Abstract
Aberrant receptor tyrosine kinase (RTK) signaling is a well-documented driver of disease onset and progression across myriad cancer types, where the MET RTK contributes to tumor progression, ...maintenance and resistance to targeted therapies. Here we explore the therapeutic potential of AZD6094, a highly potent and selective MET inhibitor, in EGFR mutant (EGFRm) non-small cell lung cancer (NSCLC). Although many EGFRm NSCLC patients receiving first-line EGFR Tyrosine Kinase Inhibitors (TKI) benefit from therapy initially, the majority of patients will acquire resistance in 9-14 months1,2. Of this patient population, ∼10-15% of patients with emerging resistance to early generation EGFRm-TKI will have MET amplification3. Using xenograft models (HCC827) of resistance to erlotinib or gefitinib, both first-generation EGFRm-TKI, we assessed the efficacy of AZD6094 in models with varying copy number gain for MET. We demonstrate that the combination of AZD6094 with gefitinib, or AZD9291, an irreversible, selective (EGFRm/T790M) EGFR TKI, results in tumor growth inhibition (TGI) of >100% in 3 models (HCC827-ER1, PCS030 clone 1 and 2)4, suggesting that the combination is necessary and sufficient to address acquired resistance due to MET gene amplification. Moreover, we explore efficacy of AZD6094 in models representative of resistance to first-line treatment with EGFRm-TKIs, harboring MET amplification and T790M EGFR mutations. In NCI-H820 xenografts (EGFRm/T790M/MET), we demonstrate for the first time that combining MET and EGFRm/T790M TKIs (AZD6094 with AZD9291) induces tumor regressions (TGI% >100%, 94% regressions) and the loss of palpable tumors in 5/7 animals as compared to AZD9291 (TGI 48%) or AZD6094 treatment alone (TGI >100%). Pharmacodynamic analysis of tumor lysates demonstrated potent and durable inhibition of pMET in all AZD6094 treatment groups. Due to the clinical importance of understanding acquired resistance to targeted TKIs, we then generated a model of resistance to AZD6094 in MET-amplified NSCLC NCI-H1993 cells and analyzed several resistant clones (H1993R). Interestingly, MET phosphorylation remains strongly inhibited in AZD6094-treated H1993R cells, while EGFR protein expression is upregulated and leads to co-dependency between both pathways. Enhanced expression and phosphorylation of EGFR, as well as AKT, MEK and ERK activation were commonly observed in H1993R cells. Taken together, our data support the potential of AZD6094 as a novel combination therapy for MET-driven NSCLC in the context of EGFRm TKI resistance, and highlight the clinical relevance of EGFR and MET signaling in the context of emerging TKI resistance mechanisms and coordinated pathways.
1. Mok et al. N Engl J Med 2009;361:947-957.
2. Rosell et al. Lancet Oncol 2012;13:239-246.
3. Engelman et al. Science 2007;316:1039-1043.
4. Models from Precos Ltd
Citation Format: Celina D'Cruz, Evan Barry, Ryan Henry, Lillian Castriotta, Alwin Schuller, Garry Beran, Susan Ashton, Cath Eberlein, Corinne Reimer, Melanie Frigault, Michael Zinda, Darren Cross, Stephen Fawell. Changing the paradigm for treating drug resistance in NSCLC: Novel combinations of AZD6094, a selective MET inhibitor, and an irreversible, selective (EGFRm/T790M) EGFRTKI, AZD9291. 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 761. doi:10.1158/1538-7445.AM2015-761
Abstract
The bromodomain and extra-terminal (BET) protein, BRD4 functions as a transcriptional co-activator through recruitment of regulatory complexes to acetylated chromatin. Among all ...malignancies, the most focal, and recurrent BRD4 gene amplification occurs in high-grade serous ovarian cancer (HGSOC). Despite the elucidation of BRD4 dependencies in cancer, it is unclear whether BRD4 gene amplification can result in oncogenic activity, and hence serve as a patient selection strategy for BET bromodomain inhibitors. To assess the oncogenic potential of BRD4 amplification in HGSOC, we assayed enforced BRD4 expression in non-transformed immortalized surface ovarian cells (IOSE). Physiologically relevant expression of either long or short BRD4 spliced isoforms in IOSE (BRD4-IOSE) resulted in marked phenotypic changes indicative of cellular transformation, including altered growth kinetics, chromatin reorganization, and acquisition of colony formation potential. Transcriptional profiling of BRD4-IOSE and BRD4-amplified HGSOC patients revealed shared expression patterns, including enriched MYC, E2F1, and VEGF gene signatures. Interrogation of BRD4 inhibitor pharmacodynamic markers, and transcripts upregulated through enforced BRD4 expression uncovered Neuregulin-1 as a critical BRD4 transcriptional effector. RNAi-mediated Neuregulin-1 depletion was capable of abolishing the oncogenic activity of BRD4-IOSE, and exogenous Neuregulin-1 was sufficient to transform IOSE without BRD4. A novel BRD4-SWI/SNF interaction was required for driving the oncogenic activity of BRD4-IOSE through maintaining NRG1 expression. The sensitivity of patient-derived HGSOC xenografts to a novel clinical candidate BRD4 inhibitor, AZD5153, correlated with BRD4 amplification and a pharmacodynamic NRG1 response. This study demonstrates the oncogenic potential of BRD4 amplification, defines a relationship between BRD4 and NRG1, and further establishes HGSOC as an appropriate patient population in which to test the therapeutic potential of BET bromodomain inhibitors.
