Marian M. Deuker

Targeting RAF kinases for cancer therapy: BRAF mutated melanoma and beyond

The identification of mutationally activated BRAF in many cancers altered our conception of the part 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 the tissue of origin and the 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 who are diagnosed annually with BRAF-mutated metastatic melanoma but also the larger patient population with malignancies harbouring mutationally activated RAF genes that are ineffectively treated with the current generation of BRAF kinase inhibitors.

Differential AKT dependency displayed by mouse models of BRAFV600E-initiated melanoma

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 PTENNull 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 BRAFV600E-induced melanomagenesis using mouse models, cultured melanoma cells, and PI3K pathway-targeted inhibitors. These experiments revealed that mutationally activated PIK3CAH1047R cooperates with BRAFV600E for melanomagenesis in mice. Moreover, pharmacological inhibition of PI3Ks prevented growth of BRAFV600E/PTENNull melanomas in vivo and in tissue culture. Combined inhibition of BRAFV600E and PI3K had more potent effects on the regression of established BRAFV600E/PTENNull melanomas and cultured melanoma cells than individual blockade of either pathway. Surprisingly, growth of BRAFV600E/PIK3CAH1047R melanomas was dependent on the protein kinase AKT; however, AKT inhibition had no effect on growth of BRAFV600E/PTENNull melanomas. These data indicate that PTEN silencing contributes a PI3K-dependent, but AKT-independent, function in melanomagenesis. Our findings enhance our knowledge of how BRAFV600E and PI3K signaling cooperate in melanomagenesis and provide preclinical validation for combined pathway-targeted inhibition of PI3K and BRAFV600E in the therapeutic management of BRAFV600E/PTENNull melanomas.

Cancer biology: Enzyme meets a surprise target.

An enzyme previously implicated in gene regulation has now been found to have a role in cancer progression, potentiating an intracellular signalling pathway that is driven by a mutated K-Ras protein. See Letter p.283

PIK3CA-mutated melanoma cells rely on cooperative signaling through mTORC1/2 for sustained proliferation

Malignant conversion of BRAF‐ or NRAS‐mutated melanocytes into melanoma cells can be promoted by PI3′‐lipid signaling. However, the mechanism by which PI3′‐lipid signaling cooperates with mutationally activated BRAF or NRAS has not been adequately explored. 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 antiproliferative 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 antiproliferative 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.

Rational targeting of BRAF and PI3-Kinase signaling for melanoma therapy.

ABSTRACT Although mitogen-activated protein kinase (MAPK) inhibitors elicit initial regression of BRAF-mutated melanoma, drug resistance is an inevitable and fatal event. We recently reported that in genetically engineered mouse models of BRAF-mutated melanoma, isoform-selective phosphatidylinositol 3-kinase inhibition cooperates with MAPK pathway inhibition to forestall the onset of MAPK pathway inhibitor resistance.

Abstract 29: MEK and PI3K signaling cooperate through mTORC1/2 to promote PIK3CA mutant melanoma cell proliferation

Oncogenic transformation of mutationally activated BRAF or NRAS melanocytes often requires the cooperation of genetic aberrations in the phosphoinositide 3-kinase (PI3K) pathway to promote melanomagenesis. Activating point mutations in the p110α catalytic subunit of PI3K are detected at a low frequency in both BRAF and NRAS mutant melanomas, yet the principle mechanism of this cooperation remains elusive. Thus, using BRAFV600E/PIK3CAH1047R, NRASQ61H/PIK3CAH1047R, and PIK3CAE545K mutant melanoma cells derived from metastatic melanoma patients treated with pathway-targeted inhibitors, we examined the contribution of mutational activation of PIK3CA to melanoma maintenance and signaling and the consequential response of these PIK3CA mutant cells to α-specific PI3K inhibition. Combined MEK and isoform-selective PI3K inhibition elicited more potent anti-proliferative effects and greater suppression of S-phase progression of the cell cycle compared to single-agent inhibition of either pathway. Analysis of signaling downstream of MEK or PI3K revealed that these pathways cooperated to regulate PIK3CA cell proliferation through mTORC1-mediated effects on ribosomal protein S6 and 4E-BP1 phosphorylation in an AKT-dependent manner. Despite the profound anti-proliferative and biochemical effects of α-specific or PI3Kβ-sparing class I PI3K inhibition in vitro, these agents elicited largely cytostatic effects on PIK3CA xenograft tumors. However, combined inhibition of PI3Kβ-sparing class I PI3K and MEK did significantly cooperate to reduce tumor growth compared to the corresponding monotherapies. Furthermore, this study provides a biochemical mechanism to explain how mTORC1/2 moderates the cooperation of MEK and PI3K signaling for the maintenance of PIK3CA mutant melanoma cell proliferation. Citation Format: Jillian M. Silva, Marian M. Deuker, Bruce C. Baguley, Martin McMahon. MEK and PI3K signaling cooperate through mTORC1/2 to promote PIK3CA mutant melanoma cell proliferation. [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 29.

