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PDF file - 614K, Cellular effects of TAK-632, vemurafenib, and TAK-733 on MAPK signaling (S1); Model describing RAF paradoxical activation (S2); Effects of TAK-632 and vemurafenib on BRAF kinase activity (S3); Reversibility of TAK-632 and vemurafenib in living cells (S4); Effects of TAK-632 and vemurafenib on RAF kinase activity in NRAS-mutated cells expressing dimerization-prone RAF mutants (S5); Effect of TAK-632 on MAPK signaling in cells expressing NRASQ61K or deltaN-BRAF (S6) Combined effect of TAK-632 and TAK-733 on cell proliferation in cells bearing wild-type RAF and RAS (S7); Combined effect of TAK-632 and TAK-733 on MAPK signaling in cells expressing NRASQ61K or deltaN-BRAF (S8); Model describing the mechanism of action of a pan-RAF inhibitor (S9).

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Introduction KRAS mutations have been recognized as undruggable for many years. Recently, novel KRAS G12C inhibitors, such as sotorasib and adagrasib, are being developed in clinical trials and have revealed promising results in metastatic NSCLC. Nevertheless, it is strongly anticipated that acquired resistance will limit their clinical use. In this study, we developed in vitro models of the KRAS G12C cancer, derived from resistant clones against sotorasib and adagrasib, and searched for secondary KRAS mutations as on-target resistance mechanisms to develop possible strategies to overcome such resistance. Methods We chronically exposed Ba/F3 cells transduced with KRASG12C to sotorasib or adagrasib in the presence of N-ethyl-N-nitrosourea and searched for secondary KRAS mutations. Strategies to overcome resistance were also investigated. Results We generated 142 Ba/F3 clones resistant to either sotorasib or adagrasib, of which 124 (87%) harbored secondary KRAS mutations. There were 12 different secondary KRAS mutations. Y96D and Y96S were resistant to both inhibitors. A combination of novel SOS1 inhibitor, BI-3406, and trametinib had potent activity against this resistance. Although G13D, R68M, A59S and A59T, which were highly resistant to sotorasib, remained sensitive to adagrasib, Q99L was resistant to adagrasib but sensitive to sotorasib. Conclusions We identified many secondary KRAS mutations causing resistance to sotorasib, adagrasib, or both, in vitro. The differential activities of these two inhibitors depending on the secondary mutations suggest sequential use in some cases. In addition, switching to BI-3406 plus trametinib might be a useful strategy to overcome acquired resistance owing to the secondary Y96D and Y96S mutations.

A high-throughput screening campaign helped us to identify an initial lead compound (1) as a protein kinase C-θ (PKCθ) inhibitor. Using the docking model of compound 1 bound to PKCθ as a model, structure-based drug design was employed and two regions were identified that could be explored for further optimization, i.e., (a) a hydrophilic region around Thr442, unique to PKC family, in the inner part of the hinge region, and (b) a lipophilic region at the forefront of the ethyl moiety. Optimization of the hinge binder led us to find 1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one as a potent and selective hinge binder, which resulted in the discovery of compound 5. Filling the lipophilic region with a suitable lipophilic substituent boosted PKCθ inhibitory activity and led to the identification of compound 10. The co-crystal structure of compound 10 bound to PKCθ confirmed that both the hydrophilic and lipophilic regions were fully utilized. Further optimization of compound 10 led us to compound 14, which demonstrated an improved pharmacokinetic profile and inhibition of IL-2 production in a mouse.

The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells. Somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, the BRAF inhibitors vemurafenib and dabrafenib have emerged as promising agents for the treatment of melanoma patients with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild-type BRAF (including NRAS-mutant melanoma) are clinical concerns. Here, we demonstrate that the selective pan-RAF inhibitor TAK-632 suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. Our analysis using RNAi and TAK-632 in preclinical models reveals that the MAPK pathway of NRAS-mutated melanoma cells is highly dependent on RAF. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. As a result, TAK-632 demonstrates potent antiproliferative effects both on NRAS-mutated melanoma cells and BRAF-mutated melanoma cells with acquired resistance to BRAF inhibitors through NRAS mutation or BRAF truncation. Furthermore, we demonstrate that the combination of TAK-632 and the MAPK kinase (MEK) inhibitor TAK-733 exhibits synergistic antiproliferative effects on these cells. Our findings characterize the unique features of TAK-632 as a pan-RAF inhibitor and provide rationale for its further investigation in NRAS-mutated melanoma and a subset of BRAF-mutated melanomas refractory to BRAF inhibitors. Cancer Res; 73(23); 7043–55. ©2013 AACR.

