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

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

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.

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.

KINOMEScan assay results for RAF709

KiNative assay results for RAF709

[Image: see text] The selective inhibition of RET kinase as a treatment for relevant cancer types including lung adenocarcinoma has garnered considerable interest in recent years and prompted a variety of efforts toward the discovery of small-molecule therapeutics. Hits uncovered via the analysis of archival kinase data ultimately led to the identification of a promising pyrrolo[2,3-d]pyrimidine scaffold. The optimization of this pyrrolo[2,3-d]pyrimidine core resulted in compound 1, which demonstrated potent in vitro RET kinase inhibition and robust in vivo efficacy in RET-driven tumor xenografts upon multiday dosing in mice. The administration of 1 was well-tolerated at established efficacious doses (10 and 30 mg/kg, po, qd), and plasma exposure levels indicated a minimal risk of KDR or hERG inhibition in vivo, as evaluated by Miles assay and free plasma concentrations, respectively.

RET (REarranged during Transfection) kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarcinoma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent optimization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against KDR (Kinase insert Domain Receptor) in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, however, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and indicative of the narrow therapeutic windows seen with this scaffold.

The mitogen-activated protein kinase (MAPK) signaling pathway is frequently activated in human cancers due to genetic alterations that can occur at multiple nodes, the most prevalent of which are mutations in RAS or BRAF. While BRAFV600 mutant tumors are responsive to RAF inhibitors such as dabrafenib and vemurafenib, these drugs are ineffective in RAS mutant cancers and tumors expressing other RAF mutations. CRAF kinase functions as a critical effector in mutant RAS and Class II/III BRAF mutant tumors and plays a role in feedback-mediated pathway reactivation following MEK inhibition. Thus, selective inhibitors that potently inhibit the activity of CRAF could be both effective in blocking mutant RAS and BRAF signaling and in inhibiting feedback-mediated activation in combination with a MEK inhibitor. LXH254 is a type II ATP-competitive inhibitor that inhibits both B- and CRAF kinase activities at picomolar concentrations with a high degree of selectivity against a panel of 456 human kinases and in cell-based assays. LXH254 not only inhibits MAPK signaling activity in tumor models harboring BRAFV600 mutation, but also inhibits mutant N- and KRAS-driven signaling due to its ability to inhibit both RAF monomers and dimers with similar potencies. LXH254 is orally bioavailable, demonstrates a direct PK/PD relationship and causes tumor regression in multiple cell line and primary human tumor derived xenograft models at well-tolerated doses. LXH254 represents a next generation RAF inhibitor that is differentiated from other RAF inhibitors in this class due to the high degree of selectivity. In preclinical efficacy and toxicology studies, LXH254 demonstrated a relatively wide therapeutic index which should enable effective interrogation of RAF inhibition in patients with decreased risk for off-target toxicity. LXH254 is currently in a Phase I trial in patients with solid tumors expressing MAPK pathway mutations. Citation Format: Darrin D. Stuart, Wenlin Shao, Yuji Mishina, Yun Feng, Giordano Caponigro, Vesselina G. Cooke, Stacey Rivera, Fang Shen, Joshua Korn, Lesley A. Mathews Griner, Giselle Nishiguchi, Benjamin Taft, Lifeng Wan, Sharadha Subramanian, Yan Lou, Lina Setti, Matthew Burger, Victor Tamez, Alice Rico, Robert Aversa, John Tellew, Jacob R. Haling, Valery Polyakov, Amy Lambert, Richard Zang, Ann Van Abbema, Mohamad Hekmat-Nejad, Payman Amiri, Mallika Singh, Nicholas Keen, Michael P. Dillon, Emma Lees, William R. Sellers, Savithri Ramurthy. Pharmacological profile and anti-tumor properties of LXH254, a highly selective RAF kinase inhibitor [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 DDT01-04.

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.

RAS oncogenes have been implicated in30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

