1] The Broad Institute of Harvard University and Massachusetts Institute of Technology, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA  Department of Medical Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA  Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, USA.
Malignant melanomas harbouring point mutations (Val600Glu) in the serine/threonine-protein kinase BRAF (BRAF(V600E)) depend on RAF-MEK-ERK signalling for tumour cell growth. RAF and MEK inhibitors show remarkable clinical efficacy in BRAF(V600E) melanoma; however, resistance to these agents remains a formidable challenge. Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations. Here we carried out systematic gain-of-function resistance studies by expressing more than 15,500 genes individually in a BRAF(V600E) melanoma cell line treated with RAF, MEK, ERK or combined RAF-MEK inhibitors. These studies revealed a cyclic-AMP-dependent melanocytic signalling network not previously associated with drug resistance, including G-protein-coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB). Preliminary analysis of biopsies from BRAF(V600E) melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF-MEK inhibition but restored in relapsing tumours. Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF. Combined treatment with MAPK-pathway and histone-deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance. Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF-MEK-ERK inhibition, which may be overcome by combining signalling- and chromatin-directed therapeutics.
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"These novel predicted genes that regulate NFκB via NR4A2 suggest a key role of this nuclear receptor within the NFκB pathway. NR4A2 is a family member of AR (NR3C4), which is known to be activated downstream of the MAPK pathway in cancer  and directly interacts with NFκB (specifically the REL subunit), as correctly predicted for NR4A2 . Along with these genes (ATF3, JUNB, and NR4A2) that we predicted to be regulated by the MAP kinase MAP3K8, a known oncogene involved in prostate cancer growth , we additionally suggest the dual specificity phosphatase 5 (DUSP5) as another upstream regulator of NFκB in prostate cancer: DUSP5 was not only predicted to be highly functionally related to NFκB in the positive regulation of NFκB transcription factor activity (Table 1), but also correctly predicted to be regulated by MAP3K8 (Fig 2) . "
[Show abstract][Hide abstract]ABSTRACT: Molecular research in cancer is one of the largest areas of bioinformatic investigation, but it remains a challenge to understand biomolecular mechanisms in cancer-related pathways from high-throughput genomic data. This includes the Nuclear-factor-kappa-B (NFκB) pathway, which is central to the inflammatory response and cell proliferation in prostate cancer development and progression. Despite close scrutiny and a deep understanding of many of its members' biomolecular activities, the current list of pathway members and a systems-level understanding of their interactions remains incomplete. Here, we provide the first steps toward computational reconstruction of interaction mechanisms of the NFκB pathway in prostate cancer. We identified novel roles for ATF3, CXCL2, DUSP5, JUNB, NEDD9, SELE, TRIB1, and ZFP36 in this pathway, in addition to new mechanistic interactions between these genes and 10 known NFκB pathway members. A newly predicted interaction between NEDD9 and ZFP36 in particular was validated by co-immunoprecipitation, as was NEDD9's potential biological role in prostate cancer cell growth regulation. We combined 651 gene expression datasets with 1.4M gene product interactions to predict the inclusion of 40 additional genes in the pathway. Molecular mechanisms of interaction among pathway members were inferred using recent advances in Bayesian data integration to simultaneously provide information specific to biological contexts and individual biomolecular activities, resulting in a total of 112 interactions in the fully reconstructed NFκB pathway: 13 (11%) previously known, 29 (26%) supported by existing literature, and 70 (63%) novel. This method is generalizable to other tissue types, cancers, and organisms, and this new information about the NFκB pathway will allow us to further understand prostate cancer and to develop more effective prevention and treatment strategies.
"Importantly, the cells that had been exposed to the drug in the tumors of treated mice were more tolerant to BRAF inhibition than cells isolated from untreated tumors, with an over 10-fold increase in the concentration causing 50% of maximal growth inhibition (GI 50 ) (Figure 1E). Moreover, in agreement with the previously described function of MITF in resistance to MAPK inhibitors (Haq et al., 2013b; Johannessen et al., 2013; Muller et al., 2014; Smith et al., 2013 ), depletion of MITF rendered drug-tolerant A375- GFP cells sensitive again (Figure 1F). Notably, after drug removal the upregulation of MITF seen in A375-GFP cells was reversible in vivo as well as in vitro (Figure 1G). "
[Show abstract][Hide abstract]ABSTRACT: Once melanomas have progressed with acquired resistance to mitogen-activated protein kinase (MAPK)-targeted therapy, mutational heterogeneity presents a major challenge. We therefore examined the therapy phase before acquired resistance had developed and discovered the melanoma survival oncogene MITF as a driver of an early non-mutational and reversible drug-tolerance state, which is induced by PAX3-mediated upregulation of MITF. A drug-repositioning screen identified the HIV1-protease inhibitor nelfinavir as potent suppressor of PAX3 and MITF expression. Nelfinavir profoundly sensitizes BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors. Moreover, nelfinavir is effective in BRAF and NRAS mutant melanoma cells isolated from patients progressed on MAPK inhibitor (MAPKi) therapy and in BRAF/NRAS/PTEN mutant tumors. We demonstrate that inhibiting a driver of MAPKi-induced drug tolerance could improve current approaches of targeted melanoma therapy.
"Analysis for combination-specific effects on gene expression, provided evidence for selective modulation of genes belonging to the ERK/MAPK and PI3K/AKT canonical pathways by the AZD6244-BEZ235, compared to PLX4720-BEZ235 and to AZD6244-AZD8055. The inhibitory effects on c-FOS, and on genes (p90RSK) encoding proteins that affect CREB phosphorylation, suggest that AZD6244-BEZ235 is a potentially effective approach to overcome recently described mechanisms mediating melanoma resistance to MAPK inhibition . In agreement with this interpretation, caspase 3/7 activation assays and cell death assays indicated that AZD6244-BEZ235 was significantly more effective in the promotion of apoptosis on melanoma cell lines of all susceptibility groups. "
[Show abstract][Hide abstract]ABSTRACT: Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 μM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.