T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. We show that loss of PTEN in tumor cells in preclinical models of melanoma inhibits T cell-mediated tumor killing and decreases T cell trafficking into tumors. In patients, PTEN loss correlates with decreased T cell infiltration at tumor sites, reduced likelihood of successful T cell expansion from resected tumors, and inferior outcomes with PD-1 inhibitor therapy. PTEN loss in tumor cells increased the expression of immunosuppressive cytokines, resulting in decreased T cell infiltration in tumors, and inhibited autophagy, which decreased T cell-mediated cell death. Treatment with a selective PI3Kβ inhibitor improved the efficacy of both anti-PD-1 and anti-CTLA4 antibodies in murine models. Together these findings demonstrate that PTEN loss promotes immune resistance and support the rationale to explore combinations of immunotherapies and PI3K-AKT pathway inhibitors.

There is a critical need to improve our understanding of the pathogenesis of melanoma brain metastases (MBM). Thus, we performed RNA sequencing on 88 resected MBMs and 42 patient-matched extracranial metastases; tumors with suffi cient tissue also underwent wholeexome sequencing, T-cell receptor sequencing, and IHC. MBMs demonstrated heterogeneity of immune infi ltrates that correlated with prior radiation and post-craniotomy survival. Comparison with patientmatched extracranial metastases identifi ed signifi cant immunosuppression and enrichment of oxidative phosphorylation (OXPHOS) in MBMs. Gene-expression analysis of intracranial and subcutaneous xenografts, and a spontaneous MBM model, confi rmed increased OXPHOS gene expression in MBMs, which was also detected by direct metabolite profi ling and [U-13 C]-glucose tracing in vivo. IACS-010759, an OXPHOS inhibitor currently in early-phase clinical trials, improved survival of mice bearing MAPK inhibitor-resistant intracranial melanoma xenografts and inhibited MBM formation in the spontaneous MBM model. The results provide new insights into the pathogenesis and therapeutic resistance of MBMs. SIGNIFICANCE: Improving our understanding of the pathogenesis of MBMs will facilitate the rational development and prioritization of new therapeutic strategies. This study reports the most comprehensive molecular profi ling of patient-matched MBMs and extracranial metastases to date. The data provide new insights into MBM biology and therapeutic resistance.

The majority of melanomas demonstrate constitutive activation of the RAS-RAF-MEK-MAPK pathway. AZD6244 is a selective MEK1/2 inhibitor which markedly reduces tumor P-MAPK levels, but it produced few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the PI3K-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines demonstrated AZD6244-induced activation of AKT. In contrast, sensitive cell lines demonstrated AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or IGF1R blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells, the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients.

Purpose: Activation of the phosphoinositide 3-kinase (PI3K)-AKT pathway has been implicated in melanoma based primarily on the prevalence of mutations in PTEN and NRAS. To improve our understanding of the regulation and clinical significance of the PI3K-AKT pathway in melanoma, we quantitatively measured the levels of phosphorylated AKT, its substrate GSK3α/β, and its negative regulator PTEN in clinical metastases. Results were compared with mutational status, clinical outcomes, and sites of metastasis. Experimental Design: DNA and protein were isolated from dissected frozen melanoma metastases (n = 96). Activating mutations of BRAF, NRAS, AKT, PIK3CA, and KIT were detected by mass spectroscopy genotyping. Phosphorylated AKT (Ser473 and Thr308), P-GSK3α/β, and PTEN protein expression were measured by reverse-phase protein array. A panel of human melanoma cells lines (n = 58) was analyzed for comparison. Results: BRAF-mutant tumors had higher levels of P-AKT-Ser473 (P = 0.01), P-AKTThr308 (P = 0.002), and P-GSK3α/β (P = 0.08) than NRAS-mutant tumors. Analysis of individual tumors showed that almost all tumors with elevated P-AKT had low PTEN levels; NRAS-mutant tumors had normal PTEN and lower P-AKT. Similar results were observed in melanoma cell lines. Stage III melanoma patients did not differ in overall survival based on activation status of the PI3K-AKT pathway. Brain metastases had significantly higher P-AKT and lower PTEN than lung or liver metastases. Conclusions: Quantitative interrogation of the PI3K-AKT pathway in melanoma reveals unexpected significant differences in AKT activation by NRAS mutation and PTEN loss, and hyperactivation of AKT in brain metastases. These findings have implications for the rational development of targeted therapy for this disease. (Clin Cancer Res 2009;15(24):7538-46) Melanoma is the most aggressive form of skin cancer. It is estimated that 68,720 patients will be diagnosed with melanoma, and 8,650 patients will die of this disease, in 2009 (1). Clinical trials of chemotherapy, immunotherapy, and biochemotherapy have failed to significantly improve outcomes in melanoma (2). Thus, new therapeutic approaches are needed.Somatic mutations in BRAF, a serine-threonine kinase that is a component of the RAS-RAF-mitogen-activated protein kinase (MAPK) signaling pathway, are detected in ∼50% of melanomas (3). These mutations result in constitutive activation of BRAF, with resultant activation of MAPK in vitro (4). Unfortunately, the pan-RAF inhibitor sorafenib failed to improve outcomes in patients with metastatic melanoma (5). It is possible that the lack of efficacy was due to insufficient inhibition of the RAS-RAF-MAPK pathway, as is suggested by the recent promising, albeit preliminary, results with the more potent, selective inhibitor of mutant BRAF, PLX4032 (6). However, there is also evidence that activating mutations in BRAF alone cannot fully

