Huang is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. Y. Hu is an employee of Takeda. F. Li is a former employee of ARIAD. M.T. Greenfield is a former employee of ARIAD. S.G. Zech is an employee and shareholder of Amgen Inc. and a former employee of ARIAD. B. Das is a former employee of ARIAD. N.I. Narasimhan is a former employee of ARIAD. T. Clackson is an employee and shareholder of Xilio Therapeutics and is a former employee of ARIAD. D. Dalgarno is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. W.C. Shakespeare is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. M. Fitzgerald is a former employee Research.
Although targeted therapies have revolutionized cancer treatment, overcoming acquired resistance remains a major clinical challenge. EZH2 inhibitors (EZH2i), EPZ-6438 and GSK126, are currently in the early stages of clinical evaluation and the first encouraging signs of efficacy have recently emerged in the clinic. To anticipate mechanisms of resistance to EZH2i, we used a forward genetic platform combining a mutagenesis screen with next generation sequencing technology and identified a hotspot of secondary mutations in the EZH2 D1 domain (Y111 and I109). Y111D mutation within the WT or A677G EZH2 allele conferred robust resistance to both EPZ-6438 and GSK126, but it only drove a partial resistance within the Y641F allele. EZH2 mutants required histone methyltransferase (HMT) catalytic activity and the polycomb repressive complex 2 (PRC2) components, SUZ12 and EED, to drive drug resistance. Furthermore, D1 domain mutations not only blocked the ability of EZH2i to bind to WT and A677G mutant, but also abrogated drug binding to the Y641F mutant. These data provide the first cellular validation of the mechanistic model underpinning the oncogenic function of WT and mutant EZH2. Importantly, our findings suggest that acquired-resistance to EZH2i may arise in WT and mutant EZH2 patients through a single mutation that remains targetable by second generation EZH2i.
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.
Disclosure of Potential Conflicts of InterestM. Fitzgerald is a former employee of Takeda. J. Chouitar is an employee of Takeda.T.E. Baker is an employee and shareholder of MOMA Therapeutics and a former employee of ARIAD. F. Gonzalvez is an employee and shareholder of Aligos Therapeutics and a former employee of ARIAD. V.M. Rivera is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. R. Brake is an employee and shareholder of Takeda. S. Vincent is an employee and shareholder of Takeda. K.K.W. is a founder of and equity holder in G1 Therapeutics. He also has sponsored Research Agreements with Takeda, BMS, Mirati, Merus, Alkermes, Ansun
Background: Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) indicated for patients with T315I positive or treatment-refractory CML and Ph+ ALL. To develop hypotheses regarding molecular and cellular targets of ponatinib that could contribute to arterial thrombotic events observed in some patients we are undertaking a broad comparative profiling analysis of ponatinib and other TKIs. Methods: Cellular activities of ponatinib and additional BCR-ABL (imatinib, nilotinib, dasatinib, bosutinib) and VEGFR2/multi-targeted (sunitinib, regorafenib) TKIs were examined in panels of Ba/F3 cell lines expressing activated kinase variants (N=61), tumor cell lines (N=246), and primary lines derived from human vasculature (aortic smooth muscle cells [ASMC] and umbilical vein [HUVEC], aortic [HAEC] and pulmonary artery [HPAEC] endothelial cells). Human steady-state Cave concentrations of 45 mg ponatinib (101 nM) were corrected for the functional effects of protein binding (3.6-fold) to derive the clinically-effective concentration. Results: Ponatinib inhibits the in vitro activity of multiple kinases with IC50s within 10-fold of ABL, including members of the VEGFR, PDGFR, FGFR, EPH receptor and SRC families of kinases, KIT, RET, TIE2, and FLT3. This profile was largely recapitulated in cellular assays using engineered Ba/F3 cells, with ponatinib demonstrating substantially greater potency against VEGFRs, FGFRs, TIE2, RET and FLT3 than other ABL TKIs. Across a broad panel of tumor cell lines, ponatinib inhibited viability with a median IC50 of 598 nM. Ponatinib only inhibited 16 cell lines (6.5%) with IC50s below its clinically effective concentration (28 nM) with the 5 most sensitive lines (IC50 <1 nM) all being BCR-ABL positive. Within the vasculature-derived cell panel, ponatinib inhibited viability of HUVECs grown in full serum with an IC50 of 261 nM, with all of the other ABL and non-ABL TKIs tested having IC50s >2000 nM. Effects of ponatinib on HAECs, HPAECs and ASMCs were more modest (IC50s 1533, 490 and 750 nM, respectively). Finally, ponatinib (IC50 20 nM) and other VEGFR2 inhibitors potently inhibited survival of HUVECs grown in VEGF-dependent conditions, while other BCR-ABL inhibitors, except dasatinib (IC50 14 nM), did not. Conclusions: Ponatinib is a potent BCR-ABL inhibitor that also inhibits VEGFR2 and other kinases at clinically achievable concentrations in vitro. Modest effects of ponatinib on endothelial cells have been observed that warrant further exploration in vivo. Developing a precise understanding of the mechanism by which ponatinib contributes to arterial thrombotic events should facilitate development of strategies to optimize its benefit/risk in patients. Disclosures Rivera: ARIAD Pharmaceuticals Inc: Employment, Equity Ownership. Pritchard:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Gonzalvez:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Baker:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Gozgit:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership. Hodgson:ARIAD Pharmaceuticals, Inc.: Employment, Equity Ownership.
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