THE RISE AND FALL OF GAKEKEEPER MUTATIONS? THE BCR-ABL1^T315I PARADIGM

Abstract

The use of tyrosine kinase inhibitors (TKIs) has become an integral component of cancer therapy. Imatinib mesylate (Gleevec), a BCR-ABL1 inhibitor, was the first TKI approved in cancer medicine and has served as a model for the development of similar agents for other cancers. An important drawback of TKI therapy is development of resistance, frequently through the acquisition of mutations. Mutations at the gatekeeper residues of BCR-ABL1 (T315I) and other oncogenic kinases have proven highly resistant to currently available TKIs. Advances in the structural biology of oncogenic kinases have facilitated the rational development of TKIs active against gatekeeper mutations.

The paradigmatic T315I gatekeeper mutation

Phosphorylation of specific serine, threonine, or tyrosine residues is germane to cellular growth and its control relies on the proper regulation of protein kinases. Protein kinases are plastic molecular switches that oscillate between “on” and “off” conformations according to diametrically opposite catalytical activity states.(1) All protein kinases catalyze the transfer of the γ-phosphate of ATP to the hydroxyl group of serine, threonine, or tyrosine. In order to do so, they must adopt a catalytically active “on” conformation, which involves changes in the orientation of the C helix in the small N-terminal lobe and the activation area in the larger C-terminal lobe.(2) Kinase activation in the wrong temporospatial context can result in unbridled cell proliferation and malignant transformation. Such is the case in chronic myeloid leukemia (CML), a clonal malignancy characterized by unchecked myeloid proliferation driven by the constitutively active kinase activity of the BCR-ABL1 fusion oncoprotein.(1) The critical role of BCR-ABL1 in the pathogenesis of CML fueled the development of ATP mimetics capable of antagonizing its kinase activity. Tyrosine kinase inhibitors (TKIs) can be divided in type I, which directly compete with ATP for the ATP-binding site, and type II, which not only bind to the ATP binding site but also occupy an adjacent hydrophobic space only accessible when the kinase is in its inactive conformation.(3) The type II BCR-ABL1 kinase inhibitor imatinib mesylate was the first TKI to be approved in cancer medicine and propelled the development of similar therapeutics for other cancer types. After 8 years of follow-up in the phase III IRIS trial, imatinib therapy produced cumulative rates of complete cytogenetic response (CCyR; i.e. no evidence of the Philadelphia chromosome in the bone marrow) and major molecular response (MMR; i.e. BCR-ABL1/ABL1 ratio ≤0.1%) of 83% and 86%, respectively among patients with CML in chronic phase (CP). These responses translated into event-free, progression-free, and overall survival rates of 81%, 92%, and 85% (93% considering only CML-related deaths), respectively.(4) In spite of these remarkable results, it is estimated that 20%−30% of patients will eventually develop resistance to imatinib, which in 40% of them will be associated with the acquisition of BCR-ABL1 kinase domain mutations.(5) Although more than 100 BCR-ABL1 point mutations have been reported,(1, 6–11) those at residues Gly250, Tyr253, Glu255, Thr315, Met351, and Phe359 account for more than 60% of all mutations.(12) Most troublesome amongst all mutations is the substitution of isoleucine at the 315 position of the ABL1 kinase for threonine (T315I). Thr315 is known as the gatekeeper residue as it maps to the periphery of the nucleotide-binding site of ABL1.(1) T315I is associated with an overall survival of 22 months in patients with CML-CP.(13) Second generation TKIs such as nilotinib or dasatinib, when used in patients with CML resistant to imatinib, render CCyR rates of 45–50%.(14, 15) Notwithstanding their remarkable activity against most BCR-ABL1 mutants, neither nilotinib nor dasatinib are active in patients carrying the T315I mutation,(14, 15) supporting the notion that this mutation represents an important escape mechanism for CML cells withstanding high levels of selection pressure imposed by TKI therapy.

Why is T315I (and for that matter, any gatekeeper mutation) so difficult to target with ATP-mimetic TKIs?