Metabolic heterogeneity is a key factor in cancer pathogenesis. We found that a subset of BRAF and NRAS mutant human melanomas resistant to the MEK inhibitor selumetinib displayed increased oxidative phosphorylation (OxPhos) mediated by the transcriptional co-activator PGC1α. Notably, all selumetinib-resistant cells with elevated OxPhos could be re-sensitized by co-treatment with the mTORC1/2 inhibitor AZD8055, whereas this combination was ineffective in resistant cell lines with low OxPhos. In both BRAF- and NRAS-mutant melanoma cells, MEK inhibition increased MITF expression which in turn elevated levels of PGC1α. In contrast, mTORC1/2 inhibition triggered cytoplasmic localization of MITF, decreasing PGC1α expression and inhibiting OxPhos. Analysis of tumor biopsies from BRAF-mutant melanoma patients progressing on BRAF inhibitor {plus minus} MEK inhibitor revealed that PGC1α levels were elevated in approximately half of the resistant tumors. Overall, our findings highlight the significance of OxPhos in melanoma and suggest that combined targeting of the MAPK and mTORC pathways may offer an effective therapeutic strategy to treat melanomas with this metabolic phenotype.

Summary Cancer cell lines are major model systems for mechanistic investigation and drug development. However, protein expression data linked to high-quality DNA, RNA and drug screening data have not been available across a large number of cancer cell lines. Using reverse-phase protein arrays, we measured expression levels of ∼230 key cancer-related proteins in >650 independent cell lines, many of which have publically available genomic, transcriptomic and drug screening data. Our dataset recapitulates the effects of mutated pathways on protein expression observed in patient samples, and demonstrates that proteins and particularly phosphoproteins provide information for predicting drug sensitivity that is not available from the corresponding mRNAs. We also developed a user-friendly bioinformatic resource, MCLP, to help serve the biomedical research community.

Although interactions between estrogen and growth factor signaling pathways have been studied extensively, how growth factors and progesterone regulate each other is less clear. In this study, we found that IGF-I sharply lowers progesterone receptor (PR) mRNA and protein levels in breast cancer cells. Other growth factors, such as epidermal growth factor, also showed the same effect. The decrease of PR levels was associated with reduced PR activity. Unlike progestins, IGF-I does not utilize the proteasome for down-regulating PR. Instead, the IGF-I-mediated decrease in PR levels is via an inhibition of PR gene transcription. In addition, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was found to be specifically involved in this IGF-I effect. Our data also suggest that the IGF-I down-regulation of PR is not mediated via a reduction of estrogen receptor (ER) levels or activity. First, IGF-I induced ligand-independent ER activity while reducing ER-dependent PR levels. Second, whereas PR and cyclin D1 are both ER up-regulated, IGF-I increased cyclin D1 levels while decreasing PR levels. Third, constitutively active PI3K or Akt induced ER activity but reduced PR levels and activity. Taken together, our data indicate that IGF-I inhibits PR expression in breast cancer cells via the PI3K/Akt/mTOR pathway. Because low or absent PR in primary breast cancer is associated with poor prognosis and response to hormone therapy, our results suggest that low PR status may serve as an indicator of activated growth factor signaling in breast tumor cells, and therefore of an aggressive tumor phenotype and resistance against hormonal therapy.

Purpose An improved understanding of the molecular pathogenesis of brain metastases, one of the most common and devastating complications of advanced melanoma, may identify and prioritize rational therapeutic approaches for this disease. In particular, the identification of molecular differences between brain and extracranial metastases would support the need for the development of organ-specific therapeutic approaches. Experimental Design Hotspot mutations, copy number variations (CNV), global mRNA expression patterns, and quantitative analysis of protein expression and activation by reverse phase protein array (RPPA) analysis were evaluated in pairs of melanoma brain metastases and extracranial metastases from patients who had undergone surgical resection for both types of tumors. Results The status of 154 previously reported hotspot mutations, including driver mutations in BRAF and NRAS, were concordant in all evaluable patient-matched pairs of tumors. Overall patterns of CNV, mRNA expression, and protein expression were largely similar between the paired samples for individual patients. However, brain metastases demonstrated increased expression of several activation-specific protein markers in the PI3K/AKT pathway compared to the extracranial metastases. Conclusions These results add to the understanding of the molecular characteristics of melanoma brain metastases and support the rationale for additional testing of the PI3K/AKT pathway as a therapeutic target in these highly aggressive tumors.