc. Natl. Acad. – covery and clinical development of BCR-ABL inhibitors. Drug Discov. Today 18, Sci. U.S.A. 111, 3550 3555 (2014). 992–1000 (2013). 34. N. P. Shah et al., Multiple BCR-ABL kinase domain mutations confer polyclonal re- 9. M. W. N. Deininger, J. M. Goldman, J. V. Melo, The molecular biology of chronic sistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast – myeloid leukemia. Blood 96, 3343–3356 (2000). crisis chronic myeloid leukemia. 2, 117 125 (2002). 35. R. V. Agafonov, C. Wilson, R. Otten, V. Buosi, D. Kern, Energetic dissection of Glee- 10. N. Iqbal, N. Iqbal, Imatinib: A breakthrough of in cancer. Chemo- vec’s selectivity toward human tyrosine kinases. Nat. Struct. Mol. Biol. 21, 848–853 ther. Res. Pract. 2014, 357027 (2014). (2014). 11. M. Laurent et al., Adjuvant therapy with imatinib in gastrointestinal stromal tumors 36. I. J. Griswold et al., Kinase domain mutants of Bcr-Abl exhibit altered transformation (GISTs)—review and perspectives. Transl. Gastroenterol. Hepatol. 4, 24 (2019). potency, kinase activity, and substrate utilization, irrespective of sensitivity to im- 12. S. Soverini et al., Implications of BCR-ABL1 kinase domain-mediated resistance in BIOCHEMISTRY atinib. Mol. Cell. Biol. 26, 6082–6093 (2006). chronic myeloid leukemia. Leuk. Res. 38,10–20 (2014). 37. B. J. Skaggs et al., Phosphorylation of the ATP-binding loop directs oncogenicity of 13. C. Chandrasekhar, P. S. Kumar, P. V. G. K. Sarma, Novel mutations in the kinase do- drug-resistant BCR-ABL mutants. Proc. Natl. Acad. Sci. U.S.A. 103, 19466–19471 (2006). main of BCR-ABL gene causing imatinib resistance in chronic myeloid leukemia pa- 38. S. Roumiantsev et al., Clinical resistance to the kinase inhibitor STI-571 in chronic tients. Sci. Rep. 9, 2412 (2019). myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. Proc. Natl. 14. M. Prieto-Vila, R.-U. Takahashi, W. Usuba, I. Kohama, T. Ochiya, Drug resistance Acad. Sci. U.S.A. 99, 10700–10705 (2002). driven by cancer stem cells and their niche. Int. J. Mol. Sci. 18 , 2574 (2017). 39. A. S. Corbin, E. Buchdunger, F. Pascal, B. J. Druker, Analysis of the structural basis of 15. M. Al-Hajj, M. S. Wicha, A. Benito-Hernandez, S. J. Morrison, M. F. Clarke, Prospective specificity of inhibition of the Abl kinase by STI571. J. Biol. Chem. 277, 32214–32219 identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U.S.A. 100, (2002). 3983–3988 (2003). 40. C.-H. Yun et al., Structures of lung cancer-derived EGFR mutants and inhibitor com- 16. T. Saleh, P. Rossi, C. G. Kalodimos, Atomic view of the energy landscape in the allo- plexes: Mechanism of activation and insights into differential inhibitor sensitivity. – steric regulation of Abl kinase. Nat. Struct. Mol. Biol. 24, 893 901 (2017). Cancer Cell 11, 217–227 (2007). 17. E. J. Jabbour, J. E. Cortes, H. M. Kantarjian, Resistance to tyrosine kinase inhibition 41. T. O’Hare et al., In vitro activity of Bcr-Abl inhibitors AMN107 and BMS-354825 therapy for chronic myelogenous leukemia: A clinical perspective and emerging against clinically relevant imatinib-resistant Abl kinase domain mutants. Cancer Res. – treatment options. Clin. Myeloma Leuk. 13, 515 529 (2013). 65, 4500–4505 (2005). 18. Y.-F. Chen, L.-W. Fu, Mechanisms of acquired resistance to tyrosine kinase inhibitors. 42. A. Hochhaus et al., Molecular and chromosomal mechanisms of resistance to imatinib – Acta Pharm. Sin. B 1, 197 207 (2011). (STI571) therapy. Leukemia 16, 2190–2196 (2002). 19. S. W. Cowan-Jacob et al., Structural biology contributions to the discovery of drugs to 43. A. S. Corbin, P. La Rosée, E. P. Stoffregen, B. J. Druker, M. W. Deininger, Several Bcr- – treat chronic myelogenous leukaemia. Acta Crystallogr. D Biol. Crystallogr. 63,80 93 Abl kinase domain mutants associated with imatinib mesylate resistance remain (2007). sensitive to imatinib. Blood 101, 4611–4614 (2003). 20. N. M. Levinson et al., A Src-like inactive conformation in the Abl tyrosine kinase 44. Z. H. Foda, Y. Shan, E. T. Kim, D. E. Shaw, M. A. Seeliger, A dynamically coupled al- domain. PLoS Biol. 4, e144 (2006). losteric network underlies binding cooperativity in Src kinase. Nat. Commun. 6, 5939 21. R. Barouch-Bentov, K. Sauer, Mechanisms of drug resistance in kinases. Expert Opin. (2015). Investig. Drugs 20, 153–208 (2011). 45. T. P. Braun, C. A. Eide, B. J. Druker, Response and resistance to BCR-ABL1-targeted 22. J. Schoepfer et al., Discovery of asciminib (ABL001), an allosteric inhibitor of the ty- therapies. Cancer Cell 37, 530–542 (2020). rosine kinase activity of BCR-ABL1. J. Med. Chem. 61, 8120–8135 (2018). 46. S. Lovera et al., Towards a molecular understanding of the link between imatinib 23. C. Wilson et al., Kinase dynamics. Using ancient protein kinases to unravel a modern resistance and kinase conformational dynamics. PLoS Comput. Biol. 11, e1004578 cancer drug’s mechanism. Science 347, 882–886 (2015). (2015). 24. T. Schindler et al., Structural mechanism for STI-571 inhibition of Abelson tyrosine 47. C. Miething et al., The Bcr-Abl mutations T315I and Y253H do not confer a growth kinase. Science 289, 1938–1942 (2000). advantage in the absence of imatinib. Leukemia 20, 650–657 (2006). 25. J. S. Khorashad et al., In vivo kinetics of kinase domain mutations in CML patients 48. M. Azam, R. R. Latek, G. Q. Daley, Mechanisms of autoinhibition and STI-571/imatinib treated with after failing imatinib. Blood 111, 2378–2381 (2008). resistance revealed by mutagenesis of BCR-ABL. Cell 112, 831–843 (2003). 26. S. Kamasani et al., Computational analysis of ABL kinase mutations allows predicting 49. P. B. Allen, L. M. Wiedemann, An activating mutation in the ATP of the drug sensitivity against selective kinase inhibitors. Tumour Biol. 39, 1010428317701643 ABL kinase domain. J. Biol. Chem. 271, 19585–19591 (1996). (2017). 50. M. Newville et al., lmfit/lmfit-py 0.9.12 (Version 0.9.12, Zenodo, Geneva, Switzerland).

Hoemberger et al. PNAS Latest Articles | 7of7 Downloaded by guest on September 27, 2021