The identification of mutationally activated BRAF in many cancers altered our conception of the role played by the RAF family of protein kinases in oncogenesis. In this review we describe the development of BRAF inhibitors and the results that have emerged from their analysis in both the laboratory and the clinic. We discuss the spectrum of RAF mutations in human cancer and the complex interplay between tissue of origin and response to RAF inhibition. Finally, we enumerate mechanisms of resistance to BRAF inhibition that have been characterized and postulate how strategies of RAF pathway inhibition may be extended in scope to benefit, not only the thousands of patients diagnosed annually with BRAF-mutated metastatic melanoma, but also the larger patient population with malignancies harboring mutationally activated RAF genes that is ineffectively treated with the current generation of BRAF kinase inhibitors.

Malignant melanoma is frequently driven by mutational activation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) accompanied by silencing of the phosphatase and tensin homology (PTEN) tumor suppressor. Despite the implied importance of PI3K signaling in PTEN Null melanomas, mutational activation of the gene encoding the catalytic subunit of PI3Kα (PIK3CA), is rarely detected. Since PTEN has both PI3-lipid phosphatase-dependent and -independent tumor suppressor activities, we investigated the contribution of PI3K signaling to BRAF V600E -induced melanomagenesis using mouse models, cultured melanoma cells, and PI3K pathway-targeted inhibitors. These experiments revealed that mutationally activated PIK3CA H1047R cooperates with BRAF V600E for melanomagenesis in mice. Moreover, pharmacological inhibition of PI3Ks prevented growth of BRAF V600E /PTEN Null melanomas in vivo and in tissue culture. Combined inhibition of BRAF V600E and PI3K had more potent effects on the regression of established BRAF V600E /PTEN Null melanomas and cultured melanoma cells than individual blockade of either pathway. Surprisingly, growth of BRAF V600E /PIK3CA H1047R melanomas was dependent on the protein kinase AKT; however, AKT inhibition had no effect on growth of BRAF V600E /PTEN Null melanomas. These data indicate that PTEN silencing contributes a PI3K-dependent, but AKT-independent, function in melanomagenesis. Our findings enhance our knowledge of how BRAF V600E and PI3K signaling cooperate in melanomagenesis and provide preclinical validation for combined pathway-targeted inhibition of PI3K and BRAF V600E in the therapeutic management of BRAF V600E /PTEN Null melanomas.

Phosphatidylinositide 3′ (PI3′)-lipid signaling cooperates with oncogenic BRAFV600E to promote melanomagenesis. Sustained PI3′-lipid production commonly occurs via silencing of the PI3′-lipid phosphatase PTEN or, less commonly, through mutational activation of PIK3CA, encoding the 110kDa catalytic subunit of PI3′-kinase-α (PI3Kα). To define the PI3K catalytic isoform dependency of BRAF-mutated melanoma, we utilized pharmacologic, isoform-selective PI3K inhibitors in conjunction with melanoma-derived cell lines and genetically engineered mouse (GEM) models. While BRAFV600E/PIK3CAH1047R melanomas were sensitive to the anti-proliferative effects of selective PI3Kα blockade, inhibition of BRAFV600E/PTENNull melanoma proliferation required combined blockade of PI3Kα, δ and γ, and was insensitive to PI3Kβ blockade. In GEM models, isoform-selective PI3K inhibition elicited cytostatic effects, but significantly potentiated melanoma regression in response to BRAFV600E pathway-targeted inhibition. Interestingly, PI3K inhibition forestalled the onset of MEK inhibitor resistance in two independent GEM models of BRAFV600E-driven melanoma. These results suggest that combination therapy with PI3K inhibitors may be a useful strategy to extend the duration of clinical response of BRAF-mutated melanoma patients to BRAFV600E pathway-targeted therapies. (Words: 165)

SUMMARY Malignant conversion of BRAF- or NRAS-mutated melanocytes into melanoma cells can be promoted by PI3’-lipid signaling. Using human NRAS- or BRAF-mutated melanoma cells that co-express mutationally activated PIK3CA, we explored the contribution of PI3′-lipid signaling to cell proliferation. Despite mutational activation of PIK3CA, melanoma cells were more sensitive to the biochemical and anti-proliferative effects of broader spectrum PI3K inhibitors than to an α-selective PI3K inhibitor. Combined pharmacological inhibition of MEK1/2 and PI3K signaling elicited more potent anti-proliferative effects and greater inhibition of the cell division cycle compared to single-agent inhibition of either pathway alone. Analysis of signaling downstream of MEK1/2 or PI3K revealed that these pathways cooperate to regulate cell proliferation through mTORC1-mediated effects on ribosomal protein S6 and 4E-BP1 phosphorylation in an AKT-dependent manner. Although PI3K inhibition resulted in cytostatic effects on xenografted NRASQ61H/PIK3CAH1047R melanoma, combined inhibition of MEK1/2 plus PI3K elicited significant melanoma regression. This study provides insights as to how mutationally activated PIK3CA acts in concert with MEK1/2 signaling to cooperatively regulate mTORC1/2 to sustain PIK3CA-mutated melanoma proliferation.

