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PI3Kα is the most mutated oncogene with a high mutation rate in breast, lung, colorectal, gastric, bladder, and other tumor types. However, current inhibitors have had little efficacy in the clinic due to their inability to achieve maximal PI3Kα target engagement in a highly specific and durable manner. Contributing to this is that most PI3Kα mutant cancer cells are prone to robust pathway feedback and re-activation of remaining wild type PI3Kα signaling upon initial inhibition. We have analyzed pathway feedback in a variety of PI3Kα mutant cancer cell lines across multiple diverse indications and found this to be a common trait among PI3Kα mutant cancer cells. Additionally, when the mutant PI3Kα protein alone is targeted in these cells using mutant-specific inhibitors, we observed robust pathway re-activation and tumor cell proliferation. In contrast, TOS-358, a highly selective first-in-class covalent PI3Kα inhibitor, achieved complete inhibition of both PI3Kα mutant and wild type signaling and this correlated with the induction of cell cycle arrest and cell death across a wide variety of PI3Kα mutant tumor types. Furthermore, stimulation of PI3Kα signaling with serum rapidly re-activated the PI3K-AKT pathway in the presence of reversible and mutant specific inhibitors. Covalent molecules (namely TOS-358) achieved complete inhibition of pathway signaling in this format. Overall, this reveals the need to achieve sustained inhibition of both PI3Kα mutant and wild type signaling to achieve durable efficacy in PI3Kα mutant tumors. Citation Format: John R. MacDougall, Mengqi Zhong, Hanne Merritt, Joselyn S. Del Cid, John Bradley, Raymond Mak, Neil Dhawan, Wei Chen. Inhibition of wild-type PI3Kα signaling is required for durable efficacy in PI3Kα mutant cancer cells due to robust re-activation of wild-type PI3Kα signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4945.

PI3 kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. The PI3 Kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe how the potency of a benzothiazole fragment hit was quickly improved based on structural information and how this early chemotype was further optimized through scaffold hopping. This effort led to the identification of a series of 2-acetamido-5-heteroaryl imidazopyridines showing potent in vitro activity against all class I PI3Ks and attractive pharmacokinetic properties.

The MAPK signaling cascade, comprised of the RAS GTPases, the RAF, MEK1/2 and ERK1/2 kinases is frequently deregulated in cancer. ERK1 and ERK2 transmit signals generated by mutant BRAF, Ras or by activated receptor tyrosine kinases to a wide range of nuclear and cytoplasmic substrates, resulting in signal amplification, cell growth, migration and survival. ERK1 and ERK2 have been considered as redundant because of their high homology, large number of overlapping substrates, and ability to substitute for each other in genetically engineered mouse models. Nevertheless, several investigators have identified non-redundant roles for ERK isoforms in oncogenesis; for instance, ERK2, but not ERK1, appears to be responsible for RASmut induced epithelial-to-mesenchymal transformation. Besides, each of the ERK isoforms employs spatially distinct substrate docking domains, DEF (docking site for ERK FXFP) and D (docking domain), to signal to different subsets of substrates and differentially transmit signals downstream. We set out to determine the roles of ERK isoforms as well as DEF- and D-domain dependent signaling in the survival of melanoma tumor cells expressing activating BRAF mutations which are highly sensitive to pharmacological inhibitors of RAF, MEK1/2 and ERK1/2. We designed ERK1 and ERK2 mutants resistant to ATP-competitive ERK1/2 inhibitors and employed auto-activating, MEK-independent, ERK1 and ERK2 mutants to ask if BRAFmut melanoma survival is dependent on either or both ERK isoforms. In addition, we used RNAi and zinc-finger nucleases’ to knockdown or delete each of ERK isoforms. These experimental approaches consistently demonstrated that ERK2, but not ERK1, was the sole driver of cell survival in multiple BRAFmut melanoma cell lines. Moreover, genome-wide gene expression analysis indicated that ERK2, but not ERK1, was largely responsible for transcriptional effects imposed by pharmacological RAF, MEK1/2 or ERK1/2 inhibitors. Thus, in BRAFmut melanoma, functions of ERK1 and 2 are not redundant, and ERK1 cannot substitute for a disabled ERK2. Next, we introduced DEF- and D-substrate docking domain mutations into an ERK inhibitor resistant ERK2 to investigate whether signaling through either domain is sufficient to support melanoma survival. We observed that signaling through D- or DEF- domains of ERK2 had differential effects on gene expression and substrate phosphorylation. Consequently, we have found that a subset of melanoma cell lines was sensitive to elimination of DEF- docking domain interactions, whereas another subset of cell lines tolerated mutations in the DEF-site. Interactions and signaling through ERK D-docking site were dispensable for survival of all melanoma cell lines tested. These data suggest potential novel approaches to target oncogenic MAPK pathway. Citation Format: Tatiana Zavorotinskaya, Upasana Mehra, Yumin Dai, Michel Faure, Ken Crawford, Karen Yu, Jan Marie Cheng, Xiaolei Ma, Jan Xuan, Kelly Yan, Mohammad Hekmat-Nejad, Hanne Merritt, Darrin Stuart, Charles Voliva. Dissecting MAPK pathway in BRAFmut melanoma: Intricacies of ERK1 and ERK2. [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 LB-121. doi:10.1158/1538-7445.AM2014-LB-121

Preclinical studies have demonstrated that BRAF wild-type cancer cells are not only refractory but paradoxically activate the MAPK pathway when treated with RAF inhibitors. Potentially through a related mechanism, multiple point mutations in the activation loop and Phosphate binding loop (P. loop) domains of BRAF have been characterized in human cancers, which often render BRAF catalytically impaired, yet stimulate phosphorylation of downstream targets in cells. However, the underlying biochemistry causing these phenomena has yet to be fully understood. In this study we investigate the biochemical mechanism of wild-type RAF activation in response to RAF inhibitor treatment and identify a novel auto-phosphorylation site within the well conserved BRAF/CRAF P. loop. Disruption of the auto-phosphorylation, either through pharmacologic or genetic alterations, results in activation of the MAPK pathway. This work offers new insight into RAF biochemistry by uncovering a previously unrecognized regulatory mechanism of RAF kinases that is bypassed by the BRAF oncoproteins. Citation Format: Matthew Holderfield, Hanne Merritt, Julie Lin, Zenhai Gao, Darrin Stuart, Frank McCormick. Identification of a RAF inhibitory auto-phosphorylation site. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-54. doi:10.1158/1538-7445.AM2013-LB-54

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.