Supplemental Materials from Computational and Functional Analyses of HER2 Mutations Reveal Allosteric Activation Mechanisms and Altered Pharmacologic Effects

Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplars exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant that rapidly generated bispecific nanomolar degraders of ERα, with PROTACs inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization. Citation Format: Anthony D. Keefe, Jeremy S. Disch, Jennifer Duffy, Esther C. Lee, Diana Gikunju, Betty Chan, Benjamin D. Levin, Michael I. Monteiro, Sarah A. Talcott, Anthony Lau, Fei Zhou, Anton Kozhushnyan, Neil E. Westlund, Patrick B. Mullins, Yan Yu, Moritz von Rechenberg, Junyi Zhang, Yelena Arnautova, Yanbin Liu, Ying Zhang, Andrew J. McRiner, Anna Kohlmann, Matthew A. Clark, John W. Cuozzo, Christelle Huguet, Shilpi Arora. Discovery of new targeted protein degraders using DNA-encoded chemistry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5346.

Supplementary methods, patient information and figure legends

Supplementary Tables. Table S1. In Vitro Kinase IC50 Values (nM) for Native and Mutant Recombinant KIT. Table S2. Summary of Ba/F3 KIT Cell Lines Generated in this Study. Table S3. Summary of IC50 Viability Values in Ba/F3 KIT Cells. Table S4. Summary of KIT Driven Ba/F3 In Vivo Studies.

Supplementary Figures. Figure S1. Chemical structures of imatinib, sunitinib, regorafenib and ponatinib. Figure S2. Expression and activation of KIT in engineered Ba/F3 cells. Figure S3. Ponatinib inhibits the phosphorylation of exon 11primary activating, and secondary resistant mutant forms of KIT. Figure S4. Secondary mutants reduce ponatinib potency in Ex9 ins and V560D cell lines. Figure S5. Illustration of the optimal fit of ponatinib to KIT. Figure S6. Impact of compound treatment on KIT signaling in GIST-derived cell lines.

Abstract Amplification of HER2 can drive the proliferation of cancer cells, and several inhibitors of HER2 have been successfully developed. Recent advances in next-generation sequencing now reveal that HER2 is subject to mutation, with over 2,000 unique variants observed in human cancers. Several examples of oncogenic HER2 mutations have been described, and these primarily occur at allosteric sites outside the ATP-binding site. To identify the full spectrum of oncogenic HER2 driver mutations aside from a few well-studied mutations, we developed mutation-allostery-pharmacology (MAP), an in silico prediction algorithm based on machine learning. By applying this computational approach to 820 single-nucleotide variants, a list of 222 known and potential driver mutations was produced. Of these 222 mutations, 111 were screened by Ba/F3-retrovirus proliferation assays; 37 HER2 mutations were experimentally determined to be driver mutations, comprising 15 previously characterized and 22 newly identified oncogenic mutations. These oncogenic mutations mostly affected allosteric sites in the extracellular domain (ECD), transmembrane domain, and kinase domain of HER2, with only a single mutation in the HER2 orthosteric ATP site. Covalent homodimerization was established as a common mechanism of activation among HER2 ECD allosteric mutations, including the most prevalent HER2 mutation, S310F. Furthermore, HER2 allosteric mutants with enhanced covalent homodimerization were characterized by altered pharmacology that reduces the activity of existing anti-HER2 agents, including the mAb trastuzumab and the tyrosine kinase inhibitor lapatinib. Overall, the MAP-scoring and functional validation analyses provided new insights into the oncogenic activity and therapeutic targeting of HER2 mutations in cancer. Significance: This study identified new oncogenic HER2 allosteric mutations, including ECD mutations that share covalent dimerization as a mechanism of oncogenicity, suggesting the need for novel inhibitors to treat HER2-mutant cancers.

