Andrew Paul Garner

An antibody that locks HER3 in the inactive conformation inhibits tumor growth driven by HER2 or neuregulin.

HER2/HER3 dimerization resulting from overexpression of HER2 or neuregulin (NRG1) in cancer leads to HER3-mediated oncogenic activation of phosphoinositide 3-kinase (PI3K) signaling. Although ligand-blocking HER3 antibodies inhibit NRG1-driven tumor growth, they are ineffective against HER2-driven tumor growth because HER2 activates HER3 in a ligand-independent manner. In this study, we describe a novel HER3 monoclonal antibody (LJM716) that can neutralize multiple modes of HER3 activation, making it a superior candidate for clinical translation as a therapeutic candidate. LJM716 was a potent inhibitor of HER3/AKT phosphorylation and proliferation in HER2-amplified and NRG1-expressing cancer cells, and it displayed single-agent efficacy in tumor xenograft models. Combining LJM716 with agents that target HER2 or EGFR produced synergistic antitumor activity in vitro and in vivo. In particular, combining LJM716 with trastuzumab produced a more potent inhibition of signaling and cell proliferation than trastuzumab/pertuzumab combinations with similar activity in vivo. To elucidate its mechanism of action, we solved the structure of LJM716 bound to HER3, finding that LJM716 bound to an epitope, within domains 2 and 4, that traps HER3 in an inactive conformation. Taken together, our findings establish that LJM716 possesses a novel mechanism of action that, in combination with HER2- or EGFR-targeted agents, may leverage their clinical efficacy in ErbB-driven cancers.

Ponatinib inhibits polyclonal drug-resistant KIT oncoproteins and shows therapeutic potential in heavily pretreated gastrointestinal stromal tumor (GIST) patients.

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.

Acquisition of a single EZH2 D1 domain mutation confers acquired resistance to EZH2-targeted inhibitors

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.

Abstract 5655: AP26113 possesses pan-inhibitory activity versus crizotinib-resistant ALK mutants and oncogenic ROS1 fusions.

AP26113 is a potent, reversible inhibitor of ALK fusions and mutant (but not native) EGFR. To overcome mutation-based resistance, AP26113 was designed to maintain activity against crizotinib-resistant ALK variants such as the gatekeeper ALK mutant L1196M. In an ongoing phase 1 dose-escalation study, AP26113 has achieved steady-state trough concentrations in excess of 1 μM and demonstrated promising clinical activity in both crizotinib-resistant and naive ALK-positive NSCLC patients and preliminary evidence of activity in patients with mutant EGFR (#439O, ESMO 2012). To further assess the activity of AP26113 against crizotinib-resistant ALK mutants, and the structurally related ROS1 fusions recently identified in NSCLC, we engineered Ba/F3 cells to express the appropriate oncogenic drivers and evaluated their sensitivities to crizotinib and AP26113. Ba/F3 cell lines expressing clinically-identified EML4-ALK mutants (T1151T insertion, S1206Y, D1203N, L1196M, G1202R, F1174C and C1156Y) had substantially reduced sensitivity to crizotinib (viability IC50= 363-1296 nM) compared with native EML4-ALK (IC50= 137 nM). These data are consistent with the identification of these mutants in patients with acquired crizotinib resistance. AP26113 potently inhibited both native (IC50= 21 nM) and crizotinib-resistant ALK mutants (IC50= 26-254 nM) at concentrations substantially below the clinically achievable trough levels of AP26113 (1 μM), suggesting it may possess a pan-ALK inhibitory profile. AP26113 also effectively inhibited the viability of Ba/F3 cells expressing CD74-ROS1 (IC50= 18 nM), FIG-ROS1 (IC50= 31 nM), SDC4-ROS1 (IC50= 16 nM) and EZR-ROS1 (IC50= 41 nM) thus demonstrating that it is an equipotent inhibitor of ALK and ROS1. In a Ba/F3 CD74-ROS1 xenograft model, AP26113 inhibited tumor growth in a dose-dependent manner, with 50 mg/kg AP26113 inducing substantial tumor regression. Since ROS1 and ALK are structurally related and similarly sensitive to crizotinib, we reasoned that acquired drug resistance in ROS1-driven tumors may also occur via mutation. We therefore generated Ba/F3 cells driven by L2026M gatekeeper mutant forms of CD74-ROS1 and FIG-ROS1 and tested their drug sensitivity. Interestingly, the inhibitory capacity of AP26113 was unaffected by L2026M. In contrast, crizotinib potencies were reduced approximately 4-fold. In conclusion, we have demonstrated that AP26113 inhibits clinically relevant crizotinib- resistant ALK mutants and oncogenic ROS1 fusions recently identified in NSCLC irrespective of the fusion partner. Importantly, since AP26113 maintains potent activity against mutant forms of ALK and ROS1 at concentrations substantially below its human trough concentrations these data suggest that AP26113 may be able to prevent the emergence of drug-resistant mutants in NSCLC patients. Citation Format: Rachel M. Squillace, Rana Anjum, David Miller, Sadanand Vodala, Lauren Moran, Frank Wang, Tim Clackson, Andrew P. Garner, Victor M. Rivera. AP26113 possesses pan-inhibitory activity versus crizotinib-resistant ALK mutants and oncogenic ROS1 fusions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5655. doi:10.1158/1538-7445.AM2013-5655

Abstract A98: An in vitro mutagenesis screen identifies L1951R and G2032R as drug-resistant mutants of CD74-ROS1.

