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Mcl-1, a member of the Bcl/Mcl family, is a key protein involved in evasion of apoptosis in a wide variety of tumors. Its amplification and overexpression have also been implicated in innate and acquired resistance to anticancer drugs. Mcl-1 is capable of preventing induction of apoptosis, both by binding and inactivating the pro-apoptotic executioner Bcl-2 protein, Bak, as well as by sequestering other pro-apoptotic BH3-only proteins such as Bim and Noxa. AZD5991 is a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1. It demonstrates all the hallmarks of a true Mcl-1 inhibitor: 1. potent, selective, and rapid apoptosis in Mcl-1-dependent cell lines (e.g., GI50 as low as 10 nM in multiple myeloma cell lines); 2. loss of activity upon overexpression of Bcl-xL or siRNA-mediated knockout of Bak; 3. Mcl-1:Bak complex disruption as demonstrated by co-immunoprecipitation. AZD5991 is active in vivo, with complete (100%) tumor regression demonstrated in several mouse xenograft models after a single tolerated dose. We have also demonstrated synergistic in vivo efficacy in combination with standard-of-care agents. Analysis of ex vivo activity in primary samples from leukemia patients indicates that a high percentage of leukemia patients should respond to drug treatment, which supports our plan for a phase I trial of AZD5991 in patients with hematologic cancers. Citation Format: Alexander W. Hird, J. Paul Secrist, Ammar Adam, Matthew A. Belmonte, Eric Gangl, Frank Gibbons, David Hargreaves, Jeffrey W. Johannes, Stephen L. Kazmirski, Jason G. Kettle, Stephen E. Kurtz, Michelle L. Lamb, Martin J. Packer, Bo Peng, Craig R. Stewart, Jeffrey W. Tyner, Wenzhan Yang, Qing Ye, XiaoLan Zheng, Edwin A. Clark. AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers [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 DDT01-02. doi:10.1158/1538-7445.AM2017-DDT01-02

Mcl-1 is a member of the Bcl/Mcl family of proteins that promotes cell survival by preventing induction of apoptosis in a broad range of cancers. High expression of Mcl-1 has been linked to tumor development and resistance to anticancer therapies, underscoring the potential of Mcl-1 inhibitors as anticancer drugs. We have previously shown that AZD5991, a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1 and high selectivity, induces rapid and irreversible commitment to apoptosis in Mcl-1-dependent cancer cells in a manner dependent on proapoptotic BAK. Here, we demonstrate that AZD5991 exhibits cytotoxic activity (GI50 Citation Format: Adriana E. Tron, Matthew A. Belmonte, Steven Criscione, Edwin A. Clark, Eric Gangl, Francis D. Gibbons, Jeffrey W. Tyner, Stephen E. Kurtz, Qing Ye, Alexander W. Hird, Alwin Schuller, J. Paul Secrist. Selective Mcl-1 inhibition by AZD5991 induces on-target cell death and achieves antitumor activity in multiple myeloma and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 302.

Apoptosis is a normal cellular process that is regulated by the dynamic interaction of pro- and anti-apoptotic proteins of the B-cell lymphoma 2 (Bcl-2) family. Cancers, however, have evolved mechanisms to hijack this process and tip the balance in favor of anti-apoptotic proteins, conferring a survival advantage for tumor cells as well as a means of resistance to anti-cancer therapies. Indeed, the Bcl-2 family are some of the most frequently amplified genes and over-expressed proteins across various tumor types. As a result, tumor cells can become addicted to Bcl-2 family members and, hence, vulnerable to targeted BH3 mimetics. Clinical validation of this concept has been demonstrated by venetoclax with its approval for treatment of R/R CLL patients with 17p deletion. Given the great potential that directly targeting the apoptotic machinery holds in treating cancer, developing BH3 mimetics is an attractive proposition. To that end, we have developed a potent small molecule, AZD4320,1 that has nanomolar affinity for Bcl-2 and Bcl-xL, similar to navitoclax, but has physicochemical properties suitable for IV administration. This will help mitigate toxicities observed with oral administration of navitoclax (e.g. allow recovery of platelets), thus improving therapeutic index. AZD4320 also displays the hallmarks of a bona fide BH3 mimetic, most notably the ability to disrupt the complex formation of Bcl-2 with BH3-only proteins and the necessity for intact BAK and BAX to propagate the apoptotic cascade. A kinetic study was also conducted to explore apoptosis induction in the Bcl-2-addicted