In the treatment of non-small cell lung cancer (NSCLC), patients harboring exon 20 insertion mutations in the epidermal growth factor receptor (EGFR) gene (EGFR) have few effective therapies because this subset of mutants is generally resistant to most currently approved EGFR inhibitors. This report describes the structure-guided design of a novel series of potent, irreversible inhibitors of EGFR exon 20 insertion mutations, including the V769_D770insASV and D770_N771insSVD mutants. Extensive structure-activity relationship (SAR) studies led to the discovery of mobocertinib (compound 21c), which inhibited growth of Ba/F3 cells expressing the ASV insertion with a half-maximal inhibitory concentration of 11 nM and with selectivity over wild-type EGFR. Daily oral administration of mobocertinib induced tumor regression in a Ba/F3 ASV xenograft mouse model at well-tolerated doses. Mobocertinib was approved in September 2021 for the treatment of adult patients with advanced NSCLC with EGFR exon 20 insertion mutations with progression on or after platinum-based chemotherapy.

Inhibition of hydroxy acid oxidase 1 (HAO1) is a strategy to mitigate the accumulation of toxic oxalate that results from reduced activity of alanine-glyoxylate aminotransferase (AGXT) in primary hyperoxaluria 1 (PH1) patients. DNA-Encoded Chemical Library (DECL) screening provided two novel chemical series of potent HAO1 inhibitors, represented by compounds 3-6. Compound 5 was further optimized via various structure-activity relationship (SAR) exploration methods to 29, a compound with improved potency and absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) properties. Since carboxylic acid-containing compounds are often poorly permeable and have potential active glucuronide metabolites, we undertook a brief, initial exploration of acid replacements with the aim of identifying non-acid-containing HAO1 inhibitors. Structure-based drug design initiated with Compound 5 led to the identification of a nonacid inhibitor of HAO1, 31, which has weaker potency and increased permeability.

Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library (DECL) screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function (GOF) mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplar 7 exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant of 7 rapidly generated bispecific nanomolar degraders of ERα, with PROTACs 18 and 21 inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization.

Activating mutations in RAS proteins occur in ~1/3 of human cancers. These mutations impair the ability of the protein to hydrolyze GTP to GDP. As a result, mutant RAS proteins exist predominantly in the GTP-bound state, which directly activates aberrant downstream signaling via interaction with effectors such as RAF. Most RAS mutations occur at glycine 12 of the KRAS isoform. One such mutation, KRAS G12C, is particularly common in non-small cell lung cancer where it is found in ~15% of lung adenocarcinomas. Recent efforts have targeted KRAS G12C in the GDP-bound state; however, direct pharmacologic inhibition of active, GTP-bound KRAS G12C has proved challenging. Here, we deployed a novel SMARTTM (Small Molecule Assisted Receptor Targeting) platform to advance covalent compounds that selectively inhibit GTP-bound KRAS G12C. Using a mechanism reminiscent of the natural products rapamycin and cyclosporine, these compounds promote formation of a novel inhibitory ternary complex consisting of cyclophilin A (CypA, an abundant immunophilin present in all human cells), the SMART inhibitor, and GTP-KRAS G12C. Structure-based design of the SMART inhibitor yielded potent covalent inhibitors of GTP-KRAS G12C that exhibit >100-fold selectivity for mutant KRAS G12C over WT KRAS. Structural analysis of the ternary complex revealed that the covalent linkage between the SMART inhibitor and the mutant cysteine of KRAS occurred in the context of extensive interactions between CypA, the SMART inhibitor, and GTP-KRAS G12C that provide significant binding affinity (KI = 2.5 μM). The GTP-KRAS G12C|Inhibitor|CypA complex directly occluded effector binding, and as such, the compounds disrupted the KRAS-RAF interaction in biochemical assays. This activity was dependent on CypA, underlining the importance of the KRAS G12C| CypA protein-protein interaction in driving target engagement. In cell-based studies, SMART inhibitors crosslinked KRAS G12C and potently inhibited ERK phosphorylation and cell growth in G12C mutant tumor cell lines but had no effect on non-G12C bearing tumor cells. CRISPR knockout of cellular CypA confirmed that these activities were dependent on the presence of endogenous CypA. Importantly, SMART inhibitors bind directly to active, GTP-KRAS G12C and thus, their activity does not rely on trapping KRAS G12C in the inactive GDP-bound state. As a result, the cellular potency of SMART inhibitors with respect to crosslinking, pERK inhibition, and growth inhibition was maintained in the presence of growth factor treatments that reduce the cellular GDP-KRAS G12C pool. In contrast, we found that the activity of a previously described GDP-KRAS G12C targeting inhibitor was attenuated by growth factor treatment. To our knowledge, these are the first examples of mutant-selective KRAS inhibitors that target the active, GTP-bound state of KRAS G12C. We are currently optimizing the drug-like properties of these SMART inhibitors and evaluating their activity in in vivo models. Citation Format: Michelle L. Stewart, Nicholas R. Perl, Seung-Joo Lee, Linlong Xue, Minyun Zhou, Jonah Simon, Kathryn M. Luly, Siminia Grigoriu, Alex Yuzhakov, Alec Silver, Jason T. Lowe, Cindy C. Benod, Alan S. Mann, Gregory L. Verdine, Alan C. Rigby, Mark J. Mulvihill, Earl W. May, Anna Kohlmann, Sharon A. Townson, Roy M. Pollock, Meizhong Jin. Development of inhibitors of the activated form of KRAS G12C [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B37.

