Matthew John Meyer

Functional epigenetics approach identifies BRM/SMARCA2 as a critical synthetic lethal target in BRG1-deficient cancers

Significance Mammalian SWI/SNF (mSWI/SNF) alterations are highly prevalent, now estimated to occur in 20% of cancers. The inactivating nature of mSWI/SNF mutations presents a challenge for devising strategies to target these epigenetic lesions. By performing a comprehensive pooled shRNA screen of the epigenome using a unique deep coverage design shRNA (DECODER) library across a large cancer cell line panel, we identified that BRG1/SMARCA4 mutant cancer cells are highly sensitive to BRM/SMARCA2 depletion. Our study provides important mechanistic insight into the BRM/BRG1 synthetic lethal relationship, shows this finding translates in vivo, and highlights BRM as a promising therapeutic target for the treatment BRG1-mutant cancers. Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficient cancer cells leads to a cell cycle arrest, induction of senescence, and increased levels of global H3K9me3. We further demonstrate the selective dependency of BRG1-mutant tumors on BRM in vivo. Genetic alterations of the mSWI/SNF chromatin remodeling complexes are the most frequent among chromatin regulators in cancers, with BRG1/SMARCA4 mutations occurring in ∼10–15% of lung adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM function as mutually exclusive catalytic subunits of the mSWI/SNF complex, we propose that such synthetic lethality may be explained by paralog insufficiency, in which loss of one family member unveils critical dependence on paralogous subunits. This concept of “cancer-selective paralog dependency” may provide a more general strategy for targeting other tumor suppressor lesions/complexes with paralogous subunits.

Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

Inhibition of casein kinase 1 alpha prevents acquired drug resistance to erlotinib in EGFR-mutant non-small cell lung cancer

Patients with lung tumors harboring activating mutations in the EGF receptor (EGFR) show good initial treatment responses to the EGFR tyrosine kinase inhibitors (TKI) erlotinib or gefitinib. However, acquired resistance invariably develops. Applying a focused shRNA screening approach to identify genes whose knockdown can prevent and/or overcome acquired resistance to erlotinib in several EGFR-mutant non-small cell lung cancer (NSCLC) cell lines, we identified casein kinase 1 α (CSNK1A1, CK1α). We found that CK1α suppression inhibits the NF-κB prosurvival signaling pathway. Furthermore, downregulation of NF-κB signaling by approaches independent of CK1α knockdown can also attenuate acquired erlotinib resistance, supporting a role for activated NF-κB signaling in conferring acquired drug resistance. Importantly, CK1α suppression prevented erlotinib resistance in an HCC827 xenograft model in vivo. Our findings suggest that patients with EGFR-mutant NSCLC might benefit from a combination of EGFR TKIs and CK1α inhibition to prevent acquired drug resistance and to prolong disease-free survival.

Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight.

MELK kinase has been implicated in playing an important role in tumorigenesis. Our previous studies suggested that MELK is involved in the regulation of cell cycle and its genetic depletion leads to growth inhibition in a subset of high MELK-expressing basal-like breast cancer cell lines. Herein we describe the discovery and optimization of novel MELK inhibitors 8a and 8b that recapitulate the cellular effects observed by short hairpin ribonucleic acid (shRNA)-mediated MELK knockdown in cellular models. We also discovered a novel fluorine-induced hydrophobic collapse that locked the ligand in its bioactive conformation and led to a 20-fold gain in potency. These novel pharmacological inhibitors achieved high exposure in vivo and were well tolerated, which may allow further in vivo evaluation.

Discovery and Optimization of HKT288, a Cadherin-6 Targeting ADC for the Treatment of Ovarian and Renal Cancer.