B-ALL cell line, RS4;11, which revealed both a dose- and time-dependent increase in cleaved caspase-3 (CC3) and corresponding reduction in cell viability. In an expanded panel of human cancer cell lines, AZD4320 rapidly induced CC3 (6h) and loss of viability (24h) in a diverse set of hematological lines with a median EC50 of 182nM. Solid tumor cell lines, however, were much less responsive (median EC50 >30μM). A comparison to venetoclax from the same cell line panel screen revealed that many more hematological tumor cell lines were sensitive to AZD4320, highlighting the utility and promise of a dual Bcl-2/xL inhibitor. Furthermore, in a venetoclax-resistant derived ABC-DLBCL cell line, AZD4320 was equally potent when compared to the parental cell line whereas venetoclax exhibited a >20-fold reduction in activity. Lastly, for in vivo efficacy studies with RS4;11 xenograft tumors, regressions with corresponding induction of CC3 were observed following a single dose of AZD4320. Together, these results highlight the therapeutic potential of a dual Bcl-2/xL inhibitor to be used as a foundation therapy across a broad range of hematological tumor types as well as combat resistance to other BH3 mimetics and targeted therapies. 1Hennessy, E; et al. ACS National Meeting 24 (2015). Citation Format: Justin Cidado, J Paul Secrist, Francis D. Gibbons, Edward J. Hennessy, Stephanos Ioannidis, Edwin A. Clark. AZD4320 is a potent, dual Bcl-2/xLinhibitor that rapidly induces apoptosis in preclinical hematologic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 311.

Alterations in the MET oncogene occurs across a broad range of tumor indications. Amplification or mutations in MET lead to increased activity of downstream pathways including PI3K and MAPK, eventually resulting in tumor formation. Several small molecule inhibitors are currently in clinical trials, including the selective inhibitor Savolitinib (HMP-504, Volitinib, AZD6094), which shows single digit nanomolar activity in MET-amplified cell lines. Newly emerging data suggest mutations in MET causing complete skipping of Exon 14 occur in approximately 4% of non-small cell lung cancer (NSCLC), and are more rare in other indications [1, 2]. MET exon 14 skipping mutations were shown to be mutually exclusive from EGFRm, ALK and KRAS and can occur in the context of MET gene amplification [3]. Exon 14 harbors the CBL binding site (Y1003), which is critical for receptor degradation after binding of its ligand, HGF, and suppression of downstream signaling events. Clinical trial results with less potent, pan RTK inhibitors Crizotinib (31nM GI50 vs 3nM for Savolitinib) and Cabozantinib show promising early results, but fall short in long term responses. Therefore, better therapies targeting MET are needed. Human cell line models with Exon 14 deletions are rare. Therefore, we used engineered cell lines to test the effect of Savolitinib on these mutations. To do this, we expressed MET-Y1003F mutants in NIH-3T3 and HEK293T cells. We found that Savolitinib potently inhibited phospho-MET in both models expressing this mutant (100% phospho-MET inhibition). In addition, we tested whether or not Savolitinib could inhibit HGF-dependent signaling and growth of a NSCLC cell line, NCI-H596. In the presence of FBS (10%), Savolitinib had no effect on the growth rate of these cells, however was highly efficient at blocking HGF-dependent growth in the absence of FBS. To test the effect of this mutation in the background of amplification, we also tested the gastric cancer cell line Hs746T, which harbors exon 14 skipping in addition to MET amplification. Savolitinib was highly efficacious at blocking the growth of this cell line. Future studies are aimed at looking at the in vivo effect of Savolitinib targeting exon 14 mutants. These data provide a platform of evidence for using Savolitinib to target exon 14 mutant MET in patients. 1. Paik, P.K., et al., Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping. Cancer Discov, 2015. 5(8): p. 842-9. 2. Frampton, G.M., et al., Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. Cancer Discov, 2015. 5(8): p. 850-9. 3. Cancer Genome Atlas Research, N., Comprehensive molecular profiling of lung adenocarcinoma. Nature, 2014. 511(7511): p. 543-50. Citation Format: Evan Barry, Elizabeth Maloney, Ryan Henry, Alexandra Borodovsky, Edwin Clark, Melanie Frigault, Michael Zinda, Celina D’Cruz. Targeting MET Exon 14 mutations with the selective small molecule inhibitor Savolitinib. [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 1150.

Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with non-localized disease. The central role of the hepatocyte growth factor (HGF) receptor MET in PRCC has been explored, demonstrating that MET aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 (the location of MET and HGF) occur in the majority of PRCC cases. We sought to evaluate AZD6094 (HMPL-504), a potent and selective small molecule MET kinase inhibitor, in this disease setting. However, the development of effective therapies targeting MET and other targets in PRCC has been hampered in part by a lack of available preclinical models to test novel targeted therapies. Here we describe for the first time the pharmacodynamic (PD) response and anti-tumor activity of the selective MET inhibitor AZD6094 in two preclinical patient derived xenograft (PDX) models of PRCC (RCC-43b and RCC-47). Both PDX models have increased MET copy number of 8 and 9 copies by FISH in RCC-43b, and RCC-47 respectively, and robust MET protein staining by IHC. AZD6094 treatment resulted in dose dependent anti-tumor responses reaching ∼85% tumor growth inhibition (TGI) when dosed at 2.5 mg/kg daily (qd), stasis when dosed 10 mg/kg qd, and ∼20% regression when dosed at 25 mg/kg qd in the RCC-43b model and ∼63% TGI, ∼89% TGI, ∼64% regression, and ∼96% regression in the RCC-47 model when dosed 0.5, 2.5, 10, and 25 mg/kg qd respectively. The standard of care for RCC, sunitinib, showed no activity in RCC-43b when dosed at 10 mg/kg qd (∼10% TGI, p>0.05 vs vehicle) and ∼60% TGI when dosed at 80 mg/kg qd. Pharmacodynamic analysis of RCC-47 tumors revealed that two hours after an acute dose of AZD6094 pMET levels were reduced >95% at all dose levels tested (0.5 - 25 mg/kg). Eight hours after dosing, pMET levels returned to ∼50% in the 0.5 and 2.5 mg/kg dose groups whereas pMET was still inhibited >90% in the 10 and 25 mg/kg dose groups indicating that the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose and time dependent induction of cleaved PARP, a marker of cell death. The finding that lower, sub-optimal doses of AZD6094 showed return of pMET 8 hours after a single administration, raised the question whether splitting the dose over a longer duration would increase anti-tumor activity. Indeed, twice a day dosing (bid, 8;16 hours) of AZD6094 at 1.25 mg/kg was more efficacious than daily administration of 2.5 mg/kg resulting in 8% regression compared to 89% TGI (p Citation Format: Alwin Schuller, Evan Barry, Rhys Jones, Melanie Frigault, Garry Beran, Ryan Henry, David Linsenmayer, Maureen Hattersley, Aaron Smith, Joanne Wilson, Ammar Adam, Michael Zinda, Corinne Reimer, Stephen Fawell, Edwin Clark, Celina D'Cruz. Pharmacodynamic response and anti-tumor activity of the MET inhibitor AZD6094 in papillary renal cell carcinoma patient derived xenograft 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 1477. doi:10.1158/1538-7445.AM2015-1477

MET is a transmembrane tyrosine kinase receptor that is deregulated (gene amplification, mutation and over-expression) across multiple cancer types. Signaling through MET is normally activated through interactions with its specific ligand, hepatocyte growth factor (HGF). Aberrant MET/HGF activation can stimulate tumor growth, promote angiogenesis, induce metastasis and may contribute to resistance mechanisms in several tumor types. Several non-selective MET inhibitors have entered clinical development; results have been mixed based on potency, selectivity, and/or patient selection. Volitinib is a potent (IC50 4 nM) and selective (>650 fold selectivity over 265 kinases), small molecule inhibitor of MET. Recent evaluation of Volitinib across a panel of cancer cell lines demonstrated selectivity for MET-driven disease, with MET amplified cell lines being most sensitive (IC50s of 1nM) and also suggesting limited off target activity. Volitinib resistant cell lines with MET amplification were identified and used to better understand the relationship of concurrent mutations with response. In addition to cancer cell line selectivity, we are analyzing preclinical models of NSCLC (non small cell lung cancer) that are representative of key patient segments, namely MET amplification and