Structural studies have shown that the location of the Thr315 residue plays a big role.(16) Thr315 locates at the periphery of the nucleotide-binding site of ABL1 kinase within the hinge region of the enzymatic cleft and stabilizes imatinib binding through hydrogen-bond interactions and regulates access to a deep hydrophobic pocket in the active site.(1, 7) Importantly, although imatinib and nilotinib bind BCR-ABL1 kinase in the inactive conformation and dasatinib does so in the active conformation, all three TKIs make a critical hydrogen bond with the side chain hydroxyl group of Thr315.(1, 17, 18) A mutation of the threonine gatekeeper residue to isoleucine prevents the formation of this critical hydrogen bond. Second, such mutation also causes steric hindrance between the large hydrophobic isoleucine residue and any of the three TKIs, thus blocking the access of the latter to the hydrophobic pocket in the proximity of Thr315.(16) Third, Thr315 participates in a network of hydrophobic interactions when the kinase is in the active conformation. Its mutation to isoleucine promotes the assembly of an enzymatically active kinase conformation through the stabilization a series of hydrophobic interactions.(2, 19) As a consequence, T315I results in complete insensitivity to imatinib, nilotinib, dasatinib, and bosutinib.(16, 17, 20–24)

Is it possible to target BCR-ABL1^T315I with an ATP competitive small molecule?

In recent years, a series of dual inhibitors of BCR-ABL1^T315I and Aurora kinases have been tested in clinical trials, including MK-0457,(25) danusertib (PHA-739538),(26, 27) or XL-228.(28) Unfortunately, their administration involved inconvenient intravenous infusions, responses were limited and typically short-lived, coinciding with the periods of drug administration. Furthermore, all of them have been associated with very high rates of grade 3–4 cytopenia as well as significant non-hematologic toxicities due to their activity against both malignant as well as normal cells, as Aurora kinases are involved in normal cell signaling. The interest to develop T315I inhibitors has been reignited with the irruption of ponatinib (AP24534) in the clinical arena. Ponatinib is a TKI obtained through a structure-guided drug design strategy aimed at targeting the inactive conformation of the ABL1 kinase and at avoiding the interaction with the side chain of 315I.(29) Ponatinib potently inhibits both native (IC₅₀ 0.37nM) and T315I (IC₅₀ 2.0nM) ABL1 kinases, as well as multiple BCR-ABL1 mutations that confer high levels of resistance to other TKIs, SRC family of kinases, vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor 1 (FGFR1), and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, but not Aurora kinases.(30) Structural analysis of the co-crystal structure of the ponatinib analog AP24589 with BCR-ABL1^T315I indicate that the ability of ponatinib to inhibit the gatekeeper mutation resides in its slender linear triple carbon ethynyl linker that a) prevents direct contact with the isoleucine residue and b) promotes an extended conformation of the unbound inhibitor that favors binding to the inactive conformation that T315I adopts.(29)

We have conducted in silico molecular simulations that have confirmed the ability of ponatinib to bind in a highly efficient manner to the T315I mutant isoform of BCR-ABL1 by virtue of its ethynyl linker, which minimizes steric clash with the bulky isoleucine residue (Figure 1A–C). Indeed, ponatinib derivatives with altered ethynyl linkers exhibit a markedly diminished activity against T315I in cellular assays, thus highlighting the tremendous importance of this region of the molecule in avoiding the steric hindrance posed by the mutant isoleucine residue.(29) In cell-based mutagenesis screens, ponatinib completely abrogated the emergence of resistant CML clones at concentrations ≥40nM in cell-based mutagenesis screens, and prolonged the survival of mice previously injected intravenously with Ba/F3 cells expressing BCR-ABL1^T315I.(30) In a recently completed phase I study, ponatinib was administered orally at doses ranging from 2 to 60 mg daily to 74 patients, of whom 60 had CML (44 in CP, 7 in accelerated phase and 9 in blastic phase), 4 with BCR-ABL1+ acute lymphoblastic leukemia (ALL), and 10 with other myeloid malignancies. Of the 64 patients with Ph+ leukemia, 95% had failed at least 2 TKIs (65% at least 3 TKIs), 63% carried at least one BCR-ABL1 mutation, including the T315I mutation in 28% of patients.(31) The dose limiting toxicity was pancreatic, which was reported in 4 of the 12 evaluable patients treated at 60 mg daily. The most frequent grade 3–4 treatment-related grade 3–4 non-hematologic toxicities were thrombocytopenia (16%), neutropenia (7%), and elevation of lipase (7%) but only 4% of patients discontinued ponatinib therapy due to toxicity. Doses over 30 mg/d consistently rendered plasma concentrations predicted to prevent the emergence of all BCR-ABL1 mutations in vitro (>40nM).(31) Among patients with CML-CP, the CCyR rate was 53% (89% in T315I) and the MMR rate was 42% (78% in patients with T315I), with most responses being maintained after 12 months of follow-up.(31) These results, while preliminary, are remarkable and if confirmed in an ongoing phase II study, will establish ponatinib as the drug of choice for patients carrying the T315I mutation. Furthermore, patients failing sequential TKI therapy are known to accumulate more than one BCR-ABL1 mutation within the same malignant clone, which has been shown to increase their oncogenicity.(10, 32) In addition to single point mutants, our in silico experiments have also shown that ponatinib binds with high affinity several double mutant isoforms of this protein, mainly by virtue of substantially reduced steric clashes and preserved favorable interaction with the mutant residues as well as other amino acids lining the drug binding site (Figure 1D). A recently published drug resistance screen with AP24163, a TKI related to ponatinib, using randomly mutagenised BCR-ABL1^T315I has shown that compound mutations involving T315I can still be recovered.(33) However, structural analyses of some of the most frequently encountered compound mutants found in patients with imatinib-resistant CML treated at our institution in complex with ponatinib have shown the latter to be able to bind to all double mutant BCR-ABL1 isoforms with IC₅₀ values that, while several-fold higher compared with the single point-mutants, can be easily reached at 45mg daily, the dose currently being tested in phase II studies.

