The Benefit of Reactivating p53 under MAPK Inhibition on the Efficacy of Radiotherapy in Melanoma.
ABSTRACT: Radiotherapy (RT) in patients with melanoma historically showed suboptimal results, because the disease is often radioresistant due to various mechanisms such as scavenging free radicals by thiols, pigmentary machinery, or enhanced DNA repair. However, radiotherapy has been utilized as adjuvant therapy after the complete excision of primary melanoma and lymph nodes to reduce the rate of nodal recurrences in high-risk patients. The resistance of melanoma cells to radiotherapy may also be in relation with the constitutive activation of the MAPK pathway and/or with the inactivation of p53 observed in about 90% of melanomas. In this study, we aimed to assess the potential benefit of adding RT to BRAF-mutated melanoma cells under a combined p53 reactivation and MAPK inhibition in vitro and in a preclinical animal model. We found that the combination of BRAF inhibition (vemurafenib, which completely shuts down the MAPK pathway), together with p53 reactivation (PRIMA-1Met) significantly enhanced the radiosensitivity of BRAF-mutant melanoma cells. This was accompanied by an increase in both p53 expression and activity. Of note, we found that radiation alone markedly promoted both ERK and AKT phosphorylation, thus contributing to radioresistance. The combination of vemurafenib and PRIMA-1Met caused the inactivation of both MAPK kinase and PI3K/AKT pathways. Furthermore, when combined with radiotherapy, it was able to significantly enhance melanoma cell radiosensitivity. Interestingly, in nude mice bearing melanoma xenografts, the latter triple combination had not only a synergistic effect on tumor growth inhibition, but also a potent control on tumor regrowth in all animals after finishing the triple combination therapy. RT alone had only a weak effect. In conclusion, we provide a basis for a strategy that may overcome the radioresistance of BRAF-mutated melanoma cells to radiotherapy. Whether this will translate into a rational to use radiotherapy in the curative setting in BRAF-mutated melanoma patients deserves consideration.
Project description:Targeting MAPK pathway in mutant BRAF melanoma with the specific BRAF inhibitor vemurafenib showed robust initial responses in the majority of patients followed by relapses due to acquired resistance to the drug. In <sup>V600E</sup>BRAF melanoma cell lines, senescence-associated ?-galactosidase activity is often encountered in a constitutive manner or induced after MAPK inhibition. However, the link between the senescence-like phenotype and the resistance to BRAF inhibition is not fully understood yet. Our data validate a senescence-like phenotype (low cell proliferation, high cell volume, and high ?-Gal activity) in mutant BRAF cells. Vemurafenib increased ?-Gal activity in 4 out of 5 sensitive lines and in 2 out of 5 lines with intrinsic resistance to the drug. Interestingly, the 3 lines with acquired resistance to vemurafenib became depending on the drug for proliferation. In absence of drug, these lines showed a lower cell proliferation rate together with a substantial increase of ?-Gal activity both <i>in vitro</i> and <i>in vivo</i>. In all settings, the senescence-like phenotype was significantly associated with an inhibition of pRB and cyclin D1, explaining the inhibition of cell proliferation. In conclusion, ?-Gal activity is increased by <sup>V600E</sup>BRAF inhibition in the majority of sensitive and intrinsically resistant melanoma cells. Acquired resistance to vemurafenib is associated with a dependence to the drug for cell proliferation and tumor growth, and, in this case, drug removal stimulate ?-Gal activity suggesting that the senescence-like phenotype could contribute to the acquired resistance to BRAF inhibition.
