The Broad Stroke of Hsp90 Inhibitors: Painting over the RAF Inhibitor Paradox.

The novel Hsp90 inhibitor XL888 is undergoing clinical investigation for use in conjunction with the rapidly accelerated fibrosarcoma (RAF) kinase inhibitor vemurafenib to treat unresectable melanoma. The addition of XL888 to current regimens may serve an additional purpose by blocking the RAF inhibitor paradox. Such activity could reduce adverse events in patients and provide a biomarker for the successful inhibition of Hsp90 target proteins

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

The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma.

Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma

Mechanisms Underlying RAF Inhibitor Resistance by BRAF Splice Variants in Melanoma

The serine/threonine kinase, BRAF, is mutated in 7% of cancers and 50% of melanomas. The majority of the mutations result in constitutive protein activation due to a valine to glutamic acid substitution (V600E). FDA approved RAF inhibitors prolong survival for patients with BRAF V600E melanoma; however, acquired resistance mechanisms limit the long-term efficacy of such therapies. One of the most common mechanisms of resistance to RAF inhibitors in melanoma is the expression of aberrantly spliced BRAF. We study the mechanisms underlying BRAF splice variant mediated resistance to RAF inhibitors in melanoma. Prior studies have focused on the observation that BRAF splice variants homodimerize to a greater degree than full-length BRAF V600E. We demonstrate that low-dose treatment with the RAF inhibitor, vemurafenib, disrupts BRAF dimers at concentrations that do not affect BRAF signaling. Instead, we observe enhanced association with BRAF’s substrate, MEK1/2, and measure association levels that correlate with the response to vemurafenib. Point mutagenesis of BRAF’s MEK-binding interface demonstrates that enhanced BRAF/MEK association is required for splice variant mediated resistance. Aberrant BRAF splicing removes one of the binding sites for the 14-3-3 family of scaffolding proteins at serine 365 (S365). The other binding, serine 729 (S729), is always retained. 14-3-3 binding sites regulate BRAF activity in the physiologic setting but their involvement in oncogenic signaling and response to RAF inhibitor are not fully appreciated. We demonstrate that loss of S365 in full-length BRAF is sufficient to increase dimerization and reduce sensitivity to vemurafenib. Mutation of S729 in BRAF splice variants abrogates protein dimerization and MEK association and increases the sensitivity to vemurafenib. These studies demonstrate that the 14-3-3 binding sites, and S729 in particular, play an integral role in the response to RAF inhibitors. In total, we expand the model of splice variant mediated resistance to incorporate enhanced BRAF/MEK association and 14-3-3 binding site regulation. These studies inform the characterization of next-generation RAF inhibitors that may have unappreciated effects on BRAF substrate association and dimerization. Finally, we suggest that the regulation of S729 may represent a future therapeutic target for patients with RAF driven malignancies

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