Concurrent MEK2 Mutation and BRAF Amplification Confer Resistance to BRAF and MEK Inhibitors in Melanoma

SummaryAlthough BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it

A Comprehensive Patient-Derived Xenograft Collection Representing the Heterogeneity of Melanoma

Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups

BRAF Inhibitor Unveils Its Potential against Advanced Melanoma

SummaryUnresponsiveness to therapy is a hallmark feature of advanced metastatic melanoma. However, the discovery of BRAF-activating mutations in approximately 50% of human melanomas has provided an attractive therapeutic target. Here, we discuss two recent publications focusing on the mutant BRAF kinase inhibitor PLX4032 that validate oncogene-targeted melanoma therapy

Examination of gap junctional, intercellular communication by in situ electroporation on two co-planar indium-tin oxide electrodes

Gap junctions are plasma membrane channels between neighboring cells. We previously described a powerful technique where gap junctional, intercellular communication (GJIC) of adherent cells can be examined by in situ electroporation on a slide, part of which is coated with electrically conductive and transparent indium-tin oxide. An electric pulse is applied through an electrode placed on the cells in the presence of the tracking dye, Lucifer yellow (LY). The pulse causes LY's penetration into the cells growing on the conductive part of the slide, and the subsequent migration of the dye to the non-electroporated cells growing on the non-conductive area is microscopically observed under fluorescence illumination. Although this technique is adequate for a number of cell lines, the turbulence generated as the electrode is removed can cause cell detachment, which makes GJIC examination problematic. In this communication, we describe a slide configuration where junctional communication can be examined in the absence of an upper electrode: Cells are grown on two co-planar electrodes separated by a barrier which diverts the electric field, rendering it vertical to the cell layer. The elimination of an upper electrode is especially valuable for the electroporation of sensitive cells, such as terminally differentiated adipocytes. This technique can also be used for the introduction of other non-permeant molecules such as peptides or siRNA, followed by examination of the cellular phenotype or gene expression levels in situ

Differential effects of c-Ras upon transformation, adipocytic differentiation, and apoptosis mediated by the simian virus 40 large tumor antigen

To investigate the functional relationship between the ability of the simian virus 40 large tumor antigen (TAg) to transform and its ability to block adipocytic differentiation and induce apoptosis, we expressed TAg in C3H10T1/2 (10T1/2)-derived preadipocytes. The results demonstrated that differentiation could be suppressed at lower TAg levels than at the levels required for full neoplastic conversion. Progressively higher TAg levels were accompanied by apoptosis induction in this system. To further examine the role of the cellular Ras protooncogene product (Ras) in TAg function, TAg was expressed in 10T1/2-derived preadipocytes rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs. The results indicated that Ras is required for TAg-mediated transformation and for suppression of adipocytic differentiation, while TAg-mediated apoptosis following serum starvation was independent from Ras action. Unexpectedly, our results further demonstrated a dramatic reduction in the levels of the TAg protein itself as differentiation progressed in Ras-knockdown cells, with a concomitant reduction in TAg’s ability to induce apoptosis as a result. These findings suggest that Ras, although cytoplasmic, is an integral component of the pathway whereby TAg, an oncoprotein believed to have primarily nuclear targets, suppresses differentiation or induces neoplastic conversion of murine preadipocytes

The role of Orai-STIM calcium channels in melanocytes and melanoma

Sucesos entre Mexico y los Estados Unidos de America, relacionados con Texas y otros estados limitrofes. Limites originales, colonizaciones, inmigraciones, invasiones, incidentes y otros diversos asuntos anteriores a la Independecia de Texas. Idiomas cubiertos: español, inglés y francés. - Events between Mexico and the United States of America, related to Texas and other bordering states. Original boundaries, colonizations, immigration, invasions, incidents, and various other issues prior to Texas Independence. The Mexican Embassy in United States of America and the Consulate of Mexico in New Orleans sent reports to the Secretary of Foreign Affairs on the arrest of the Mexican ship Mail of Mexico by the American warship San Felipe off the coast of Texas for alleged smuggling. (press clippings.) Languages covered: Spanish, English, and French.https://scholarworks.utrgv.edu/guerra/1004/thumbnail.jp

Stat3 Is Required for Full Neoplastic Transformation by the Simian Virus 40 Large Tumor Antigen

To investigate the role of Stat3 (signal transducer and activator of transcription-3) in neoplastic transformation by the Large Tumor antigen of Simian Virus 40 (TAg), murine fibroblasts were rendered deficient in Stat3 activity through expression of a Stat3-specific siRNA or a Cre-loxP recombination system. The results demonstrate that growth rate, formation of foci overgrowing a monolayer of normal cells and colony formation in soft agar were dramatically reduced in Stat3-deficient cells. In addition, TAg expression led to increased Stat3 tyrosine phosphorylation, DNA binding, and transcriptional activity, suggesting that Stat3 is required for TAg-mediated neoplasia. Stat3 activation was prevented by blocking the binding of TAg to pRb (retinoblastoma-susceptibility gene product), whereas genetic ablation of pRb increased Stat3 activity, suggesting that pRb inactivation by TAg might be responsible for the observed Stat3 activation. Stat3 activation by TAg was suppressed after inhibition of c-Src, JAKs or the insulin-like growth factor receptor. On the other hand, targeted disruption of the Fer kinase or pharmacological inhibition of Abl had no effect. Inhibition of Src activity led to Stat3 down-regulation as well as apoptosis of sparsely growing, TAg-transformed cells. However, Src inhibition was relatively ineffective in confluent cells, consistent with previous results indicating that cell to cell adhesion activates Stat3 by a Src-independent mechanism. Direct Stat3 inhibition on the other hand induced apoptosis very effectively in confluent cells, which could have significant therapeutic implications. Taken together, our results suggest that Stat3 is an important component of a pathway emanating from TAg and leading to neoplastic conversion

Beyond structure, to survival: activation of Stat3 by cadherin engagement

Cells in normal tissues or in tumors have extensive opportunities for adhesion to their neighbors and the importance of cell to cell contact in the study of fundamental cellular processes in beginning to emerge. In this review, we discuss recent evidence of dramatic changes in the activity of an important signal transducer found to be profoundly affected by cell to cell adhesion, the signal trasducerand activator of transcription-3 (Stat3). Direct cadherin engagement, growth of cells to postconfluence, or formation of multicellular aggregates were found to induce a striking increase in the levels of Stat3 activity, Rac1/Cdc42, and members of the IL6 receptor family in different settings. This activation was specific to Stat3, in that the levels of the extracellular signal regulated kinase (Erk1/2)), a signal transducer often coodinately activated with Stat3 by a number of growth factors or oncogenes, remained unaffected by cell density. Density-dependent Stat3 activation may play a key role in survival, and could contribute to the establishment of cell polarity. It is clear that at any given time the total Stat3 activity levels in a cell are the sum of the effects of cell to cell adhesion plus the conventional Stat3 ac tivating factors present