ABSTRACT: Variable clinical responses, tumor heterogeneity, and drug resistance reduce long-term survival outcomes for metastatic melanoma patients. To guide and accelerate drug development, we characterized tumor responses for five melanoma patient derived xenograft models treated with Vemurafenib. Three BRAF(V600E) models showed acquired drug resistance, one BRAF(V600E) model had a complete and durable response, and a BRAF(V600V) model was expectedly unresponsive. In progressing tumors, a variety of resistance mechanisms to BRAF inhibition were uncovered, including mutant BRAF alternative splicing, NRAS mutation, COT (MAP3K8) overexpression, and increased mutant BRAF gene amplification and copy number. The resistance mechanisms among the patient derived xenograft models were similar to the resistance pathways identified in clinical specimens from patients progressing on BRAF inhibitor therapy. In addition, there was both inter- and intra-patient heterogeneity in resistance mechanisms, accompanied by heterogeneous pERK expression immunostaining profiles. MEK monotherapy of Vemurafenib-resistant tumors caused toxicity and acquired drug resistance. However, tumors were eradicated when Vemurafenib was combined the MEK inhibitor. The diversity of drug responses among the xenograft models; the distinct mechanisms of resistance; and the ability to overcome resistance by the addition of a MEK inhibitor provide a scheduling rationale for clinical trials of next-generation drug combinations.
Project description:Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, with??50% of tumours expressing the BRAF(V600E) oncoprotein. Moreover, the marked tumour regression and improved survival of late-stage BRAF-mutated melanoma patients in response to treatment with vemurafenib demonstrates the essential role of oncogenic BRAF in melanoma maintenance. However, as most patients relapse with lethal drug-resistant disease, understanding and preventing mechanism(s) of resistance is critical to providing improved therapy. Here we investigate the cause and consequences of vemurafenib resistance using two independently derived primary human melanoma xenograft models in which drug resistance is selected by continuous vemurafenib administration. In one of these models, resistant tumours show continued dependency on BRAF(V600E)?MEK?ERK signalling owing to elevated BRAF(V600E) expression. Most importantly, we demonstrate that vemurafenib-resistant melanomas become drug dependent for their continued proliferation, such that cessation of drug administration leads to regression of established drug-resistant tumours. We further demonstrate that a discontinuous dosing strategy, which exploits the fitness disadvantage displayed by drug-resistant cells in the absence of the drug, forestalls the onset of lethal drug-resistant disease. These data highlight the concept that drug-resistant cells may also display drug dependency, such that altered dosing may prevent the emergence of lethal drug resistance. Such observations may contribute to sustaining the durability of the vemurafenib response with the ultimate goal of curative therapy for the subset of melanoma patients with BRAF mutations.
Project description:Rationale: Melanoma is an aggressive tumor of the skin and drug resistance is still a major problem in melanoma therapy. Novel targets and effective agents to overcome drug resistant melanoma are urgently needed in clinical therapy. Methods: Gene Expression Omnibus (GEO) database analysis, pathway enrichment analysis, and survival rate analysis were utilized to identify a candidate target. An anchorage-independent cell growth assay, flow cytometry, Western blot, and a xenograft mouse model were used to study the function of Aurora kinase B (AURKB) in both drug-sensitive and drug-resistant melanoma. Next, HI-511, a novel dual-target inhibitor targeting both AURKB and BRAF V600E, was designed and examined by an in vitro kinase assay. Methods as indicated above in addition to a BRAF V600E/PTEN-loss melanoma mouse model were used to demonstrate the effect of HI-511 on melanoma development in vitro and in vivo. Results: AURKB is highly expressed in melanoma and especially in vemurafenib-resistant melanoma and the expression was correlated with patient survival rate. Knocking down AURKB inhibited cell growth and induced apoptosis in melanoma, which was associated with the BRAF/MEK/ERKs and PI3-K/AKT signaling pathways. Importantly, we found that HI-511, a novel dual-target inhibitor against AURKB and BRAF V600E, suppresses both vemurafenib-sensitive and vemurafenib-resistant melanoma growth in vitro and in vivo by inducing apoptosis and mediating the inhibition of the BRAF/MEK/ERKs and PI3K/AKT signaling pathways. Conclusion: AURKB is a potential target for melanoma treatment. HI-511, a novel dual-target inhibitor against both AURKB and BRAF V600E, could achieve durable suppression of melanoma growth, even drug-resistant melanoma growth.
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:Vemurafenib and dabrafenib block MEK-ERK1/2 signaling and cause tumor regression in the majority of advanced-stage BRAF(V600E) melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next-generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF(V600E) melanoma cells without paradoxical effects in wild-type cells. Furthermore, PLX7904 blocks the growth of vemurafenib-resistant BRAF(V600E) cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF(V600E) splice variant-mediated vemurafenib-resistant cells. We show that paradox-breaker RAF inhibitors potently block MEK-ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720-resistant cells harboring distinct BRAF(V600E) splice variants. These data support the further investigation of paradox-breaker RAF inhibitors as a second-line treatment option for patients failing on vemurafenib or dabrafenib.