Project description:<h4>Background</h4>RAF inhibitors are an effective therapy for patients with BRAF-mutant melanoma and brain metastasis. Efficacy data are derived from clinical studies enriched with physiologically fit patients; therefore, it is of interest to assess the real-world experience of vemurafenib in this population. Tumor-specific genetic variants that influence sensitivity to RAF kinase inhibitors also require investigation.<h4>Methods</h4>Records of patients with BRAF-mutant melanoma and brain metastases who were treated with vemurafenib were reviewed. Clinical data were extracted to determine extracranial and intracranial objective response rates, progression-free survival (PFS), overall survival (OS), and safety. A bait-capture, next-generation sequencing assay was used to identify mutations in pretreatment tumors that could explain primary resistance to vemurafenib.<h4>Results</h4>Among patients with intracranial disease treated with vemurafenib, 27 were included in survival analyses and 22 patients were assessable for response. The extracranial and intracranial objective response rates were 71% and 50%, respectively. Discordant responses were observed between extracranial and intracranial metastatic sites in 4 of 19 evaluable patients. Median PFS was 4.1 months (95% confidence interval [CI]: 2.6-7.9); median intracranial PFS was 4.6 months (95% CI: 2.7-7.9), median OS was 7.5 months (95% CI: 4.3-not reached), with a 30.4% 1-year OS rate. Outcomes were influenced by performance status. Vemurafenib was tolerable, although radiation-induced dermatitis occurred in some patients who received whole-brain radiotherapy. Adequate samples for next-generation sequencing analysis were available for seven patients. Melanomas categorized as "poorly sensitive" (?20% tumor growth, new lesions, or ?50% shrinkage for <4 months) harbored co-occurring mutations in genes predicted to activate the phosphatidylinositol 3-kinase-AKT (PI3K-AKT) pathway.<h4>Conclusion</h4>Vemurafenib is highly active in BRAF-mutant melanoma brain metastases but has limited activity in patients with poor performance status. The safety and efficacy of concurrent radiotherapy and RAF inhibition requires careful clinical evaluation. Combination strategies blocking the MAPK and PI3K-AKT pathway may be warranted in a subset of patients.<h4>Implications for practice</h4>Vemurafenib is active for BRAF-mutant intracranial melanoma metastases in an unselected patient population typical of routine oncologic practice. Patients with poor performance status appear to have poor outcomes despite vemurafenib therapy. Preliminary data indicate that co-occurring or secondary alterations in the phosphatidylinositol 3-kinase-AKT (PI3K-AKT) pathway are involved in resistance to RAF inhibition, thus providing a rationale for dual MAPK and PI3K-AKT pathway inhibition in this patient population.
Project description:In melanoma, dysregulation of the MAPK pathway, usually via BRAF(V600) or NRAS(Q61) somatic mutations, leads to constitutive ERK signaling. While BRAF inhibitors are initially effective for BRAF-mutant melanoma, no FDA-approved targeted therapies exist for BRAF-inhibitor-resistant BRAF(V600), NRAS mutant, or wild-type melanoma.The 50% inhibitory concentration (IC50) of SCH772984, a novel inhibitor of ERK1/2, was determined in a panel of 50 melanoma cell lines. Effects on MAPK and AKT signaling by western blotting and cell cycle by flow cytometry were determined.Sensitivity fell into three groups: sensitive, 50% inhibitory concentration (IC50) < 1 ?M; intermediately sensitive, IC50 1-2 ?M; and resistant, >2 ?M. Fifteen of 21 (71%) BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH772984. All three (100%) BRAF/NRAS double mutants, 11 of 14 (78%) NRAS mutants and 5 of 7 (71%) wild-type melanomas were sensitive. Among BRAF(V600) mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH772984. SCH772984 caused G1 arrest and induced apoptosis.Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines and significantly delayed the onset of acquired resistance in long term in vitro assays. Therefore, SCH772984 may be clinically applicable as a treatment for non-BRAF mutant melanoma or in BRAF-mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors.
Project description:Melanoma is a type of malignant tumor derived from melanocytes, most of which occur in the skin, and a few occur in the mucosa and choroid. BRAF mutations occur in approximately 50% of melanoma patients. Vemurafenib is a specific and potent BRAF inhibitor that significantly prolongs progression-free survival in patients with BRAF mutant melanoma. But most patients have tumor recurrence after 7-9 months. Drug resistance severely limits the long-term clinical effects of targeted drugs. To explore the mechanism of melanoma resistance to Vemurafenib, the transcripts of Vemurafenib-resistant melanoma A375R cells and the parental A375 cells were sequenced. For more insight please see Transcripts 202 and 205 of IL-6 confer resistance to Vemurafenib by reactivating the MAPK pathway in BRAF(V600E) mutant melanoma cells . RNA-seq data has been uploaded to Sequence Read Archive (SRA), which allows researchers to obtain RNA sequence data for these cells.