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Abstract 1176: Preclinical characterization of the pharmacokinetic-pharmacodynamics-efficacy relationship of novel allosteric SHP2 inhibitors

SHP2 is a non-receptor protein tyrosine phosphatase downstream of receptor tyrosine kinases (RTK). Mutations yielding constitutive activation of SHP2 primarily lead to activation of the MAPK pathway and have been found in multiple tumor types. These observations make SHP2 a potentially promising therapeutic target for the treatment of cancers with RTK dependence. Recently, a novel allosteric mechanism of SHP2 inhibition was identified where the autoinhibited form of SHP2 is stabilized via small molecule binding. Herein we describe efforts to characterize the pharmacokinetic (PK)/pharmacodynamic (PD)/efficacy relationship of orally bioavailable novel allosteric SHP2 inhibitors. Single dose PK/PD studies were conducted in nude mice engrafted with the EGFR amplified esophageal squamous cell carcinoma cell line, KYSE520. The allosteric SHP2 inhibitor SHP099 achieved time and dose dependent increases in plasma concentrations and concomitant reductions in tumor pERK that could be described by an Emax model. Tumor pERK levels were reduced by 50 to 70% when SHP099 unbound plasma concentrations exceeded the in vitro cellular PD IC50, suggesting that exposure above this threshold was required for pathway inhibition in vivo. A second allosteric SHP2 inhibitor, SHP065, was also profiled in vivo and yielded data consistent with this hypothesis. Two additional allosteric SHP2 inhibitors (SHP156, SHP393) that achieved unbound plasma concentrations approximating their cellular IC50s failed to modulate tumor pERK. The totality of these data support the hypothesis that unbound plasma concentrations in excess of the cellular IC50 is required for allosteric SHP2 inhibitors to inhibit the MAPK pathway in vivo. We further demonstrated that SHP099 achieves dose dependent inhibition of KYSE520 tumor xenograft growth in nude mice. Integration of the antitumor efficacy data and pERK inhibition data revealed a direct linear relationship between tumor growth inhibition and the fraction of time between dosing intervals in which pERK is inhibited by at least 50%. To test this model, SHP099 PK data from nude rats was applied to the Emax model and the resulting predicted PD responses were applied to the PD/efficacy model to predict SHP099 anti-tumor efficacy in nude rats. Data generated from a SHP099 KYSE520 efficacy study in nude rats demonstrated that the exposure/response model was remarkably robust. Doses of 8 mg/kg qd, 25 mg/kg qd, or 75 mg/kg q2d yielded observed T/C of 70, 14, and 18%, respectively; versus a model predicted T/C of 75, 6, and 6%, respectively. In summary, we describe for the first time successful efforts to characterize the PK/PD/efficacy relationship of novel allosteric SHP2 inhibitors. These exposure/response models served as a basis for further allosteric SHP2 inhibitor drug discovery efforts and begin to inform rational approaches to dose and schedule selection in clinic. Citation Format: Minying Pu, Laura R. La Bonte, Stan Spence, Kathy Hsiao, Shumei Liu, Brant Firestone, Ping Wang, Pascal D. Fortin, Ying-Nan P. Chen, Matthew J. LaMarche, Matthew J. Meyer. Preclinical characterization of the pharmacokinetic-pharmacodynamics-efficacy relationship of novel allosteric SHP2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1176. doi:10.1158/1538-7445.AM2017-1176

Abstract 872: In vivo activity of a novel CDH6 targeting antibody-drug conjugate, including population-scale ovarian PDX clinical trial

