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Antiproliferative effect of RAF709 compared to dabrafenib, trametinib, RAF265 and sorafenib across a tumor cell panel

Legends for supplemental figures and tables

Table S1: Kinase hits from KinomeScan panel. Table S4: Antiproliferative activity of RAF709 in a Ba/F3 cell kinase panel.

Ovarian cancer cell lines do not reproduce the clinical association between BRCA loss of function and sensitivity to olaparib, but patient-derived ovarian tumor xenografts do.

Ovarian PDX raw growth and sequencing data.

Candidate biomarkers are not independently prognostic of survival in primary ovarian tumor samples from TCGA.

Genomic landscape analysis of epithelial ovarian carcinoma of PDXs, patient tumors (TCGA) and cell lines (CCLE).

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor–derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537–48. ©2018 AACR.

Advanced ovarian cancers are a leading cause of cancer-related death in women and are currently treated with surgery and chemotherapy. This standard of care is often temporarily successful but exhibits a high rate of relapse, after which, treatment options are few. Here we investigate whether biomarker-guided use of multiple targeted therapies, including small molecules and antibody–drug conjugates, is a viable alternative. A panel of patient-derived ovarian cancer xenografts (PDX), similar in genetics and chemotherapy responsiveness to human tumors, was exposed to 21 monotherapies and combination therapies. Three monotherapies and one combination were found to be active in different subsets of PDX. Analysis of gene expression data identified biomarkers associated with responsiveness to each of the three targeted therapies, none of which directly inhibits an oncogenic driver. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. The distribution of biomarker positivity in The Cancer Genome Atlas data suggests the potential for a similar precision approach in human patients.Significance:This study exploits a panel of patient-derived xenografts to demonstrate that most ovarian tumors can be matched to effective biomarker-guided treatments.

Mean tumor volume change difference between best monotherapy and each of 9 combinations. Hazard ratio for combination versus independent action simulation PFS.

Most ovarian tumors respond to one of four biomarker-guided treatments.

Figure S1: pMEK and pERK suppression in KRAS mutant and BRAFV600E cells by dabrafenib or RAF709. Figure S2: pMEK and pERK suppression in NRAS mutant and BRAFV600E cells by dabrafenib or RAF709. Figure S3: Cell cycle and apoptotic effects of RAF709 in KRASmut cells. Figure S4: Antiproliferative activity of RAF709 in HPAFII cells.

Supplementary Material PDF, including supplementary methods and supplementary figures 1-4

KINOMEScan assay results for RAF709

Compound dose, dosing schedule, administration route, target and drug type.

KiNative assay results for RAF709

Ovarian PDX response category by therapy.

Advanced ovarian cancers are a leading cause of cancer-related death in women and are currently treated with surgery and chemotherapy. This standard of care is often temporarily successful but exhibits a high rate of relapse, after which, treatment options are few. Here we investigate whether biomarker-guided use of multiple targeted therapies, including small molecules and antibody–drug conjugates, is a viable alternative. A panel of patient-derived ovarian cancer xenografts (PDX), similar in genetics and chemotherapy responsiveness to human tumors, was exposed to 21 monotherapies and combination therapies. Three monotherapies and one combination were found to be active in different subsets of PDX. Analysis of gene expression data identified biomarkers associated with responsiveness to each of the three targeted therapies, none of which directly inhibits an oncogenic driver. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. The distribution of biomarker positivity in The Cancer Genome Atlas data suggests the potential for a similar precision approach in human patients. Significance: This study exploits a panel of patient-derived xenografts to demonstrate that most ovarian tumors can be matched to effective biomarker-guided treatments.

Advanced ovarian cancers are a leading cause of cancer-related death in women. Such cancers are currently treated with surgery and chemotherapy which is often temporarily successful but exhibits a high rate of relapse after which treatment options are few. Here we assess the responses of a panel of patient-derived ovarian cancer xenografts (PDXs) to 19 mono and combination therapies, including small molecules and antibody-drug conjugates. The PDX panel aimed to mimic the heterogeneity of disease observed in patients, and exhibited a distribution of responsiveness to standard of care chemotherapy similar to human clinical data. Three monotherapies and one drug combination were found to be active in different subsets of PDXs. By analyzing gene expression data we identified gene expression biomarkers predictive of responsiveness to each of three novel targeted therapy regimens. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. Biomarker frequency was similar in human patients, suggesting the possibility of a new therapeutic approach to ovarian cancer and demonstrating the potential power of PDX-based trials in broadening the reach of precision cancer medicine.

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAFV600 alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in in vivo tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor–derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.Significance: In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and in vivo properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. Cancer Res; 78(6); 1537–48. ©2018 AACR.

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 naïve 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.