Project description:Approximately 5-10% of metastatic colorectal cancers harbor a BRAF-V600E mutation, which is correlated with resistance to EGFR-targeted therapies and worse clinical outcome. Vice versa, targeted inhibition of BRAF-V600E with the selective inhibitor PLX 4032 (Vemurafenib) is severely limited due to feedback re-activation of EGFR in these tumors. Mounting evidence indicates that upregulation of the ErbB-3 signaling axis may occur in response to several targeted therapeutics, including Vemurafenib, and NRG-1?-dependent re-activation of the PI3K/AKT survival pathway has been associated with therapy resistance.Here we show that colon CSCs express, next to EGFR and ErbB-2, also significant amounts of ErbB-3 on their membrane. This expression is functional as NRG-1? strongly induces AKT/PKB and ERK phosphorylation, cell proliferation, clonogenic growth and promotes resistance to Vemurafenib in BRAF-V600E mutant colon CSCs. This resistance was completely dependent on ErbB-3 expression, as evidenced by knockdown of ErbB-3. More importantly, resistance could be alleviated with therapeutic antibody blocking ErbB-3 activation, which impaired NRG-1?-driven AKT/PKB and ERK activation, clonogenic growth in vitro and tumor growth in xenograft models. In conclusion, our findings suggest that targeting ErbB-3 receptors could represent an effective therapeutic approach in BRAF-V600E mutant colon cancer.
Project description:<h4>Background</h4>Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed.<h4>Methodology</h4>In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.<h4>Results</h4>Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.<h4>Conclusions</h4>Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.
Project description:The BT-40 low-grade childhood astrocytoma xenograft model expresses mutated BRAF(V600E) and is highly sensitive to the MEK inhibitor selumetinib (AZD6244). In this study, we developed and characterized selumetinib resistance and explored approaches to circumventing the mechanisms of acquired resistance.BT-40 xenografts were selected in vivo for selumetinib resistance. Resistant tumors were obtained and characterized, as were tumors that reverted to sensitivity. Characterization included expression profiling, assessment of MEK signature and compensatory pathways, MEK inhibition, BRAF expression, and cytokine levels. Combination treatment of BT-40/AZD-resistant tumors with the MEK inhibitor and a STAT3 inhibitor (LLL12) was assessed.Resistance was unstable, tumors reverting to selumetinib sensitivity when passaged in untreated mice, and MEK was equally inhibited in sensitive and resistant tumors by selumetinib. Drug resistance was associated with an enhanced MEK signature and increased interleukin (IL)-6 and IL-8 expression. Selumetinib treatment induced phosphorylation of STAT3 (Y705) only in resistant xenografts, and similar results were observed in BRAF(V600E) astrocytic cell lines intrinsically resistant to selumetinib. Treatment of BT-40-resistant tumors with selumetinib or LLL12 had no significant effect, whereas combined treatment induced complete regressions of BT-40/AZD-resistant xenografts.Resistance to selumetinib selected in vivo in BT-40 tumor xenografts was unstable. In resistant tumors, selumetinib activated STAT3, and combined treatment with selumetinib and LLL12 induced complete responses in resistant BT-40 tumors. These results suggest dual targeting BRAF (V600E) signaling and STAT3 signaling may be effective in selumetinib-resistant tumors or may retard or prevent onset of resistance.
Project description:Expression of aberrantly spliced BRAF V600E isoforms (BRAF V600E ?Ex) mediates resistance in 13%-30% of melanoma patients progressing on RAF inhibitors. BRAF V600E ?Ex confers resistance, in part, through enhanced dimerization. Here, we uncoupled BRAF V600E ?Ex dimerization from maintenance of MEK-ERK1/2 signaling. Furthermore, we show BRAF V600E ?Ex 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 ?Ex. Mutation of S729 to a non-phosphorylatable residue reduced BRAF V600E ?Ex-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 ?Ex. These findings may provide opportunities to target resistance driven by aberrantly spliced forms of BRAF V600E.
Project description:BACKGROUND:Genetics-based basket trials have emerged to test targeted therapeutics across multiple cancer types. However, while vemurafenib is FDA-approved for BRAF-V600E melanomas, the non-melanoma basket trial was unsuccessful, suggesting mutation status is insufficient to predict response. We hypothesized that proteomic data would complement mutation status to identify vemurafenib-sensitive tumors and effective co-treatments for BRAF-V600E tumors with inherent resistance. METHODS:Reverse Phase Proteomic Array (RPPA, MD Anderson Cell Lines Project), RNAseq (Cancer Cell Line Encyclopedia) and vemurafenib sensitivity (Cancer Therapeutic Response Portal) data for BRAF-V600E cancer cell lines were curated. Linear and nonlinear regression models using RPPA protein or RNAseq were evaluated and compared based on their ability to predict BRAF-V600E cell line sensitivity (area under the dose response curve). Accuracies of all models were evaluated using hold-out testing. CausalPath software was used to identify protein-protein interaction networks that could explain differential protein expression in resistant cells. Human examination of features employed by the model, the identified protein interaction networks, and model simulation suggested anti-ErbB co-therapy would counter intrinsic resistance to vemurafenib. To validate this potential co-therapy, cell lines were treated with vemurafenib and dacomitinib (a pan-ErbB inhibitor) and the number of viable cells was measured. RESULTS:Orthogonal partial least squares (O-PLS) predicted vemurafenib sensitivity with greater accuracy in both melanoma and non-melanoma BRAF-V600E cell lines than other leading machine learning methods, specifically Random Forests, Support Vector Regression (linear and quadratic kernels) and LASSO-penalized regression. Additionally, use of transcriptomic in place of proteomic data weakened model performance. Model analysis revealed that resistant lines had elevated expression and activation of ErbB receptors, suggesting ErbB inhibition could improve vemurafenib response. As predicted, experimental evaluation of vemurafenib plus dacomitinb demonstrated improved efficacy relative to monotherapies. CONCLUSIONS:Combined, our results support that inclusion of proteomics can predict drug response and identify co-therapies in a basket setting.