Abstract RAS proteins are small GTPases involved in cell proliferation, survival, and differentiation, and are mutationally activated in about a third of all human cancers. These mutations drive cancer by impairing GTPase activity so that the RAS protein is found predominantly in its GTP-bound “on” conformation. Most KRAS isoform mutations are located at codon 12, a glycine in the P-loop of the GTPase active site. KRAS mutations in which the glycine is mutated to a cysteine (G12C) are particularly common in lung cancer. Despite its prevalence as an important oncogene and decades of research, the RAS protein remains an unexploited cancer target. We have developed a novel platform, SMART (Small Molecule Assisted Receptor Targeting), to disrupt the protein-protein interactions of so-called “undruggable” targets. Our compounds bind the immunophilin protein Cyclophilin A (CypA), and subsequently form a ternary complex with the target protein, KRAS. An electrophilic moiety on the ligand selectively forms a covalent bond with the cysteine of GTP-KRAS G12C, thus strengthening the stability of the ternary complex, partially occupying the effector face, and thus occluding the binding of downstream effector proteins, such as RAF. In a separate presentation, we describe a wide breadth of data to support the advancement of a small molecule specifically targeting the activated form of KRAS, with a focus on the cellular pharmacology. In this presentation, we highlight biophysical and biochemical findings that support our novel strategy for targeting GTP-KRAS G12C. SPR reveals the CypA-compound binary KD. A novel SPR protocol method was developed to examine CypA-compound-KRAS ternary complex formation, exploiting the A-B-A injection system of the Biacore 8K. LC-MS is used to characterize the crosslinking of our compounds to KRAS G12C in the presence of CypA, and full kinact/KI analysis is conducted with select compounds; in combination with SPR studies, the noncovalent KD of the ternary complex can be determined. Intrinsic warhead reactivity is measured through GSH adduct formation. TR-FRET studies probe the ability of ternary complexes to inhibit the binding of RAF to KRAS G12C. ITC and BLI are used to confirm ternary complex formation for selected compounds. In addition to guiding SAR, these studies characterize the degree of presenter (CypA) dependence, specificity of compound binding to the G12C mutant and to the GTP nucleotide-bound form of KRAS, and the ability to predict cellular efficacy. Our kinetic crosslinking experiments reveal single-digit micromolar KIs and a good kinact/KI ratio for our compounds relative to published literature on compounds targeting GDP-KRAS G12C. When these values are compared to SPR findings, the noncovalent KD contributes largely to KI, and thus noncovalent interactions play a bigger role in our compound’s interaction with RAS as compared to known GDP-KRAS G12C agents. Citation Format: Minyun Zhou, Alexander Yuzhakov, Cindy C Benod, Linlong Xue, Alec D Silver, Ganesh Iyer, Sharon A. Townson, Meizhong Jin, Nicholas R. Perl, Anna Kohlmann, Alan S. Mann, Gizem Akcay, Earl W. May. Biophysical and biochemical characterization of KRAS G12C inhibition through the SMARTTM platform [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A06.