the naked mole-rat is a subterranean rodent, approximately the size of a mouse, renowned for its exceptional longevity (>30 years) and remarkable resistance to cancer. To explore putative mechanisms underlying the cancer resistance of the naked mole-rat, we investigated the regulation and function of the most commonly mutated tumor suppressor, TP53, in the naked mole-rat. We found that the p53 protein in naked mole-rat embryonic fibroblasts (NEFs) exhibits a half-life more than ten times in excess of the protein's characterized half-life in mouse and human embryonic fibroblasts. We determined that the long half-life of the naked mole-rat p53 protein reflects protein-extrinsic regulation. Relative to mouse and human p53, a larger proportion of naked mole-rat p53 protein is constitutively localized in the nucleus prior to DNA damage. Nevertheless, DNA damage is sufficient to induce activation of canonical p53 target genes in NEFs. Despite the uniquely long half-life and unprecedented basal nuclear localization of p53 in NEFs, naked mole-rat p53 retains its canonical tumor suppressive activity. Together, these findings suggest that the unique stabilization and regulation of the p53 protein may contribute to the naked mole-rat's remarkable resistance to cancer. The naked mole-rat (NMR)is a subterranean mammal residing in the arid and semi-arid grasslands of tropical, northeastern sub-Saharan Africa. Naked mole-rats live in eusocial colonies comprising one breeding female, several breeding males, and a phalanx of subordinate non-breeding workers 1. They exhibit an exceptionally long lifespan, known to be in excess of 30 years, and show no age-associated exponential increase in risk of dying 2. This observed maximum lifespan is about five times longer in duration than that predicted by their body mass 3. The extraordinary longevity of the NMR may reflect, at least in part, their extremely low incidence of cancer 4. In contrast to mice, where more than 55% of mice die with cancerous lesions identified upon necropsy 5,6 , only a handful of cancer cases have been documented in >2500 necropsies in captive NMRs 7,8. While long-considered impervious to cancer, recent case reports of carcinogenesis in a few laboratory-housed NMRs do not diminish the fact that NMRs appear to employ remarkable anti-cancer mechanisms that merit further study. Previously, NMR skin fibroblasts cultured in vitro have been shown to display unusual sensitivity to contact inhibition, mediated by induction of p16 INK4A and triggered by secretion of high molecular mass hyaluronan 9,10. Additional studies have demonstrated that the cocktail of large T antigen and oncogenic HRAS G12V , a genetic combination that potently induces transformation of mouse and human skin fibroblasts, fails to induce transformation of NMR skin fibroblasts upon implantation in an immunodeficient mouse 11. Phylogenetic analysis reveals that the NMR genome harbors 17 copies of the phosphatase and tensin (PTEN) pseudogene, highlighting the possibility of exceptional regulation of ...

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Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with greater than 50% of tumors expressing the BRAF(V600E) oncoprotein. BRAF(V600E) is a constitutively active protein kinase that promotes sustained activation of BRAF→MEK→ERK signaling in the melanoma cell. Importantly, the marked tumor regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF(V600E) in melanoma maintenance. However, since most patients relapse with lethal vemurafenib resistant disease, understanding and preventing mechanism(s) of drug resistance will be critical to extending the durability of patients' responses. Although mutational activation of BRAF is often the initiating event, progression of BRAF mutated melanomas is frequently associated with silencing of the PTEN tumor suppressor, a negative regulator of PI3′-kinase signaling. Whilst PTEN silencing is common in human melanoma, mutational activation of PIK3CA (encoding the alpha catalytic subunit of PI3′-kinase) is extremely rare, even though PIK3CA mutations are common in other human cancers. Since PTEN is reported to have tumor suppressor functions independent of its PI3′-lipid phosphatase activity, PTEN silencing may have more profound tumor promoting activity than mutational activation of PIK3CA. To test this, we generated mice carrying a conditional knock-in allele of Pik3ca that permits Cre-induced expression of PIK3CA(H1047R), a mutationally activated form of PIK3CA detected in many cancer types. Expression of PIK3CA(H0147R) in melanocytes elicited no obvious phenotype, consistent with the absence of phenotype in PTEN(Null) melanocytes. As observed previously, melanocyte-specific expression of BRAF(V600E) combined with PTEN silencing led to metastatic melanoma. Interestingly, metastatic melanoma also arose from melanocytes expressing both BRAF(V600E) and PIK3CA(H1047R), although these tumors grew more slowly than their BRAF(V600E)/PTEN(Null) counterparts. Both BRAF(V600E)/PTEN(Null) and BRAF(V600E)/PIK3CA(H1047R) melanomas were sensitive to the tumor prevention effects of pharmacological blockade of class 1 PI3′-kinases. However, the clinically relevant subset of BRAF(V600E)/PTEN(Null) melanoma was resistant to pharmacological blockade of AKT. Using genetically engineered mouse models and melanoma-derived cell lines, we demonstrate cooperative anti-tumor effects of combining RAF and PI3′-kinase inhibitors, suggesting that combined pathway-targeted chemotherapy may maximize melanoma patients' therapeutic response while delaying the onset of lethal drug resistant disease. Using patient derived xenografts propagated in immunocompromised mice we explored the cause and consequences of drug resistance, which was selected by continuous vemurafenib administration. As observed previously, resistant tumors displayed continued dependence on BRAF(V600E)→MEK→ERK signaling due to elevated BRAF(V600E) expression. However, remarkably, we observed that vemurafenib-resistant melanomas displayed a striking drug dependency for their continued proliferation, such that cessation of drug administration led to regression of established drug-resistant tumors. We further demonstrated that a discontinuous dosing strategy, which exploits the fitness deficit displayed by drug-resistant cells in the absence of the vemurafenib, forestalls the onset of lethal drug resistant disease. These data highlight the concept that drug-resistant cells may also show dependency, such that altered drug dosing may prevent the emergence of lethal drug resistance. Citation Format: Martin McMahon, Meghna Das Thakur, Victoria Marsh, Jillian Silva, Allison Landman, Marian Deuker, Fernando Salangsang, Nancy Pryer, Wayne Phillips, Mitchell Levesque, Reinhard Dummer, Marcus Bosenberg, William R. Sellers, Darrin Stuart. Targeting BRAF and PI3′-kinase signaling for therapy of melanoma. [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 SY17-03. doi:10.1158/1538-7445.AM2013-SY17-03