Henry J. Haringsma

Hsp90 Inhibition Suppresses Mutant EGFR-T790M Signaling and Overcomes Kinase Inhibitor Resistance

The epidermal growth factor receptor (EGFR) secondary kinase domain T790M non-small cell lung cancer (NSCLC) mutation enhances receptor catalytic activity and confers resistance to the reversible tyrosine kinase inhibitors gefitinib and erlotinib. Currently, irreversible inhibitors represent the primary approach in clinical use to circumvent resistance. We show that higher concentrations of the irreversible EGFR inhibitor CL-387,785 are required to inhibit EGFR phosphorylation in T790M-expressing cells compared with EGFR mutant NSCLC cells without T790M. Additionally, CL-387,785 does not fully suppress phosphorylation of other activated receptor tyrosine kinases (RTK) in T790M-expressing cells. These deficiencies result in residual Akt and mammalian target of rapamycin (mTOR) activities. Full suppression of EGFR-mediated signaling in T790M-expressing cells requires the combination of CL-387,785 and rapamycin. In contrast, Hsp90 inhibition overcomes these limitations in vitro and depletes cells of EGFR, other RTKs, and phospho-Akt and inhibits mTOR signaling whether or not T790M is present. EGFR-T790M-expressing cells rendered resistant to CL-387,785 by a kinase switch mechanism retain sensitivity to Hsp90 inhibition. Finally, Hsp90 inhibition causes regression in murine lung adenocarcinomas driven by mutant EGFR (L858R) with or without T790M. However, efficacy in the L858R-T790M model requires a more intense treatment schedule and responses were transient. Nonetheless, these findings suggest that Hsp90 inhibitors may be effective in T790M-expressing cells and offer an alternative therapeutic strategy for this subset of lung cancers.

The major lung cancer-derived mutants of ERBB2 are oncogenic and are associated with sensitivity to the irreversible EGFR/ ERBB2 inhibitor HKI-272.

Mutations in the ERBB2 gene were recently found in approximately 2% of primary non-small cell lung cancer (NSCLC) specimens; however, little is known about the functional consequences and the relevance to responsiveness to targeted drugs for most of these mutations. Here, we show that the major lung cancer-derived ERBB2 mutants, including the most frequent mutation, A775insYVMA, lead to oncogenic transformation in a cellular assay. Murine cells transformed with these mutants were relatively resistant to the reversible epidermal growth factor receptor (EGFR) inhibitor erlotinib, resembling the resistant phenotype found in cells carrying the homologous mutations in exon 20 of EGFR. However, the same cells were highly sensitive to the irreversible dual-specificity EGFR/ERBB2 kinase inhibitor HKI-272, as were those overexpressing wild-type ERBB2. Finally, the NSCLC cell line, Calu-3, overexpressing wild-type ERBB2 owing to a high-level amplification of the ERBB2 gene were highly sensitive to HKI-272. These results provide a rationale for treatment of patients with ERBB2-mutant or ERBB2-amplified lung tumors with HKI-272.

In vitro and In vivo Characterization of Irreversible Mutant-Selective EGFR Inhibitors that are Wild-type Sparing

Patients with non–small cell lung carcinoma (NSCLC) with activating mutations in epidermal growth factor receptor (EGFR) initially respond well to the EGFR inhibitors erlotinib and gefitinib. However, all patients relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR (EGFRWT). These inhibitors have not provided compelling clinical benefit in T790M-positive patients, apparently because of dose-limiting toxicities associated with inhibition of EGFRWT. Thus, there is an urgent clinical need for therapeutics that overcome T790M drug resistance while sparing EGFRWT. Here, we describe a lead optimization program that led to the discovery of four potent irreversible 2,4-diaminopyrimidine compounds that are EGFR mutant (EGFRmut) selective and have been designed to have low affinity for EGFRWT. Pharmacokinetic and pharmacodynamic studies in H1975 tumor–bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. Importantly, evaluation of normal lung tissue from the same animals showed no inhibition of EGFRWT. Of all the compounds tested, compound 3 displayed the best efficacy in EGFRL858R/T790M-driven tumors. Compound 3, now renamed CO-1686, is currently in a phase I/II clinical trial in patients with EGFRmut-advanced NSCLC that have received prior EGFR-directed therapy. Mol Cancer Ther; 13(6); 1468–79. ©2014 AACR.