The activity of the secreted phosphodiesterase autotaxin produces the inflammatory signaling molecule LPA and has been associated with a number of human diseases including idiopathic pulmonary fibrosis (IPF). We screened a single DNA-encoded chemical library (DECL) of 225 million compounds and identified a series of potent inhibitors. Optimization of this series led to the discovery of compound 1 (X-165), a highly potent, selective, and bioavailable small molecule. Cocrystallization of compound 1 with human autotaxin demonstrated that it has a novel binding mode occupying both the hydrophobic pocket and a channel near the autotaxin active site. Compound 1 inhibited the production of LPA in human and mouse plasma at nanomolar levels and showed efficacy in a mouse model of human lung fibrosis. After successfully completing IND-enabling studies, compound 1 was approved by the FDA for a Phase I clinical trial. These results demonstrate that DECL hits can be readily optimized into clinical candidates.

In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.

Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.

Abstract In non-small cell lung cancer (NSCLC), multiple classes of activating mutations have been identified in EGFR and HER2 that vary widely in their sensitivity to available tyrosine kinase inhibitors (TKIs). Erlotinib, gefitinib, and afatinib are approved for use in patients with the most common forms of EGFR activating mutations (ie, exon 19 deletions or L858R substitutions). However, no TKIs are approved for patients with EGFR activated by any other mutation, including exon 20 insertions or other uncommon substitutions, or for patients with any class of HER2 activating mutation (including exon 20 insertions). As inhibition of wild-type (WT) EGFR is associated with dose-limiting toxicities, a TKI that inhibits oncogenic EGFR and HER2 variants more potently than WT EGFR is more likely to be able to be dosed to efficacious levels. AP32788 is a potent inhibitor of all oncogenic forms of EGFR and HER2, including exon 20 insertions, with selectivity over WT EGFR. Activity of AP32788 and other TKIs was assessed by measuring viability of Ba/F3 cell lines engineered to express 20 mutant variants of EGFR (n = 14) or HER2 (n = 6): 4 EGFR variants containing a common activating mutation with or without a T790M resistance mutation, 8 EGFR/HER2 variants containing an exon 20 activating insertion (eg, EGFR ASV, HER2 YVMA), and 8 EGFR/HER2 variants containing other uncommon activating mutations (eg, EGFR G719A, HER2 G776V). Inhibition of WT EGFR was assessed by measuring effects on EGFR phosphorylation in cells (A431) that over-express WT EGFR. Consistent with their clinical activity, erlotinib and gefitinib generally only inhibited the 2 EGFR variants with common activating mutations more potently than WT EGFR (IC50s 71 and 56 nM, respectively), and afatinib generally only inhibited EGFR with common activating mutations or uncommon substitutions more potently than WT EGFR (IC50 4 nM). In contrast, AP32788 inhibited all 14 mutant variants of EGFR (IC50s 2.4-22 nM), and all 6 mutant variants of HER2 (IC50s 2.4-26 nM), more potently than it inhibited WT EGFR (IC50 35 nM), including all 8 variants with exon 20 activating insertions. In mice implanted with a patient-derived tumor containing an EGFR exon 20 activating insertion, or with engineered Ba/F3 cells containing a HER2 exon 20 activating insertion, once daily oral dosing of AP32788 induced regression of tumors at doses that were well tolerated (30-100 mg/kg). In vivo efficacy was associated with inhibition of EGFR signaling in the tumor. AP32788 potently inhibited all activated forms of EGFR and HER2 tested, including exon 20 insertions, more potently than WT EGFR, suggesting it may have the selectivity necessary to achieve efficacious levels of exposure in patients. A phase 1/2 clinical trial of AP32788 in NSCLC patients is planned. Citation Format: Francois Gonzalvez, Xiaotian Zhu, Wei-Sheng Huang, Theresa E. Baker, Yaoyu Ning, Scott D. Wardwell, Sara Nadworny, Sen Zhang, Biplab Das, Yongjin Gong, Matthew T. Greenfield, Hyun G. Jang, Anna Kohlmann, Feng Li, Paul M. Taslimi, Meera Tugnait, Yongjin Xu, Emily Y. Ye, Willmen W. Youngsaye, Stephan G. Zech, Yun Zhang, Tianjun Zhou, Narayana I. Narasimhan, David C. Dalgarno, William C. Shakespeare, Victor M. Rivera. AP32788, a potent, selective inhibitor of EGFR and HER2 oncogenic mutants, including exon 20 insertions, in preclinical models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2644.