Figure 1. Ponatinib avoids the steric hindrance imposed by T315I.

(A) Computer-simulated ribbon depictions of imatinib (purple), nilotinib (orange) and ponatinib (green) bound to BCR-ABL1T^315I mutant. The mutant residue I315 is depicted as red sticks, respectively. Highlights of the interaction of imatinib (B) and ponatinib (C) with the mutant residue I315 of BCR-ABL. Note the difference in steric clash (portrayed as colored areas), accounting for the preserved affinity of ponatinib for the mutant protein with respect to the binding failure of imatinib. (D) Computer simulation of ponatinib bound to the BCR-ABL1T^315I/F317L double mutant. The mutant residues I315 and L317 are depicted as red and yellow sticks, respectively. Drug hydrogen atoms, water molecules and counterions are omitted for clarity. Ponatinib inhibits BCR-ABL1T^315I/F317L with an IC₅₀ of 21nM, 2.8-fold higher than BCR-ABL1T^315I (IC₅₀ 7.4nM).

Are there alternative ways to inhibit gatekeeper mutations with TKIs?

Alternate ways to inhibit BCR-ABL1 kinase activity while avoiding a direct interaction with the T315I gatekeeper mutation have been reported. One of the most attractive ones is that of targeting hydrophobic pockets remote from the catalytic domain where classical TKIs clash with gatekeeper mutant residues. DCC-2036, a novel TKI class known as switch pocket inhibitors is undergoing clinical testing in a phase I study for patients carrying T315I or who have failed more than two TKIs. DCC-2036 is a multikinase inhibitor that binds to a pocket that governs the transition between the active and the inactive states of ABL1, thus locking the kinase into its inactive state through a non-ATP competitive mechanism.(34) It has been reported that gatekeeper mutants can also be inhibited by perturbing the flexibility of the P-loop.(35) Alternatively, T315I kinase could potentially be inhibited by binding to the auto-regulatory allosteric myristate cleft at the N-terminus of ABL1, rendering a similar net effect of freezing the kinase in its inactive state in a selective, non-ATP-competitive manner.(36) Such is the mechanism of action of GNF-2, although this compound does not inhibit T315.(36) However, the GNF-2 analogue GNF-5 has been shown to synergize in vitro with nilotinib and imatinib to inhibit T315I.(37) Both DCC-2036 and GNF-5 represent attractive options that warrant clinical testing in patients with highly resistant BCR-ABL1 mutations, but ultimately, these novel agents for the treatment of CML resistant to conventional TKI therapies provide conceptual experimental templates that can be adapted to the development of targeted agents for highly resistant mutant kinase driving the growth of other malignancies. In addition to TKIs, two other alternatives to the treatment of patients with CML carrying the T315I mutation are available: first, omacetaxine, a semisynthetic alkaloid with putative activity against CML stem cells,(38, 39) has been shown to induce cytogenetic and CCyR rates of 41% and 18%, respectively among patients with CML-CP carrying the T315I mutation.(40) Second, Allogeneic stem cell transplantation, which can induce CMR, particularly in patients with CML-CP.(41)

What are the implications of these advances in CML?