over expression. In newly diagnosed NSCLC adenocarcinomas, focal MET amplification events represent ∼3% of the population while over expression of MET (without gene amplification) is observed in the majority of patients. In preclinical models, focal Met amplification in EBC-1 and NCI-H1993 caused significant tumor growth inhibition, confirming sensitivity to Volitinib. Given the prevalence of over expression in NSCLC, however, we sought to build a platform of evidence for the therapeutic use of Volitinib in preclinical models lacking amplification of the MET gene. Using a patient-derived xenograft model (PDX) of EGFR WT, KRAS WT and metastatic NSCLC disease (HLXF-036LN), we demonstrate that Volitinib induces tumor regression as monotherapy and has added therapeutic benefit when used in combination with taxotere. In addition, we show robust efficacy effects for additional preclinical models, LG0567, LG0645 and Calu-3, for either Volitinib alone or in combination with taxotere. In parallel pharmacodynamic studies, we demonstrate that Volitinib inhibits p-MET and downstream signaling in each model. Together, using an integrated platform of molecular characterization and MET FISH and IHC scores, corresponding antitumor responses to Volitinib are being evaluated for several patient segments. We are using these studies to inform the design of patient selection criteria for upcoming clinical trials in NSCLC. Citation Format: Celina D'Cruz, Melanie Frigault, Ammar Adam, Minhui Shen, Garry Beran, Evan Barry, Paul Gavine, Yongxin Ren, Shiming Fan, Feng Zhou, Weiguo Qing, Mike Zinda, Weiguo Su, Edwin Clark. Targeting MET in preclinical models to support the clinical development of Volitinib in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3114. doi:10.1158/1538-7445.AM2014-3114

Fibroblast growth factor receptor (FGFR) tyrosine kinase family members have gained increasing attention as potential therapeutic targets. Gene amplification, or gain-of-function translocations and mutations of these receptors, have been implicated in a variety of cancers including breast, gastric, bladder, ovarian, endometrial, lymphoma and myeloma. We have previously described the biological profiling of ARQ 087, a potent small-molecule pan-FGFR kinase inhibitor that shows anti-tumor activity in FGFR dysregulated tumor cells in vitro and in vivo. A number of cell lines with genetically-altered FGF receptors were identified that were sensitive in anti-proliferative assays to ARQ 087, including FGFR2 amplified gastric carcinoma lines KATO III and SNU-16, and endometrial lines AN3CA, MFE-296 and MFE-280 harboring FGFR2 mutants, as well as a breast cancer cell line with increased FGFR1 gene copy (MDA MB-157). Western blot pharmacodynamic analysis demonstrates that ARQ 087 inhibits FGFR phosphorylation and downstream pathway markers in these cell lines in a concentration-dependent manner. Using antibody arrays specific for known cytokines as well as regulatory proteins involved in angiogenesis and apoptosis, we have identified a number of protein biomarkers in cell lysates and in conditioned media of sensitive cell lines that changed in response to ARQ 087 exposure. Three proteins (cytochrome c, clusterin, and HTRA2) decreased in SNU-16 (ARQ 087-sensitive, FGFR amplified) but not DLD1 (ARQ 087-resistant, non FGFR expressing) cells. VEGF decreased and IL-1rα increased in SNU-16 but not in DLD1 conditioned media in response to ARQ 087. Furthermore, analysis of mouse plasma from animals bearing AN3CA tumors treated with ARQ 087 revealed a decrease in human VEGF, CXCL16, MMP-9, Serpin E1 and MIF and an increase in RANTES, IL-1β, IP-10, SDF-1, IL-1rα. TNFα, GM-CSF, IL-2, sTREM-1, sICAM-1, MIP-1β, IL-8. In summary, we have identified a number of cell lines from a broad cell line screening campaign that exhibited dysregulated FGFR pathway function and are sensitive to the anti-proliferative effects of ARQ 087. These cell lines, in turn, have been used as models in which to identify candidate biomarkers that may greatly facilitate subsequent clinical evaluation of this molecularly targeted anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3571. doi:10.1158/1538-7445.AM2011-3571