Abstract 793: Insulin-like growth factor 1 (IGF1R)/insulin receptor (INSR) inhibitory activity of rociletinib (CO-1686) and its metabolites in nonclinical models

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rociletinib (CO-1686) is a novel, oral, targeted irreversible inhibitor of the cancer-causing mutant forms of EGFR currently being studied for the treatment of NSCLC. Rociletinib was designed to spare wild-type EGFR signaling. Heavily-pretreated T790M+ patients treated with rociletinib at 500 or 625mg BID demonstrated a 67% objective response rate (n = 56, Soria et al., ENA 2014). Adverse events (AEs) typical of wild-type EGFR inhibition, such as cutaneous toxicities, have not been observed. The most frequent AE in patients dosed with rociletinib is hyperglycemia (32% all grades/14% grade 3), which is typically managed with oral hypoglycemic therapy. Rociletinib does not directly play a role in hyperglycemia based on kinase and cellular profiling, toxicology studies performed in Sprague-Dawley rats and beagle dogs, and an oral glucose tolerance test (OGTT) in Sprague-Dawley rats. We examined the hypothesis that hyperglycemia may result from a metabolite of rociletinib. Metabolite profiling from in vitro hepatocyte incubations, as well as the analysis of plasma samples from healthy subjects and NSCLC patients, revealed three metabolites of interest referenced according to their protonated molecular ion: M460, M502, and M544. Metabolites M460 and M502 are present in higher levels in humans than in rats and dogs, whereas M544 levels are comparable between human and rat. In patients the levels of M502 are at least 5-fold greater than that of M460. All 3 metabolites demonstrated limited potency against EGFR, T790M EGFR, and HER2. Expanded cellular profiling revealed that M460 and M502 have 2-3 fold and 3-7 fold greater potency against INSR and IGF1R, respectively, as compared to rociletinib, whereas metabolite M544 demonstrated similar or reduced potency. Metabolite profiling in rats suggests that M502 is rapidly converted to M544 by acetylation, thus a rat OGTT was performed with a single dose of M502 at 1000 mg/kg to reach exposure levels comparable to those observed in rociletinib treated NSCLC patient samples. Metabolite M502 caused significant elevations in post-prandial glucose and insulin excursion. Taken together, the increased potency of M502 towards IGF1R and INSR, its absolute exposure in humans and the rat OGTT results suggest that M502 is likely to play a causative role in the hyperglycemia observed in patients. Additional data on the clinical implications of these findings, as well as the role of IGF1R pathway activation in resistance to EGFR inhibitors, will be presented. Citation Format: Andrew D. Simmons, Sarah Jaw-Tsai, Henry J. Haringsma, Andrew Allen, Thomas C. Harding. Insulin-like growth factor 1 (IGF1R)/insulin receptor (INSR) inhibitory activity of rociletinib (CO-1686) and its metabolites in nonclinical models. [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 793. doi:10.1158/1538-7445.AM2015-793

Abstract C199: PI3K/AKT/mTOR inhibitors enhance the anti-tumor activity of the FGFR, VEGFR and PDGFR inhibitor lucitanib in FGF-dependent breast cancer models

Lucitanib (S 80881, E-3810, CO-3810) is a potent inhibitor of the fibroblast growth factor receptors 1-3 (FGFR1-3), vascular endothelial growth factor receptors 1-3 (VEGFR1-3) and platelet-derived growth factor receptors alpha and beta (PDGFRα/β). A Phase 1/2 clinical study (Soria et al., 2014) of lucitanib showed a RECIST response rate of 50% in breast cancer patients with FGF-aberrant (FGFR1 and/or FGF3/4/19 gene amplified) tumors. Here we explored potential drug partners that can be used in combination with lucitanib to increase cell cytotoxicity. Five FGF-dependent breast carcinoma cell lines were evaluated - MFM-223 (human TNBC, FGFR1 / FGFR2 amplified, PIK3CA H1047R); HCC38 (human TNBC, FGFR1 high expression, PIK3CA W386L); CAL-120 (human TNBC, FGFR1 / FRS2 amplified, PIK3CA wild-type); EFM-19 (human ER+ BC, FGFR2 K659E, PIK3CA H1047L); and 4T1 (mouse TNBC, FGFR2 high expression, PIK3CA wild-type). Single agent and lucitanib drug-drug combinations were evaluated in 2D monolayer and/or 3D spheroid culture using a broad (MFM-223; n = 350 compounds) or a more focused compound library (cell lines CAL-120, EFM-19 and MFM-223; n = 70 compounds approved or in clinical trials). Drug combinations were evaluated at a concentration range of 0.06-5000 nM and co-cultured with 0.2 μM lucitanib in a 72-hour cell viability assay. Combination efficacy was determined by comparing cell viability with and without lucitanib either by direct comparison of raw RLU or by fold change in calculated GI50. Several compounds were identified that enhanced cell killing when combined with lucitanib, including HDAC inhibitors (vorinostat, entinostat and belinostat), PI3K/Akt/mTOR pathway inhibitors (GDC-0941, GDC-0980 and BLY719), EGFR/HER2 inhibitors (afatinib and lapatinib), and CDK inhibitors (AT7519 and flavopiridol). These combinations were examined for efficacy across all five cell lines, and combination indexes (CI) were calculated to determine synergy. The PI3K/Akt/mTOR and lucitanib combination demonstrated consistent synergy across all cell lines independent of PIK3CA mutation status. For example, the PI3K inhibitor GDC-0941 showed a CI range of 0.04-0.58 at the GI50 (CI Citation Format: Minh Nguyen, Liliane Robillard, Andrew D. Simmons, Henry J. Haringsma, Thomas C. Harding. PI3K/AKT/mTOR inhibitors enhance the anti-tumor activity of the FGFR, VEGFR and PDGFR inhibitor lucitanib in FGF-dependent breast cancer models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C199.