Gatekeeper mutations represent a common mechanism of escape for cancer cells to overcome the selection pressure imposed by effective TKIs. Prime examples of the latter are PDGFR^T674I/M in hypereosinophilic syndrome,(42) EGFR^T790M in non-small cell lung cancer (NSCLC),(43–45) and KIT^T670I in gastrointestinal stromal tumors.(46) Experimental modeling of BRAF kinase gatekeeper mutations suggest that this may also be a mechanism of TKI resistance in cancer cells driven by mutant BRAF kinase.(47) Similar to CML, gatekeeper mutations in these cancers are typically detected in patients developing TKI resistance after an initial response to treatment. The development of ponatinib exemplifies how drug design driven by meticulous structural analyses can render compounds that override the hurdles imposed by gatekeeper mutations. An alternative approach is that of using functional pharmacological screens of large libraries of small molecule inhibitors guided by molecular modeling predictions.(48) By means of such approach, a covalent irreversible pyrimidine EGFR inhibitor with 30- to 100-fold higher potency against EGFR^T790M compared with native EGFR has been recently identified.(48) A novel class of ATP-competitive TKI led by HG-7–85-01 has been reported as capable of inhibiting BCR-ABL1^T315I, KIT^T670I, and PDGFR^T674I/M gatekeeper mutations due to its ability to accommodate both the native gatekeeper threonine as well as the mentioned hydrophobic mutant residues. Certainly, the prospect of a universal gatekeeper mutation inhibitor is attractive but, is it realistically possible? The surface comparison of distinct tyrosine and serine–threonine kinases has revealed a set of 30 residues whose spatial positions are highly conserved associated with similar patterns of protein kinase activition.(2) This high level of conservation across different kinases involved in cancer development might unveil a vulnerability that could be therapeutically exploited. After all, the development of a gatekeeper mutation is a rather predictable (conserved?) process arising in cancer cells driven by oncokinases exposed to high levels of selection pressure by effective TKIs. The process customarily involves the substitution of an isoleucine or a methionine residue by the native gatekeeper residue. This remarkably narrow repertoire of substitutions at the gatekeeper position has furnished structural biologists and chemists the opportunity to design and synthesize compounds such as ponatinib or HG-7–85-01 versatile enough to avoid the structural constraints imposed by the gatekeeper mutants.

Lessons learned and unsolved questions

While nilotinib and dasatinib have proven more efficacious than imatinib as frontline therapy for CML-CP, a subset of patients will continue to fail therapy, which in many cases will be associated with the acquisition of BCR-ABL1 mutations, including T315I. Ponatinib is an oral TKI that has been reported to produce high rates of cytogenetic and molecular response with a favorable toxicity profile in a phase I study for patients resistant to multiple TKIs and/or carrying the T315I mutation. It appears that almost one decade after the initial description of this gatekeeper mutation, an efficacious, safe, oral TKI will finally be clinically available. However, several questions still linger: first, what will be the role of ponatinib? Given its remarkable activity in patients with multi-TKI resistant CML, it is reasonable to think that its activity will be even higher as frontline therapy and perhaps superior to nilotinib or dasatinib. Second, will the use of ponatinib in the frontline setting prevent the clinical emergence of BCR-ABL1 mutants? The in vitro characterization of this agent appears to suggest that possibility. However, it is uncertain whether alternative mechanisms of resistance will become more relevant, such as the development of complex compound BCR-ABL1 mutations or mutations in tumor suppressors (e.g. p53, ARF, p16). In that regard, it must be emphasized that in spite of the preliminary remarkable activity shown by ponatinib, a significant proportion of patients with T315I fail to respond appropriately to ponatinib, likely due to the coexistence of alternative mechanism of resistance. Third, could ponatinib improve the outcomes of patients with BCR-ABL1-positive ALL? It is possible given its high potency against native BCR-ABL1 kinase, its mild toxicity profile, and its activity against T315I, a mutation particularly prevalent in patients with BCR-ABL1-positive ALL failing TKI therapy. Finally, can ponatinib cure patients with newly diagnosed CML-CP? It is both tantalizing and naïve in equal measure to think so.

Table 1.

Characteristics of ponatinib compared to those of nilotinib and dasatinib regarding efficacy and toxicity in patients with CML.

	Dasatinib	Nilotinib	Ponatinib
Potency relative to imatinib	325	30	400–500
Main target kinase(s)	SRC, ABL1	ABL1	SRC, ABL1, VEGF
Target ABL1 conformation	Active + Inactive	Inactive	Inactive
Resistant mutations	T315I	T315I, E255V	None
Mutations with intermediate sensitivity	E255K/V, V299L, F317L	E255K/V, Y253F/H, Q252H, F359V	T315I, E255V
Standard dose (Frontline, CP)	100 mg/d	300 mg twice daily	45 mg/d
Main toxicities	Cytopenia, pleural effusion, bleeding	Cytopenia, bilirubin & lipase elevation	Cytopenia, pancreatic
KIT inhibition (vs imatinib)	Increased	Similar	Increased
PDGFR inhibition (vs imatinib)	Increased	Similar	Increased
Clinical activity after failure to 2 TKIs	+	+	+++
Clinical activity against T315I	None	None	Highly active
Activity against CML stem cells	None	None	Unknown