NSCLC can be divided into a series of genomically-defined subsets, each generally containing a distinct molecular driver. The development of drugs that specifically target drivers such as mutant EGFR (erlotinib) and ALK fusions (crizotinib) has proven to be an effective therapeutic strategy. Despite these treatment advances, drug resistance inevitably occurs via a variety of mechanisms. Drug resistant point mutations of the driver oncogene are a common occurrence, with gatekeeper mutants often being problematic. ROS1 is rearranged in approximately 1.5% of NSCLC patients and is sensitive to crizotinib. Since ALK and ROS1 are structurally related, we generated Ba/F3 cells lines expressing CD74-ROS1 and tested their sensitivity to key ALK- targeted inhibitors currently in clinical testing. Crizotinib, AP26113, LDK378, and ASP3026 inhibited the viability of CD74-ROS1 expressing cells with IC50s significantly below their clinically-achievable plasma concentrations while CH5424802 was inactive thus highlighting that co-optimization of ROS1 activity is not a universal property of all ALK inhibitors. To investigate potential drug resistant mutant liabilities associated with each inhibitor, we performed a large scale mutagenesis screen, coupled with next generation sequencing, in Ba/F3 CD74-ROS1 cells exposed to increasing concentrations of each inhibitor. From our screens we identified a number of candidate mutants spanning the kinase domain of ROS1. L1951R and G2032R were the most frequent mutants in each screen, but mutation of the ROS1 gatekeeper residue (L2026) was not observed. To confirm the degree of resistance imparted by each mutant, we engineered CD74-ROS1 Ba/F3 cells to express the key ROS1 mutants identified and determined their sensitivities to crizotinib, AP26113, LDK378 and ASP3026. In addition to their frequent occurrence in our screens, L1951R and G2032R also conferred the highest degree of resistance (>50-fold) to each inhibitor. A ROS1 structural homology model indicates that L1951 and G2032 form part of the ATP-binding pocket of ROS1, and mutation of either residue to the larger amino acid arginine negatively impacts inhibitor activity without affecting the ability of CD74-ROS1 to transform cells. Interestingly, the analogous G2032R mutant in ALK (G1202R) has been observed as a clinical resistance mechanism induced by crizotinib, but remains sensitive to AP26113 (IC50 379 nM) in preclinical studies. In conclusion, we have demonstrated that crizotinib, AP26113, LDK378, and ASP3026 inhibit ROS1 activity. However, L1951R and G2032R ROS1 mutants are particularly resistant to each inhibitor. These preclinical data are supported by the recent identification of a G2032R mutant in a ROS1 patient who acquired resistance to crizotinib treatment, suggesting that generation of a ROS1-targeted compound that potently inhibits L1951R and G2032R mutants will ultimately be needed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A98. Citation Format: Rana Anjum, Sadanand Vodala, Anna Kohlmann, Victor M. Rivera, Andrew P. Garner. An in vitro mutagenesis screen identifies L1951R and G2032R as drug-resistant mutants of CD74-ROS1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A98.

Abstract 3394: Ponatinib, a pan-BCR-ABL inhibitor, potently inhibits key activating and drug-resistant KIT mutants found in GIST.

Ponatinib (AP24534) is a multi-targeted tyrosine kinase inhibitor with potent pan-BCR-ABL activity being investigated in patients with CML. Ponatinib also inhibits the kinase activity of KIT, a clinically proven oncogenic driver. Approximately 80% of gastrointestinal stromal tumors (GIST) contain primary activating KIT mutations, the majority of which cluster in exon 11 (V560D, Δ557-8) or more rarely in exon 9 (AY502-3ins). Imatinib is approved for the 1 st line treatment of GIST; however, patients frequently relapse due to the acquisition of secondary resistance mutations located in either the KIT ATP-binding pocket (T670I, V654A) or the activation (A) loop (D816H, D820A, N822K, A829P). Sunitinib is registered for 2 nd line treatment of GIST but does not effectively inhibit A-loop mutants. Here we explored the activity of ponatinib against major primary and secondary KIT mutants found in GIST. The drug sensitivity of KIT mutants was determined using engineered Ba/F3 cells harboring mutant forms of KIT. Imatinib potently inhibited the viability of cells expressing exon 11 mutant KIT (IC 50 50 180 nM - 10 μM). Sunitinib was highly active (IC 50 50 > 200 nM). In contrast, ponatinib was highly potent (IC 50 50 = 56 nM) or when V654A was present as a secondary mutation (IC 50 = 59 nM). Importantly, in patients dosed once daily with 45 mg ponatinib, peak (145 nM) and trough (64 nM) plasma concentrations achieved are predicted to lead to substantial inhibition of all KIT mutants tested here. To rationalize ponatinib9s activity we solved the structure of the native KIT kinase domain co-crystallized with ponatinib. Ponatinib bound the inactive (DFG-out) conformation of KIT, filling the ATP-binding pocket as well as two adjacent hydrophobic pockets, analogous to its binding to ABL. Ponatinib can accommodate the gatekeeper T670I mutation by virtue of its triple bond circumventing the steric bulk of the larger isoleucine residue. Ponatinib9s activity against A-loop mutants can be rationalized by its tight binding to the inactive conformation of KIT, even in the presence of A-loop mutations. In conclusion, ponatinib possesses potent activity versus the major clinically relevant KIT mutants, inhibiting their activity with IC 50 s that fall within clinically achievable plasma concentrations. These data therefore support the evaluation of ponatinib in patients with GIST, particularly since KIT A-loop mutants represent an unmet medical need. Citation Format: Andrew P. Garner, Rana Anjum, Sadanand Vodala, Alexa Schrock, Tianjun Zhou, Tim Clackson, Joseph M. Gozgit, Victor M. Rivera. Ponatinib, a pan-BCR-ABL inhibitor, potently inhibits key activating and drug-resistant KIT mutants found in GIST. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3394. doi:10.1158/1538-7445.AM2013-3394