Phosphorus, despite its abundance in the human body, is rarely found in drug molecules, with clinical utility limited to a few phosphonic or bisphosponic acid-based medicines and several phosphonate or phosphate-containing prodrugs. Concerns about poor cell penetration, low oral bioavailability, or biological instability have limited application of these functional classes in drug design. In our efforts to discover pharmaceuticals with novel functionality, we introduced a neutral, stable phosphine oxide moiety as a unique hydrogen-bond acceptor in the design of anaplastic lymphoma kinase (ALK) inhibitors. ALK is a receptor tyrosine kinase first identified as a chromosomal rearrangement (NPM-ALK fusion gene) in anaplastic large cell lymphoma (ALCL) and subsequently detected as an alternate fusion oncogene (EML4-ALK) in a subset of non-small cell lung cancers (NSCLC). Crizotinib, the first approved ALK inhibitor, has demonstrated impressive clinical benefit in EML4-ALK (ALK+) NSCLC patients. Drug resistance, however, emerges rapidly and point mutations within the kinase domain have been identified as a major resistance mechanism. With the specific objective to identify more potent ALK inhibitors with pan-inhibitory activity against crizotinib-resistant ALK mutants, we designed and tested a series of phosphine oxide-based compounds culminating in the identification of the clinical candidate AP26113. AP26113 exhibited 10-fold greater potency than crizotinib against ALK-positive ALCL and NSCLC cell lines, and effectively inhibited clinically relevant crizotinib-resistant mutants. AP26113 displayed ∼100-fold increased selectivity for ALK-positive cells over ALK-negative cell lines while maintaining selectivity over insulin receptor tyrosine kinase receptors. Consistent with the in vitro profile, AP26113 demonstrated oral efficacy in multiple ALK+ mouse models including Karpas-299 (ALCL), H3122 (NSCLC), and Ba/F3 cells expressing crizotinib resistant mutants including G1269S and L1196M. Finally, AP26113 exhibited excellent drug-like properties including high cell permeability and solubility, moderate cross-species protein binding, and inactivity toward major cytochrome P450 iso-enzymes and hERG ion channels. Medicinal chemistry efforts leading to the discovery of this potent pan-ALK inhibitor, including design strategy, chemical series evolution, and DMPK optimization will be presented. AP26113 is currently in a global phase 2 registration trial (NCT02094573) in patients with locally advanced or metastatic NSCLC who test positive for the ALK oncogene and were previously treated with crizotinib. Citation Format: Wei-Sheng Huang, Feng Li, Lisi Cai, Yongjin Xu, Sen Zhang, Scott D. Wardwell, Yaoyu Ning, Anna Kohlmann, Tianjun Zhou, Emily Y. Ye, Xiaotian Zhu, Narayana I. Narasimhan, Tim Clackson, Victor M. Rivera, David Dalgarno, William C. Shakespeare. Discovery of AP26113, a potent, orally active inhibitor of anaplastic lymphoma kinase and clinically relevant mutants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2827. doi:10.1158/1538-7445.AM2015-2827

Abstract Purpose: KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST. Experimental Design: The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib–KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents. Results: In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST. Conclusion:Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST. Clin Cancer Res; 20(22); 5745–55. ©2014 AACR.