The mitogen-activated protein kinase (MAPK) signaling pathway is frequently activated in human cancers due to genetic alterations that can occur at multiple nodes in the pathway, the most prevalent of which are mutations in RAS or BRAF. While BRAFV600 mutant tumors are effectively treated with existing RAF inhibitors, RAS mutant cancers and tumors expressing atypical BRAF mutants remain an unmet medical need. Emerging biology has demonstrated that the CRAF kinase functions as a critical mediator of mutant KRAS-driven cell proliferation and tumor development. CRAF was also shown to be the mediator of feedback-mediated pathway reactivation following MEK inhibitor treatment in KRAS mutant cancers. Hence selective inhibitors that potently inhibit the activity of CRAF could be both effective in blocking mutant RAS-driven tumorigenesis and in alleviating feedback activation. We have developed a type II ATP-competitive inhibitor that inhibits both B- and CRAF kinase activities at picomolar IC50 values in biochemical assays with high selectivity profile against a panel of 456 human kinases. The inhibitor not only inhibits MAPK signaling activity in tumor models harboring BRAFV600 mutation, but also inhibits mutant N- and KRAS-driven signaling with minimum paradoxical activation, likely due to its activity in inhibiting both RAF monomers and dimers with similar potencies. Correspondingly, profiling data of the inhibitor in a panel of 480 human cancer cell lines shows that it has higher antitumor activities in cell lines harboring BRAF or RAS mutations as compared to those that are wild-type. The inhibitor is orally bioavailable, it demonstrates a direct PK/PD relationship and causes tumor regression in multiple cell line and primary human tumor derived xenograft models that have BRAF, NRAS or KRAS mutations with good tolerability. Thus, we have developed a next generation RAF inhibitor with unique biochemical and cellular properties that enables its antitumor activities in RAS mutant tumors. Citation Format: Wenlin Shao, Yuji Mishina, Yun Feng, Giordano Caponigro, Savithri Ramurthy, Vesselina Cooke, Lesley Griner, Gisele Nishiguchi, Alice Rico, Ben Taft, Matthew Burger, Huw Tanner, Valery Polyakov, Brent Appleton, John Tellew, Richard Zang, Mohammad Hekmat-Nejad, Payman Amiri, Mallika Singh, Darrin Stuart. Development of a highly selective B/CRAF kinase inhibitor that exhibits antitumor activities in RAS and BRAF mutant tumors with minimal paradoxical activation. [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 330.

The biological functions of the aryl hydrocarbon receptor (AHR) can be delineated into dioxin response element (DRE)-dependent or -independent activities. Ligands exhibiting either full or partial agonist activity, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin and α-naphthoflavone, have been demonstrated to potentiate both DRE-dependent and -independent AHR function. In contrast, the recently identified selective AHR modulators (SAhRMs), e.g., 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360), bias AHR toward DRE-independent functionality while displaying antagonism with regard to ligand-induced DRE-dependent transcription. Recent studies have expanded the physiological role of AHR to include modulation of hematopoietic progenitor expansion and immunoregulation. It remains to be established whether such physiological roles are mediated through DRE-dependent or -independent pathways. Here, we present evidence for a third class of AHR ligand, “pure” or complete antagonists with the capacity to suppress both DRE-dependent and -independent AHR functions, which may facilitate dissection of physiological AHR function with regard to DRE or non-DRE-mediated signaling. Competitive ligand binding assays together with in silico modeling identify N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351) as a high-affinity AHR ligand. DRE-dependent reporter assays, in conjunction with quantitative polymerase chain reaction analysis of AHR targets, reveal GNF351 as a potent AHR antagonist that demonstrates efficacy in the nanomolar range. Furthermore, unlike many currently used AHR antagonists, e.g., α-naphthoflavone, GNF351 is devoid of partial agonist potential. It is noteworthy that in a model of AHR-mediated DRE-independent function, i.e., suppression of cytokine-induced acute-phase gene expression, GNF351 has the capacity to antagonize agonist and SAhRM-mediated suppression of SAA1. Such data indicate that GNF351 is a pure antagonist with the capacity to inhibit both DRE-dependent and -independent activity.

