A STATement on vemurafenib-resistant melanoma.

Despite recent advancements in the treatment of late-stage mutant BRAF (V600E/K) melanomas, a major hurdle continues to be acquired resistance to BRAF inhibitors such as vemurafenib. The mechanisms for resistance have proven to be heterogeneous, emphasizing the need to use broad therapeutic approaches. In this issue, the study Stat3-targeted therapies overcome the acquired resistance to vemurafenib in melanomas by Liu et al. proposes that signal transducer and activator of transcription 3 (STAT3)-paired box 3 (PAX3) signaling may be a mechanism that is used by melanomas to resist RAF inhibitors

Response and Resistance to Paradox-Breaking BRAF Inhibitor in Melanomas

FDA-approved BRAF inhibitors produce high response rates and improve overall survival in patients with BRAF V600E/K-mutant melanoma, but are linked to pathologies associated with paradoxical ERK1/2 activation in wild-type BRAF cells. To overcome this limitation, a next-generation paradox-breaking RAF inhibitor (PLX8394) has been designed. Here, we show that by using a quantitative reporter assay, PLX8394 rapidly suppressed ERK1/2 reporter activity and growth of mutant BRAF melanoma xenografts. Ex vivo treatment of xenografts and use of a patient-derived explant system (PDeX) revealed that PLX8394 suppressed ERK1/2 signaling and elicited apoptosis more effectively than the FDA-approved BRAF inhibitor, vemurafenib. Furthermore, PLX8394 was efficacious against vemurafenibresistant BRAF splice variant-expressing tumors and reduced splice variant homodimerization. Importantly, PLX8394 did not induce paradoxical activation of ERK1/2 in wild-type BRAF cell lines or PDeX. Continued in vivo dosing of xenografts with PLX8394 led to the development of acquired resistance via ERK1/2 reactivation through heterogeneous mechanisms; however, resistant cells were found to have differential sensitivity to ERK1/2 inhibitor. These findings highlight the efficacy of a paradox-breaking selective BRAF inhibitor and the use of PDeX system to test the efficacy of therapeutic agents. © 2017 American Association for Cancer Research

Molecular regulation of VEGFR-2 function and expression through post-translational modifications

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at [email protected]. Thank you.Vascular endothelial growth factor receptor-2 (VEGFR-2) is an endothelial cell receptor tyrosine kinase (RTK) whose activity is an obligate requirement for both normal development and pathological angiogenesis. A critical post-translational modification (PTM) of RTKs such as VEGFR-2 is tyrosine phosphorylation, which regulates these proteins at multiple levels including, tyrosine kinase activation, signaling, trafficking, and degradation. Similarly, growing evidence now suggests that protein methylation is another important type of PTM that plays a role in protein-protein interaction and signal transduction. In an effort to explore the possibility that methylation plays a role in regulation of VEGFR-2 function, we have employed mass spectrometry analysis coupled with pharmacological inhibitors of the methylation pathway. Our analysis revealed the presence of five methylated residues, three lysine and two arginine. Particularly, methylation of lysine 1041 (K1041), which is proximal to the conserved activation loop of the kinase domain, significantly contributed to VEGFR-2 kinase activation. Mutation of K1041 to multiple different amino acids rendered VEGFR-2 inactive and inhibited the activation of key downstream signaling proteins. Moreover, these mutations reduced VEGFR-2 mediated cell proliferation and capillary tube formation. Single mutations of R817, K856, K861 and R1115 yielded no apparent effect on tyrosine phosphorylation of VEGFR-2, however compounding the methyl deficiencies with triple and quadruple mutations markedly weakened tyrosine phosphorylation and the ligand-mediated downregulation of VEGFR-2. Furthermore, treatment of endothelial cells with global methylation inhibitors including adenosine dialdehyde (AdOx) and 3-deazaneplanocin A (DZNep) decreased ligand mediated tyrosine phosphorylation of VEGFR-2. The study presented here provides evidence that arginine and lysine methylation of VEGFR-2 through both combinatorial and non-combinatorial mechanisms regulate VEGFR-2 phosphorylation and function. This study also demonstrates that RNF121, an endoplasmic reticulum (ER) resident ubiquitin E3 ligase, binds to nascent VEGFR-2 protein and controls the abundance of cell surface VEGFR-2. Taken together, our data describes a novel role for arginine and lysine methylation in the regulation of VEGFR-2 functions and identifies a link between RNF121 ubiquitin E3 ligase and cell surface expression of VEGFR-2