The Cadherin-6 (CDH6) gene was found to be frequently overexpressed in ovarian and renal cancers, while featuring a lineage-restricted normal tissue expression pattern. We hypothesized that based on the combined observation of frequent overexpression of CDH6 in cancer and a restricted normal tissue expression, CDH6 might be an ideal tumor antigen for targeting using an antibody-drug conjugate (ADC) approach. CHD6-ADC is a fully-human anti-CDH6 IgG1, linked via sulfo-SPDB to the tubulin-binding maytansinoid payload DM4. CDH6-ADC was evaluated across multiple linker-payload combinations with the sulfo-SPDB-DM4 format being selected based on a superior combined profile pertaining to activity, selectivity and tolerability. To gain a broader understanding of CDH6-ADC activity in vivo we profiled the lead candidate against a panel of 31 unselected patient derived ovarian xenograft (PDX) models in a 1×1×1 PDX clinical trial, similar to that described in Gao et al., 2015. In this unbiased high throughput in vivo screen, CDH6-ADC demonstrated robust antitumor activity, with an overall response rate of 39%. Responses were generally durable beyond 150 days and were achieved at doses yielding exposures anticipated to be achievable in humans and observed in PDX models featuring a range of CDH6 expression level and degree of tumor heterogeneity. Retrospective analysis of individual PDX responses and molecular profiling data demonstrate that sensitivity to CDH6-ADC is highly correlated to CDH6 transcript and protein levels. These findings suggest an ability to prospectively identify patients most likely to benefit from this novel targeted therapy. Furthermore, CDH6-ADC demonstrated robust tumor regressions in a representative PDX xenograft model that was refractory to carboplatin/paclitaxel standard of care therapy. These data suggest that CDH6-ADC may benefit both treatment naive patients and patients that have progressed on prior therapy containing tubulin-targeting anti-mitotics. Extending beyond ovarian cancer, we found CDH6 to be frequently overexpressed in renal cancer. CDH6-ADC was active against RCC PDX models featuring patient relevant levels of CDH6 expression. Data described herein suggest that this novel ADC may be an effective treatment for patients with CDH6 expressing tumors, including ovarian and renal cancer - both indications with a high unmet medical need. Citation Format: Carl U. Bialucha, Scott D. Collins, Yeonju Shim, Xiamei Zhang, Roberto Velazquez, Colleen Kowal, Caroline Bullock, Hongbo Cai, Stacy M. Rivera, Julie M. Goldovitz, Esther Kurth, Alice T. Loo, Guizhi Yang, John Green, Lance Ostrom, Matthew J. Meyer, Rebecca Mosher, Hui Gao, Juliet Williams, Emma Lees. In vivo activity of a novel CDH6 targeting antibody-drug conjugate, including population-scale ovarian PDX clinical trial. [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 872.

Abstract 2974: Targeting cadherin-6 (CDH6) with an antibody-drug conjugate for the treatment of ovarian and renal cancer

In an attempt to mine tumor versus normal mRNA expression datasets for novel tumor antigens, we identified the Cadherin-6 (CDH6) gene as frequently overexpressed in ovarian and renal cancers, while featuring a lineage-restricted normal tissue expression pattern. CDH6, also known as K-(kidney)-cadherin, is a member of the cadherin superfamily of calcium-dependent cell-cell adhesion molecules. We hypothesized that based on the combined observation of frequent overexpression of CDH6 in cancer and a restricted normal tissue expression, CDH6 might be an ideal tumor antigen for targeting using an antibody-drug conjugate (ADC) approach. CDH6-ADC is a fully-human anti-CDH6 IgG1, linked via sulfo-SPDB to the maytansinoid payload DM4. The antibody component of CDH6-ADC was selected from a panel of anti-CDH6 antibodies based on a multi-factorial lead selection campaign incorporating readouts of internalization propensity, in vitro cytotoxicity, as well as in vivo PK and efficacy across multiple linker-payload formats. CDH6-ADC features potent, target-dependent in vivo activity consistent with the mechanism of the anti-mitotic, tubulin-targeting sulfo-SPDB-DM4 linker-payload combination used. Specifically, treatment of CDH6-expressing ovarian cancer xenograft models with CDH6-ADC results in the time-dependent generation of intra-tumoral ADC catabolites and concomitant induction of phospho-histone H3 and cleaved caspase-3 - markers of G2/M arrest and apoptosis, respectively. CDH6-ADC induces durable tumor regressions at clinically relevant exposures in multiple human patient-derived tumor xenografts (PDX) across both ovarian and renal cancer lineages. To gain a more thorough understanding of CDH6-ADC activity in pre-clinical models of human ovarian cancer and identify potential molecular correlates for patient stratification, we profiled CDH6-ADC in a PDX clinical trial or PCT comprising 31 individual PDX models. In this unselected population, treatment with CDH6-ADC resulted in robust anti-tumor activity. Integration of PDX response data with CDH6 target expression in both the PDX models and human clinical samples indicate CDH6 expression patterns consistent with in vivo activity are found in a substantial fraction of ovarian, renal and cholangiocarcinoma patients. Together, the encouraging pre-clinical efficacy and tolerability data support the clinical evaluation of CDH6-ADC. Citation Format: Scott D. Collins, Parmita Saxena, Xiao Y. Li, Yeonju Shim, Lance Ostrom, Nicholas C. Yoder, Kalli C. Catcott, Molly A. McShea, Xiuxia Sun, Sanela Bilic, William R. Tschantz, Meghan Flaherty, Keith Mansfield, Tiancen Hu, Vladimir Capka, Markus Kurz, Ivana Liric Rajlic, Anne Serdakowski London, Duc Nguyen, Rebecca Mosher, Matthew J. Meyer, Aaron Bourret, Jamal Saeh, Scott Cameron, Emma Lees, Carl U. Bialucha. Targeting cadherin-6 (CDH6) with an antibody-drug conjugate for the treatment of ovarian and renal cancer. [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 2974.