Abstract 3595: In vivo acquired resistance to the mutant EGFR inhibitor Rociletinib (CO-1686) is associated with activation of the c-MET pathway

Rociletinib is a novel, oral, targeted irreversible inhibitor of the cancer-causing mutant forms of EGFR currently being studied for the treatment of NSCLC. Rociletinib was designed to spare wild-type EGFR signaling. Heavily-pretreated T790M+ patients treated with rociletinib at 500 or 625mg BID demonstrated a 67% objective response rate (n = 56, Soria et al., ENA 2014). Despite these promising data, acquired resistance to rociletinib monotherapy is anticipated. To assess the in vivo mechanisms of acquired resistance to rociletinib, mice bearing PC-9 (EGFR del19) human NSCLC tumors were chronically dosed with erlotinib (50 mg/kg QD) or rociletinib (150 mg/kg BID). Rociletinib treated mice had an increased time to tumor progression as compared to erlotinib treated mice. Resistance, as defined by tumors reaching >300mm3, was observed in all (n = 10) erlotinib treated mice between days 42-61. Alternatively, resistance to rociletinib was only observed in 3/10 mice after 126 days of dosing. Emergence of the EGFR T790M resistance mutation was detected in all tumors resistant to erlotinib. Erlotinib resistant tumors (n = 7) were crossed over to rociletinib monotherapy on day 60 of the study at a mean tumor volume of 500mm3. Rociletinib treatment generated durable tumor regressions in both crossover and monotherapy treated mice, however continued dosing of rociletinib ultimately allowed for the collection of four resistant tumors. Rociletinib resistant tumors were analyzed using the SuraSeq 500 NGS panel, and all harbored copy number gains (n = 12-15) in MET and corresponding MET pathway activation as shown by RTK arrays and Western blotting. Combining rociletinib with the MET inhibitor crizotinib caused regression of the MET amplified rociletinib-resistant tumors. In separate experiments, the addition of 50 ng/ml exogenous HGF was sufficient to render several mutant EGFR cell lines immediately resistant to rociletinib, and this effect could be overcome with the combination of rociletinib and crizotinib. Finally, the combination of rociletinib and crizotinib also demonstrated potent activity in a patient derived L858R EGFR xenograft model with MET amplification (14 copies). Taken together, these data show that (1) in a PC-9 “front-line” model of mutant EGFR lung cancer, rociletinib has a longer time-to-resistance than erlotinib; and (2) MET/HGF pathway activation is a likely driver of acquired resistance to rociletinib in patients with mutant EGFR lung cancer, and can potentially be successfully prevented and/or treated with a combination of rociletinib plus a MET tyrosine kinase inhibitor. Citation Format: Henry J. Haringsma, Andrew Allen, Thomas C. Harding, Andrew D. Simmons. In vivo acquired resistance to the mutant EGFR inhibitor Rociletinib (CO-1686) is associated with activation of the c-MET pathway. [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 3595. doi:10.1158/1538-7445.AM2015-3595

Abstract B120: Preclinical assessment of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC.

Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations initially respond well to first generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, attributable in 60% of cases to a secondary mutation of EGFR at T790M. In addition, patients also experience skin and GI toxicity due to wild type (WT) EGFR inhibition. CO-1686 is a novel, irreversible and orally delivered kinase inhibitor that specifically targets the most frequently observed front-line and acquired resistance mutant forms of EGFR, while exhibiting minimal activity towards WT EGFR. Selectivity of CO-1686 was demonstrated using two separate biochemical assays in which CO-1686 was at least 10-fold more selective for mutant EGFR as compared to the WT receptor. In human cancer cell lines, CO-1686 potently inhibited in vitro proliferation of cells expressing mutant EGFR, while minimally affecting proliferation of WT EGFR-dependent cells. To confirm this selectivity in vivo, CO-1686 was evaluated in WT (A431), front line (HCC827, transgenic), and second line (NCI-H1975, LUM1868, transgenic) xenograft models. CO-1686 dosed at 50 mg/kg BID exhibited a minimal reduction in tumor growth in the A431 xenograft model with no significant alterations in body weight and EGFR WT pathway signaling. Comparatively, both erlotinib (75 mg/kg QD) and afatinib (20 mg/kg QD) administration resulted in profound tumor regression, but with concurrent body weight loss and EGFR WT pathway inhibition. CO-1686, when dosed as a single agent at 50 mg/kg BID and/or 100 mg/kg QD, caused tumor regressions in the HCC827 (del19), NCI-H1975 (L858R/T790M), and patient-derived lung tumor xenograft model LUM1868 (L858R/T790M). The efficacy of CO-1686 was also examined in EGFR-L858R and EGFR-L858R-T790M genetically engineered mouse (GEM) models that develop lung adenocarcinoma upon human mutant EGFR transgene induction in the lung epithelium. In both models, CO-1686 administration (50 mg/kg BID) resulted in potent anti-tumor activity as measured by MRI and histological assessment. Following a 3-week dosing period, animals treated with CO-1686 demonstrated a complete regression of established lesions. Comparatively, in the EGFR-L858R-T790M GEM model, afatinib dosed at the maximum tolerated dose (20 mg/kg QD) resulted in limited efficacy. In summary, CO-1686 exhibits potent and significant anti-tumor activity as a single agent in xenograft models driven by the most frequently observed front-line and acquired resistance mutant forms of EGFR, while exhibiting minimal activity towards WT EGFR. These data suggest that CO-1686 may have utility in both front-line and acquired resistance EGFR-mutated NSCLC, and CO-1686 is currently being evaluated in a phase I/II clinical trial in previously treated mutant EGFR NSCLC patients (ClinicalTrials.gov identifier: NCT01526928). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B120. Citation Format: Andrew D. Simmons, Henry J. Haringsma, Annette O. Walter, Robert Tjin Tham Sjin, Zoe Weaver, Andrew Allen, Thomas C. Harding. Preclinical assessment of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC. [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 B120.

Abstract A114: In vitro acquired resistance to the mutant selective EGFR inhibitor CO-1686 is associated with epithelial-mesenchymal transition (EMT).

Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations initially respond well to first generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance driven by the primary drug-resistance T790M mutation in EGFR. CO-1686 is a novel, irreversible and orally delivered kinase inhibitor that specifically targets the most common primary and acquired mutant forms of EGFR while exhibiting minimal activity towards the wild-type (WT) receptor, and has shown evidence of clinical efficacy in on-going phase I/II clinical trials in NSCLC. To assess the mechanisms of acquired resistance to CO-1686, we continuously exposed NCI-H1975 (L858R/T790M), HCC827 (del19) and PC-9 (del19) NSCLC cell lines to several months of increasing doses of CO-1686 until resistance (IC50 >100x over parental) developed. Drug resistance in the CO-1686 resistant (COR) clones extended to additional EGFR TKIs including erlotinib and afatinib. To determine if CO-1686 resistance in the COR cell clones was dependent on EGFR signaling, we examined the functional effects of EGFR siRNA knockdown in NCI-H1975 parental cells and CO-1686 resistant clones. Compared to the parental NCI-H1975 cell line, the resistant clones demonstrated a reduced dependence on EGFR expression for viability. Analysis of genes differentially expressed in the COR clones compared to the parental cell line demonstrated a significant enrichment of genes associated with EMT. Further genetic mutation or copy number alteration in the EGFR gene were not observed in COR clones. Consistent with a mesenchymal cell signature in the COR clones, vimentin expression was up-regulated and E-cadherin down-regulated in the CO-1686 resistant clones at both the protein and RNA level. qRT-PCR analysis of additional markers further supported EMT including the up-regulation of AXL, ZEB1, CDH5, FN1 and the down-regulation of the epithelial markers MIR200B, CLDN4, EPCAM and CLDN7. Higher basal levels of pAKT were also observed in the COR cell clones as compared to the parental NCI-H1975 cell line. Although not effective when used as a single agent, the allosteric AKT inhibitor MK-2206 restored partial drug sensitivity to one of the COR clones when used together in an equimolar fashion with CO-1686. Taken together, these preclinical data suggest that CO-1686 resistance, unlike first and second generation EGFR TKIs, is not mediated by further mutation of EGFR. Resistance to CO-1686 is associated with EMT and increased levels of pAKT and pAXL, suggesting potential actionable targeted combination therapies to be explored in the clinic. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A114. Citation Format: Henry Haringsma, Annette O. Walter, Robert Tjin Tham Sjin, Andrew Allen, Thomas C. Harding, Andrew D. Simmons. In vitro acquired resistance to the mutant selective EGFR inhibitor CO-1686 is associated with epithelial-mesenchymal transition (EMT). [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 A114.