Project description:BRAF mutations occur in 10-15% of colorectal cancers (CRCs) and confer adverse outcome. While RAF inhibitors such as vemurafenib (PLX4032) have proven effective in BRAF mutant melanoma, they are surprisingly ineffective in BRAF mutant CRCs, and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRCs expressed higher levels of phospho-EGFR than BRAF mutant melanomas, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells and markedly improved efficacy in vitro and in vivo. These findings support evaluation of combined RAF and EGFR inhibition in BRAF mutant CRC patients.BRAF valine 600 (V600) mutations occur in 10% to 15% of colorectal cancers, yet these tumors show a surprisingly low clinical response rate (~5%) to selective RAF inhibitors such as vemurafenib, which have produced dramatic response rates (60%–80%) in melanomas harboring the identical BRAF V600 mutation. We found that EGFR-mediated MAPK pathway reactivation leads to resistance to vemurafenib in BRAF-mutant colorectal cancers and that combined RAF and EGFR inhibition can lead to sustained MAPK pathway suppression and improved efficacy in vitro and in tumor xenografts.
Project description:For patients with advanced melanoma, primary and secondary resistance to selective BRAF inhibition remains one of the most critically compelling challenges. One rationale argues that novel biologically informed strategies are needed to maximally cripple melanoma cells up front before compensatory mechanisms emerge. As p53 is uncommonly mutated in melanoma, restoration of its function represents an attractive adjunct to selective BRAF inhibition.Thirty-seven BRAF(V600E)-mutated melanoma lines were subjected to synergy studies in vitro using a combination of vemurafenib and nutlin-3 (Nt-3). In addition, cellular responses and in vivo efficacy were also determined. We also analyzed changes in the levels of canonical apoptotic/survival factors in response to vemurafenib.Dual targeting of BRAF(V600E) and Hdm2 with vemurafenib and Nt-3, respectively, synergistically induced apoptosis and suppressed melanoma viability in vitro and tumor growth in vivo. Suppression of p53 in melanoma cells abrogated Nt-3's effects fully and vemurafenib's effects partially. A survey of canonical survival factors revealed that both vemurafenib and Nt-3 independently attenuated levels of the antiapoptotic protein, survivin. Genetic depletion of survivin reproduces the cytotoxic effects of the combination strategy.These results show preclinical feasibility for overcoming primary vemurafenib resistance by restoring p53 function. Moreover, it identifies survivin as one downstream mediator of the observed synergism and a potential secondary target.
Project description:Mutations in the serine/threonine kinase BRAF are found in more than 60% of melanomas. The most prevalent melanoma mutation is BRAF(V600E), which constitutively activates downstream MAPK signalling. Vemurafenib is a potent RAF kinase inhibitor with remarkable clinical activity in BRAF(V600E)-positive melanoma tumours. However, patients rapidly develop resistance to vemurafenib treatment. One resistance mechanism is the emergence of BRAF alternative splicing isoforms leading to elimination of the RAS-binding domain. Here we identify interference with pre-mRNA splicing as a mechanism to combat vemurafenib resistance. We find that small-molecule pre-mRNA splicing modulators reduce BRAF3-9 production and limit in-vitro cell growth of vemurafenib-resistant cells. In xenograft models, interference with pre-mRNA splicing prevents tumour formation and slows growth of vemurafenib-resistant tumours. Our results identify an intronic mutation as the molecular basis for a RNA splicing-mediated RAF inhibitor resistance mechanism and we identify pre-mRNA splicing interference as a potential therapeutic strategy for drug resistance in BRAF melanoma.