BRAF Splice Variant Resistance to RAF Inhibitor Requires Enhanced MEK Association

Summary: Expression of aberrantly spliced BRAF V600E isoforms (BRAF V600E ΔEx) mediates resistance in 13%–30% of melanoma patients progressing on RAF inhibitors. BRAF V600E ΔEx confers resistance, in part, through enhanced dimerization. Here, we uncoupled BRAF V600E ΔEx dimerization from maintenance of MEK-ERK1/2 signaling. Furthermore, we show BRAF V600E ΔEx association with its substrate, MEK, is enhanced and required for RAF inhibitor resistance. RAF inhibitor treatment increased phosphorylation at serine 729 (S729) in BRAF V600E ΔEx. Mutation of S729 to a non-phosphorylatable residue reduced BRAF V600E ΔEx-MEK interaction, reduced dimerization or oligomerization, and increased RAF inhibitor sensitivity. Conversely, mutation of the BRAF dimerization domain elicited partial effects on MEK association and RAF inhibitor sensitivity. Our data implicate BRAF S729 in resistance to RAF inhibitor and underscore the importance of substrate association with BRAF V600E ΔEx. These findings may provide opportunities to target resistance driven by aberrantly spliced forms of BRAF V600E. : BRAF splice variants represent a common resistance mechanism to FDA-approved RAF inhibitors in melanoma. Through co-IP and functional studies, Vido et al. demonstrate that the phospho-binding site serine 729 mediates enhanced association between splice variants and their substrate, MEK, that is required for resistance to RAF inhibitors. Keywords: BRAF, MEK, melanoma, resistance, serine 72

Multi-omics profiling shows BAP1 loss is associated with upregulated cell adhesion molecules in uveal melanoma

BRCA1-associated protein 1 (BAP1) is a tumor suppressor gene that is mutated in cancer, including uveal melanoma (UM). Loss-of-function BAP1 mutations are associated with UM metastasis and poor prognosis, but the mechanisms underlying these effects remain unclear. Upregulation of cell-cell adhesion proteins is involved with collective migration and metastatic seeding of cancer cells. Here, we show that BAP1 loss in UM patient samples is associated with upregulated gene expression of multiple cell adhesion molecules (CAMs), including E-cadherin (CDH1), cell adhesion molecule 1 (CADM1), and syndecan-2 (SDC2). Similar findings were observed in UM cell lines and scRNA seq data from UM patient samples. BAP1 re-expression in UM cells reduced E-cadherin and CADM1 levels. Functionally, knockdown of E-cadherin decreased spheroid cluster formation and knockdown of CADM1 decreased growth of BAP1 mutant UM cells. Together, our findings demonstrate that BAP1 regulates the expression of CAMs which may regulate metastatic traits. Implications: BAP1 mutations and increased metastasis may be due to upregulation of cell adhesion molecules

CADM1 is a TWIST1-regulated suppressor of invasion and survival

Abstract Metastatic cancer remains a clinical challenge; however, patients diagnosed prior to metastatic dissemination have a good prognosis. The transcription factor, TWIST1 has been implicated in enhancing the migration and invasion steps within the metastatic cascade, but the range of TWIST1-regulated targets is poorly described. In this study, we performed expression profiling to identify the TWIST1-regulated transcriptome of melanoma cells. Gene ontology pathway analysis revealed that TWIST1 and epithelial to mesenchymal transition (EMT) were inversely correlated with levels of cell adhesion molecule 1 (CADM1). Chromatin immunoprecipitation (ChIP) studies and promoter assays demonstrated that TWIST1 physically interacts with the CADM1 promoter, suggesting TWIST1 directly represses CADM1 levels. Increased expression of CADM1 resulted in significant inhibition of motility and invasiveness of melanoma cells. In addition, elevated CADM1 elicited caspase-independent cell death in non-adherent conditions. Expression array analysis suggests that CADM1 directed non-adherent cell death is associated with loss of mitochondrial membrane potential and subsequent failure of oxidative phosphorylation pathways. Importantly, tissue microarray analysis and clinical data from TCGA indicate that CADM1 expression is inversely associated with melanoma progression and positively correlated with better overall survival in patients. Together, these data suggest that CADM1 exerts tumor suppressive functions in melanoma by reducing invasive potential and may be considered a biomarker for favorable prognosis