Abstract B11: PI3-kinase inhibition forestalls the development of drug resistance in BRAFV600E/PTENNull melanoma

The phosphatidylinositol 3′-kinase (PI3K) signaling pathway is frequently dysregulated in BRAF mutated melanomas and serves to promote the growth of tumors initiated by expression of BRAF V600E . The most prevalent mechanism of PI3K pathway activation occurs via silencing expression of the phosphatase and tensin homolog ( PTEN ) tumor suppressor, but mutational activation of PIK3CA , encoding the 110kDa catalytic subunit of PI3□-kinase-α, has also been observed at low frequency. However, the PI3K catalytic isoform dependence of BRAF mutated melanoma remains poorly understood, as does the differential isoform dependence imbued by discrete nodes of PI3K pathway dysregulation. To characterize the PI3K isoform dependence of BRAF V600E /PTEN Null as well as BRAF V600E /PIK3CA H1047R melanoma, we administered a panel of isoform-selective PI3K inhibitors to cultured melanoma cells and to genetically engineered mouse (GEM) models of melanoma of both genotypes. These studies revealed that, while BRAF V600E /PIK3CA H1047R melanoma was sensitive to p110α-selective inhibition, inhibition of BRAF V600E /PTEN Null melanoma cell proliferation required combined blockade of p110α ( PIK3CA ), p110δ ( PIK3CD ) and p110γ ( PIK3CG ). Moreover, in GEM models of BRAF V600E /PIK3CA H1047R or BRAF V600E /PTEN Null melanoma, isoform-selective PI3K inhibition elicited only a cytostatic effect as monotherapy, but enhanced melanoma regression in response to BRAF V600E →MEK→ ERK pathway-targeted inhibition. Most interestingly, treatment with GDC0032, a p110β-sparing PI3K inhibitor, forestalled the onset of MEK inhibitor (GDC-973, cobimetinib) resistant disease in the BRAF V600E /PTEN Null GEM model of melanoma. Together, these data suggest that PIK3CA mutational status may be used to select for melanoma patients that will respond to a p110α-selective inhibitor. By contrast, PTEN Null melanoma patients will require a p110β sparing pan-class I PI3K inhibitor. Moreover, although monotherapy PI3K inhibition did not elicit dramatic tumor regression, blockade of this pathway may be a useful strategy to extend the duration of response to BRAF V600E pathway-targeted therapy. Citation Format: Marian M. Deuker, Victoria Marsh Durban, Wayne Phillips, Martin McMahon. PI3-kinase inhibition forestalls the development of drug resistance in BRAFV600E/PTENNull melanoma. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B11.

Abstract A09: Cooperation of the MAPK and PI3K>AKT>mTORC1 signaling pathways is required for PIK3CA mutant melanoma maintenance

Activating mutations in BRAF and NRAS are the most common genetic alterations that occur in melanoma resulting in the constitutive activation of the ERK1/2 MAP kinase (MAPK) pathway. However, oncogenic transformation of BRAF or NRAS expressing melanocytes often require the cooperation of additional genetic events, in which aberrations in the PI3K>AKT signaling pathway are consistently found in conjunction with these activating mutations. Although oncogenic NRAS is also thought to activate the PI3K pathway, activating mutations in the p110 alpha catalytic subunit of PI3K (PIK3CA) are detected at a low frequency in both BRAF and NRAS-mutated melanomas. Since the mechanism(s) underlying this cooperation still remains elusive, we explored the contribution of alpha-specific PI3K signaling to the cooperation between the MAPK and PI3K>AKT pathways using pathway-targeted inhibitors and human melanoma-derived cell lines that contain a PIK3CAH1047R and either a BRAFV600E or NRASQ61H mutation. Inhibition of MEK or either class I or alpha-specific PI3K>AKT>mTORC1 elicited antiproliferative effects against NRASQ61H, PIK3CAH1047R mutant melanoma cells, whereas the combinatorial inhibition of these signaling pathways was significantly more potent. Even though blockade of class I PI3K or mTORC1 inhibited BRAFV600E, PIK3CAH1047R-mutated melanoma proliferation, these cells were insensitive to single-agent inhibition of BRAFV600E>MEK or alpha-specific PI3K>AKT. However, only the combined inhibition of MEK and alpha-specific PI3K>AKT led to a significant decrease in proliferation. Examination of signaling downstream of BRAFV600E or NRASQ61H and PI3K>AKT revealed that these pathways cooperated to regulate the phosphorylation of the cap-dependent translation regulators, p70S6K, ribosomal protein S6, and 4E-BP1 through mTORC1-dependent effects. While the combined inhibition of MEK and either class I or alpha-specific PI3K signaling resulted in a significant regression of NRASQ61H, PIK3CAH1047R mutant xenograft tumors, the combinatorial inhibition of MEK and class I PI3K showed a greater tumor reduction than the inhibition of MEK and alpha-specific PI3K. Taken together, these data suggest that a PIK3CA mutation does not entirely predict for sensitivity to alpha-specific PI3K inhibition. Furthermore, this study demonstrates that MAPK and PI3K>AKT>mTORC1 signaling cooperates for melanoma maintenance and provides a biochemical basis for combined pathway-targeted therapy for PIK3CA and BRAF or NRAS-mutated melanoma. Citation Format: Jillian M. Silva, Marian M. Deuker, Wayne R. Joseph, Bruce C. Baguley, Martin McMahon. Cooperation of the MAPK and PI3K>AKT>mTORC1 signaling pathways is required for PIK3CA mutant melanoma maintenance. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A09.

Enzyme meets a surprise target: Cancer biology

An enzyme previously implicated in gene regulation has now been found to have a role in cancer progression, potentiating an intracellular signalling pathway that is driven by a mutated K-Ras protein. See Letter p.283

Methylation Matters In KRAS Oncogenesis

An enzyme previously implicated in gene regulation has now been found to have a role in cancer progression, potentiating an intracellular signalling pathway that is driven by a mutated K-Ras protein. See Letter p.283