Citation Format: Garrett W. Rhyasen, Yi Yao, Austin Dulak, Lillian Castriotta, Kelly Jacques, Maureen Hattersley, Gordon B. Mills, Michael Zinda, Stephen Fawell, Paul Lyne, Edwin Clark, Huawei Chen. BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian 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 1971.
Abstract
Here we report results of an independent evaluation of an automated capillary electrophoresis-immunoassay system for its utility in generating quantitative and reproducible pharmacodynamic ...(PD) biomarker data for the PI3K pathway using clinical specimens. Quantitative measurement of PD biomarkers associated with drug -target engagement in tumor tissue is an increasingly important part of evaluating new cancer therapies in early clinical trials. PD data are currently being used along with safety, pharmacokinetics, and efficacy data to make more informed clinical decisions regarding likelihood of success and progression of drugs into Phase II testing. Despite this increased focus, there are still very few assay platforms that support automated, reproducible and quantitative methods amenable to the small tumor biopsies available for these analyses. The Peggy™ (ProteinSimple) platform provides automated separation of proteins by size or charge and quantitation of analytes over a broad dynamic range from tissue samples using as little as 40 ng protein. Accompanied by the ability to process 96-samples in a single run, this platform is amenable to both pre-clinical and clinical PD determination.
Our initial studies focused on evaluating the feasibility of developing a set of robust, quantitative PD biomarker assays that could be used systematically across our oncology portfolio to monitor the PI3K pathway. Here we present results for a panel of biomarkers associated with the PI3K pathway using cell lines and tumor xenografts in which we measured responses to drug treatments using AKT and mTOR inhibitors. Further, in a set of prospectively collected breast cancer core needle biopsies, we were able to detect baseline levels of these biomarkers. Taken together, these data suggest that this platform will be useful in quantitatively measuring changes in multiple PD biomarkers in a single tumor biopsy. Going forward, we propose to utilize this approach alone or in tandem with other immunoassay formats, such as immunohistochemistry, to provide greater insight into tumor biology and the effects of drug treatment following PI3K -AKT inhibition in the clinic to help inform important clinical decisions.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A200.
Citation Format: Patricia E. Mccoon, Lourdes Pablo, Kristen McEachern, Emily Foster, Chris Womack, Lillian Castriotta, Jeff Brown, Carl Barrett. Toward quantitative PI3K pathway PD analysis in clinical samples using an automated capillary electrophoresis-immunoassay platform. 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 A200.
Abstract
Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. Aberrant receptor tyrosine kinase (RTK) signaling is a ...well-documented driver of disease onset and progression in multiple cancer types, including non-small cell lung cancer (NSCLC), where the cMET RTK contributes to tumor progression, maintenance and resistance to targeted therapies. Here, we explore the therapeutic potential of the potent and selective cMET inhibitor savolitinib (volitinib, AZD6094, HMPL-504) in NSCLC and begin to elucidate mechanisms of acquired savolitinib resistance in preclinical models. Using in vitro proliferation assays and immunoblot analysis, we determine that savolitinib rapidly inhibits cMET auto-phosphorylation/activation and reduces the viability of NSCLC cell lines NCI-H1993 and EBC-1 with a GI50 of 4.20 nM and 2.14 nM, respectively. In vivo, once daily treatment of NCI-H1993 xenografts with 3.0 mg/kg savolitinib significantly slows tumor growth, whereas treatment of EBC-1 xenografts with 30.0 mg/kg results in tumor stasis. Importantly, we observe tumor regressions in a patient-derived xenograft model of a NSCLC lymph node metastasis, HLXF-036LN, dosed with savolitinib 50.0 mg/kg once daily. Pharmacodynamic analysis of in vitro and in vivo models shows that savolitinib sensitivity correlates with blockade of PI3K/AKT and MAPK signaling, and interestingly, with cMYC (MYC) protein down-regulation. To elucidate mechanisms of acquired resistance in NSCLC, we generated savolitinib resistance in vitro using the NCI-H1993 and EBC-1 cell lines and further sub-cloned resistant NCI-H1993 cells to study the heterogeneity of resistance mechanisms. Using small-molecule screening, phospho-protein arrays and interrogation of signaling pathway activity by immunoblot, we identify 1) deregulated mTORC1/2 signaling and 2) the uncoupling of MYC expression from cMET activation as commonly contributing to resistance in all clones tested. RNA interference (siRNA) and MYC over-expression experiments confirm the novel finding that sustained MYC expression can partially drive resistance to a tyrosine kinase inhibitor such as savolitinib. Additionally, we identify clone-specific resistance mechanisms arising via a previously-described switch to EGFR dependence or by our novel finding of a de novo requirement for PIM signaling. Taken together, this work demonstrates the preclinical efficacy of savolitinib in NSCLC and provides an initial characterization of potential resistance mechanisms, identifying core resistance targets and clone-specific vulnerabilities that could be exploited to counter acquired savolitinib resistance that may emerge in the clinic.
Citation Format: Ryan E. Henry, Evan R. Barry, Brendon Ladd, Aleksandra Markovets, Garry J. Beran, Yongxin Ren, Feng Zhou, Lillian Castriotta, Ammar Adam, Weiguo Qing, Weiguo Su, Edwin Clark, Celina M. D'Cruz, Alwin Schuller. Acquired resistance to the cMET inhibitor savolitinib in lung cancer models through EGFR/mTOR/MYC deregulation and adoption of PIM signaling. 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 LB-C22.