Melanoma is one of the deadliest and most aggressive forms of skin cancer, arising from the malignant transformation of pigment-producing cells, melanocytes. The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells and somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, it has been reported that the BRAF inhibitors vemurafenib and dabrafenib showed high response rates and improved overall survival in melanomas with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild-type BRAF (including NRAS mutant melanoma) are clinical concerns. In this preclinical study, we describe the biological characterization of TAK-632 as a potent and selective pan-RAF inhibitor that suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. We used both genetic and chemical approaches to investigate the dependence of NRAS-mutated melanoma and BRAF inhibitor-resistant BRAF mutant melanoma cells on RAF. Our analysis reveals that the MAPK pathway and proliferation of these cells is highly dependent on RAF. Such dependence was not observed in several RAS/RAF-wild type and KRAS-mutated cells. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. Furthermore, we demonstrate that the combination of TAK-632 and various MAPK kinase (MEK) inhibitors exhibits synergistic anti-proliferative effects on these cells. Our findings indicate that TAK-632 has favorable characteristics in terms of suppressing the proliferation of NRAS mutant melanoma and BRAF inhibitor-resistant BRAF mutant melanoma cells. Citation Format: Akito Nakamura, Takeo Arita, Shuntarou Tsuchiya, Jill Donelan, Jouhara Chouitar, Elizabeth Carideo, Katherine Galvin, Masanori Okaniwa, Tomoyasu Ishikawa, Sei Yoshida. Characterization of the selective pan-RAF inhibitor TAK-632 with antitumor activity in BRAF inhibitor-resistant melanoma. [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 4247. doi:10.1158/1538-7445.AM2014-4247

Background: RAS mutant melanoma and colorectal cancer represent areas of significant unmet medical need. MLN2480 is an investigational class II RAF kinase inhibitor and TAK-733 is an investigational allosteric MEK kinase inhibitor; each of which is the subject of a single agent phase I clinical trial. The present studies have characterized the combination activity of these agents in BRAF mutant and RAS mutant preclinical models of melanoma and colorectal cancer. Methods: Combination effects of MLN2480 and TAK-733 on cell viability were studied using an ATP-based cell viability assay across a panel of BRAF and RAS mutant melanoma and CRC cell lines. Western blot analysis was used to compare effects on MAPK pathway signaling and response markers in cell lines showing a range of sensitivity to this combination. Pharmacodynamic responses and growth inhibitory effects of the combination were studied in xenografts of the same cell lines, as well as in primary human tumor xenografts, of RAS mutant melanoma and CRC. Results: MLN2480 inhibits MAPK pathway signaling in BRAF mutant and some RAS mutant preclinical cancer models at concentrations that are tolerated in vivo. MLN2480 is most potent in BRAF mutant melanoma models but also has single agent activity in some RAS mutant models. The combination of MLN2480 with TAK-733 inhibits the growth of a broader range of RAS mutant tumor models than single agent MLN2480, including primary human tumor xenograft models of melanoma and CRC. In vitro analysis of this drug combination in cell proliferation assays demonstrates synergistic activity. Western blot analysis demonstrated the effect of MLN2480 in reversing feedback activation of MEK in response to TAK-733, leading to more concerted MAPK pathway inhibition. Conclusions: The activity of the RAF kinase inhibitor MLN2480 in preclinical models of BRAF and RAS mutant melanoma and CRC provides a rationale for clinical testing. The combination of MLN2480 with the MEK inhibitor TAK-733 represents an additional strategy for clinical research within these tumor types. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C146. Citation Format: Elizabeth Grace Carideo Cunniff, Julie Zhang, Jouhara Chouitar, Jerome Mettetal, Kazuhide Nakamura, Takeo Arita, Akito Nakamura, Masanori Okaniwa, Tomoyasu Ishikawa, Sei Yoshida, Robyn Fabrey, Patrick Vincent, Kurt Eng, Khristofer Garcia, Deanna Borelli, Teena Vagrhese, Steve Stroud, Saurabh Menon, Mike Kuranda, Katherine Galvin. Combination treatment with the investigational RAF kinase inhibitor MLN2480 and the investigational MEK kinase inhibitor TAK-733 inhibits the growth of BRAF mutant and RAS mutant preclinical models of melanoma and CRC. [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 C146.