Project description:BRAF mutations are detected in >50% of all melanomas. These mutations impair the LKB1-AMPK signaling, an important metabolic pathway associated with cell growth, proliferation and survival. Melanoma patients with BRAF mutations are usually treated with BRAF inhibitors such as vemurafenib, but responses are short-lived as drug resistant tumors metabolically switch to mitochondrial oxidative phosphorylation (OXPHOS) to escape metabolic stress-induced BRAF inhibition. Additionally, a large subset of melanoma utilizes OXPHOS in their metabolism, which can confer de novo resistance to BRAF inhibitors. Therefore, uncoupling of OXPHOS to perturb energy homeostasis and to indirectly stimulate AMPK could be a novel treatment for melanoma and to overcome intrinsic and acquired resistance to BRAF inhibitors. Here, we investigated the effects of SR4 and niclosamide, two small molecule mitochondria uncouplers, on the growth and proliferation of treatment-naïve and vemurafenib-resistant melanomas in vitro and in vivo. SR4 and niclosamide inhibited melanoma proliferation irrespective of BRAF/NRAS status. Melanomas with greater OXPHOS phenotype (higher OCR/ECAR), with LKB1 mutation, or with acquired resistance to vemurafenib displayed greater sensitivity to both uncouplers. More importantly, SR4 and niclosamide inhibited tumor growth in both treatment-naïve and vemurafenib-resistant xenograft mice models. Mechanistic studies indicate both uncouplers induced energetic stress, modulated the AMPK-mTOR pathway, and promoted apoptosis without affecting MEK-ERK MAPK signaling. These results suggest that uncouplers such as SR4 and niclosamide may be useful as first line treatment against melanoma regardless of BRAF/NRAS status, and as an adjuvant therapy for patients failing MAPK inhibitors.
Project description:Resistance to BRAF inhibition is a major cause of treatment failure for BRAF-mutated metastatic melanoma patients. Abemaciclib, a cyclin-dependent kinase 4 and 6 inhibitor, overcomes this resistance in xenograft tumours and offers a promising drug combination. The present work aims to characterise the quantitative pharmacology of the abemaciclib/vemurafenib combination using a semimechanistic pharmacokinetic/pharmacodynamic modelling approach and to identify an optimum dosing regimen for potential clinical evaluation.A PK/biomarker model was developed to connect abemaciclib/vemurafenib concentrations to changes in MAPK and cell cycle pathway biomarkers in A375 BRAF-mutated melanoma xenografts. Resultant tumour growth inhibition was described by relating (i) MAPK pathway inhibition to apoptosis, (ii) mitotic cell density to tumour growth and, under resistant conditions, (iii) retinoblastoma protein inhibition to cell survival.The model successfully described vemurafenib/abemaciclib-mediated changes in MAPK pathway and cell cycle biomarkers. Initial tumour shrinkage by vemurafenib, acquisition of resistance and subsequent abemaciclib-mediated efficacy were successfully captured and externally validated. Model simulations illustrate the benefit of intermittent vemurafenib therapy over continuous treatment, and indicate that continuous abemaciclib in combination with intermittent vemurafenib offers the potential for considerable tumour regression.The quantitative pharmacology of the abemaciclib/vemurafenib combination was successfully characterised and an optimised, clinically-relevant dosing strategy was identified.
Project description:Usp5 is a deubiquitinase (DUB) previously shown to regulate unanchored poly-ubiquitin (Ub) chains, p53 transcriptional activity and double-strand DNA repair. In BRAF mutant melanoma cells, Usp5 activity was suppressed by BRAF inhibitor (vemurafenib) in sensitive but not in acquired or intrinsically resistant cells. Usp5 knockdown overcame acquired vemurafenib resistance and sensitized BRAF and NRAS mutant melanoma cells to apoptosis initiated by MEK inhibitor, cytokines or DNA-damaging agents. Knockdown and overexpression studies demonstrated that Usp5 regulates p53 (and p73) levels and alters cell growth and cell cycle distribution associated with p21 induction. Usp5 also regulates the intrinsic apoptotic pathway by modulating p53-dependent FAS expression. A small molecule DUB inhibitor (EOAI3402143) phenocopied the FAS induction and apoptotic sensitization of Usp5 knockdown and fully blocked melanoma tumor growth in mice. Overall, our results demonstrate that BRAF activates Usp5 to suppress cell cycle checkpoint control and apoptosis by blocking p53 and FAS induction; all of which can be restored by small molecule-mediated Usp5 inhibition. These results suggest that Usp5 inhibition can provide an alternate approach in recovery of diminished p53 (or p73) function in melanoma and can add to the targeted therapies already used in the treatment of melanoma.