Abstract 1680: In vitro and in vivo activity of a highly potent and novel FGFR2/FGFR4 dual targeting antibody-drug conjugate

The fibroblast growth receptors 2 and 4 (FGFR2, FGFR4) are overexpressed in a broad spectrum of malignancies. In a subset of breast, gastric, and esophageal cancers, increased FGFR2 expression is driven by FGFR2 copy number (CN) gain, which renders these tumors dependent on FGFR2 pathway activation and is associated with poor prognosis. FGFR4 expression is increased in response to both FGFR4 CN gain, as seen in embryonal rhabdomyosarcoma, or by the PAX3-FOXO1 fusion gene product which transcriptionally activates FGFR4, as seen in alveolar rhabdomyosarcoma. Additionally, other mechanisms drive increased FGFR2 expression, e.g. in subsets of lung and breast cancer, and increased FGFR4 expression, e.g. in subsets of hepatocellular, breast, and pancreatic cancer. This elevated expression in malignancies combined with the observation that FGFR2 and FGFR4 are efficiently internalized upon antibody binding make both receptors attractive targets for antibody-drug conjugate (ADC) therapy. To this end, a drug discovery campaign was initiated and a novel, highly potent FGFR2, FGFR4 dual targeting ADC was discovered. This ADC consists of a fully human antibody (discovered in collaboration with MorphoSys) conjugated to the potent maytansine-derived microtubule-disruptor, DM1, via an SMCC non-cleavable thioether linkage (linker payload technology licensed from ImmunoGen, Inc.). In vitro, the ADC is active against FGFR2 and FGFR4 positive cells in viability assays and is efficiently processed yielding the principle catabolite Lys-SMCC-DM1. In vivo, the ADC is highly efficacious against a variety of disease relevant xenograft models including FGFR2 amplified breast and gastric models and a PAX3-FOXO1 translocation positive alveolar rhabdomyosarcoma model. Additionally, the ADC is potent against a subset of primary tumor derived breast and lung xenograft models that lack FGFR2 CN gain or the PAX3-FOX01 translocation. Consistent with the molecule9s mode of action, anti-tumor activity is preceded by G2/M cell cycle arrest and apoptosis. Taken together these data suggest that this novel, dual targeting ADC may be an effective treatment for patients with FGFR2 or FGFR4 positive tumors including, but not limited to those with FGFR2 CN gain or the PAX3-FOXO1 fusion gene. Citation Format: Matthew J. Meyer, David Jenkins, David Batt, Rebecca Mosher, Randi Isaacs, Tiancen Hu, Vladimir Capka, Xiamei Zhang, Dongshu Chen, Lujia Tang, Mike Daley, Patrycja Nowakowski, Yeonju Shim, Wei Jiang, Seth Ettenberg, Emma Lees. In vitro and in vivo activity of a highly potent and novel FGFR2/FGFR4 dual targeting antibody-drug conjugate. [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 1680. doi:10.1158/1538-7445.AM2015-1680