The RAF family kinases play critical roles in cancer progression. Recently, BRAF selective inhibitors have shown significant clinical efficacy in melanoma patients bearing oncogenic BRAFV600E mutation. However, several studies reported that RAF inhibitors instinctively transactivate RAF homodimers (CRAF-CRAF) or heterodimers (CRAF-BRAF(wt)) and activate RAS dependent MAPK signaling. Along with this mechanism, it has been reported that selective BRAF inhibitors have not shown potent anti-proliferative activity against cancer cell lines such as NRAS mutant melanoma in which RAS dependent MAPK signaling is activated (Hong Yang et al., Cancer Res., 2010, 70, 5518-5527). However, our initial investigation using fibroblast CsFb (BRAFwt) cells indicated that phosphorylation of MEK and ERK was inhibited by some DFG-out inhibitors, but not by DFG-in inhibitors. These results led to the hypothesis: continuous inhibition of pan-RAF (BRAF and CRAF) with DFG-out type inhibitors could suppress the feedback activation. Here we report the discovery and characterization of pan-RAF inhibitor TAK-632. We designed novel 1,3-benzothiazole class derivatives using knowledge of structure-activity relationships gained from studies of our thiazolo[5,4-b]pyridine class RAF/VEGFR2 inhibitor (Masanori Okaniwa et al., J. Med. Chem., 2012, 55, 3452-3478). To enrich RAF kinase selectivity vs. VEGFR2, we utilized the cocrystal structures of our lead compound with both BRAF and VEGFR2. Eventually, we designed and selected 7-cyano derivative TAK-632 as a development candidate. Cocrystal structure analysis of BRAF bearing TAK-632 revealed that accommodation of the 7-cyano group into the BRAF-selectivity pocket and the 3-(trifluoromethyl)phenyl acetamide moiety into the hydrophobic back pocket of BRAF in the DFG-out conformation contributed to enhanced RAF inhibition and selectivity vs. VEGFR2. Reflecting its potent pan-RAF inhibition (IC50: BRAFV600E 2.4 nM, CRAF 1.4 nM) and slow dissociation (koff) profile measured by surface plasmon resonance (SPR) spectroscopy, TAK-632 demonstrated significant cellular activity against mutated BRAF or mutated NRAS cancer cell lines. Furthermore, in both A375 (BRAFV600E) and HMVII (NRASQ61K) xenograft models in rats, TAK-632 demonstrated regressive antitumor activity by twice daily, 14-day repetitive administration without significant body weight loss. In conclusion, these results raise the possibility of using slow off-rate pan-RAF inhibitors such as TAK-632 for the treatment of human cancers harboring either BRAFV600E or NRAS mutant. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C255. Citation Format: Masanori Okaniwa, Masaaki Hirose, Takeo Arita, Masato Yabuki, Akito Nakamura, Terufumi Takagi, Tomohiro Kawamoto, Noriko Uchiyama, Akihiko Sumita, Shunichirou Tsutsumi, Tsuneaki Tottori, Yoshitaka Inui, Bi-Ching Sang, Jason Yano, Kathleen Aertgeerts, Sei Yoshida, Tomoyasu Ishikawa. Discovery of TAK-632: A selective kinase inhibitor of pan-RAF with potent antitumor activity against BRAF and NRAS mutant melanomas. [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 C255.