Selective RAF inhibitors have significant activity in patients with metastatic melanoma whose tumors express BRAFV600E. However, not all patients respond equally well to treatment and the duration of response is often limited to less than 6 months. LGX818 was developed with the hypothesis that a more potent inhibitor with excellent pharmacological properties would maximize the degree and duration of patient response. LGX818 is a highly potent RAF inhibitor with selective anti-proliferative and apoptotic activity in cells expressing BRAFV600E. In the A375 (BRAFV600E) human melanoma cell line LGX818 suppresses phospho-ERK (EC50 = 3 nM) leading to potent inhibition of proliferation (EC50 = 4 nM). No significant activity was observed against a panel of 100 kinases (IC50 > 900 nM) and LGX818 did not inhibit proliferation of > 400 cell lines expressing wild-type BRAF. Contributing to the high potency of LGX818 is the extremely slow off-rate from BRAFV600E which is not observed with other RAF inhibitors. In biochemical assays the dissociation half-life was >24 hours which translated into sustained target inhibition in cells following drug wash-out. Single dose PK/PD studies in human melanoma xenograft models (BRAFV600E) indicated that LGX818 treatment at oral doses as low as 6 mg/kg resulted in strong (75%) and sustained (>24 hours) decrease in phospho-MEK, even following clearance of drug from circulation. Decreases in phospho-ERK were consistent with phospho-MEK but markers of downstream transcriptional output (DUSP6 and SPRY4) appeared to provide a more sensitive measure of pathway activation. LGX818 induced tumor regression in multiple BRAF mutant human tumor xenograft models grown in immune compromised mice and rats at doses as low as 1 mg/kg. Consistent with the in vitro data, LGX818 was inactive against BRAF wild-type tumors at doses up to 300 mg/kg bid, with good tolerability and linear increase in exposure. Efficacy was also achieved in a more disease-relevant spontaneous metastatic melanoma and a model of melanoma brain metastasis. LGX818 is a potent and selective RAF kinase inhibitor with unique biochemical properties that contribute to an excellent pharmacological profile. A Phase I clinical trial in patients with BRAF mutant tumors is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3790. doi:1538-7445.AM2012-3790

Genetic alterations in the Ras/Raf/MEK/ERK pathway are among the most common in human cancers. Up to 70% of melanomas harbor B-Raf mutations, and roughly 90% of pancreatic tumors have K-Ras mutations. To address these Raf pathway-driven cancers, small molecule Raf kinase inhibitors have been developed and are currently under clinical investigation. In B-RafV600E cells, Raf compounds inhibit signaling through MEK and ERK, resulting in the expected anti-proliferative effects. Paradoxically, in wild-type Raf cells and in mutant Ras cells, these compounds induce downstream signaling and can induce cell growth in some settings in vitro. While the induction of downstream signaling has previously been attributed to published Raf pathway feedback loops, this has not been proven directly. In fact, we show here that induction of pMEK and pERK can occur within minutes of Raf compound treatment, even before reported feedback phosphorylation events are seen on B-Raf and C-Raf. Interestingly, the induction of signaling and cell growth both occur in a biphasic pattern, with low compound concentrations (0.01-0.1 uM) causing maximal induction, and higher compound concentrations (1-10 uM) causing less profound induction. Such a biphasic pattern is also observed in biochemical assays with purified wild-type B-Raf or C-Raf. The biphasic pattern is suggestive of a mechanism involving the interaction of two signaling subunits. In addition, recent literature data (Rajakulendran, Nature, 461:542-6) has demonstrated that Raf dimerization can upregulate pMEK, not through trans-phosphorylation of Raf molecules but presumably by conformational activation of the kinase. Consistent with that model, we show that Raf compound treatment induces B/C-Raf dimer formation in cells. In addition, knockdown of A-, B- or C-Raf with siRNA does not abrogate the Raf compound induction of pMEK and pERK, suggesting that induction might be mediated by Raf homo- as well as hetero-dimerization. Notably, knockdown of K-Ras in K-RasMUT cells also does not abolish the induction, implying that this effect is not mediated by Ras. Taken together, these data suggest a model in which compound binding to one Raf molecule induces dimerization and conformational activation of a partner Raf molecule in the dimer. These observations can explain why wild-type Raf and mutant Ras tumors are insensitive to selective Raf kinase inhibitors and might also have important implications for toxicity, since induction of strong mitogenic signaling could lead to hyperproliferation of normal tissues. Understanding the Raf compound induction mechanism may lead not only to the design of improved inhibitors, but also to methods for overcoming the induction seen with current development compounds. Toward that end, we show that combining a MEK inhibitor with a Raf compound causes inhibition of both pERK and cell growth and may therefore have significant advantages in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 20.

ATP competitive inhibitors of the BRAF(V600E) oncogene paradoxically activate downstream signaling in cells bearing wild-type BRAF (BRAF(WT)). In this study, we investigate the biochemical mechanism of wild-type RAF (RAF(WT)) activation by multiple catalytic inhibitors using kinetic analysis of purified BRAF(V600E) and RAF(WT) enzymes. We show that activation of RAF(WT) is ATP dependent and directly linked to RAF kinase activity. These data support a mechanism involving inhibitory autophosphorylation of RAF's phosphate-binding loop that, when disrupted either through pharmacologic or genetic alterations, results in activation of RAF and the mitogen-activated protein kinase (MAPK) pathway. This mechanism accounts not only for compound-mediated activation of the MAPK pathway in BRAF(WT) cells but also offers a biochemical mechanism for BRAF oncogenesis.