FBXW7 inactivation in a BrafV600E -driven mouse model leads to melanoma development.
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ABSTRACT:
SUBMITTER: Aydin IT
PROVIDER: S-EPMC5668175 | biostudies-literature | 2017 Jan
REPOSITORIES: biostudies-literature
Publications
FBXW7 inactivation in a Braf<sup>V600E</sup> -driven mouse model leads to melanoma development.
Aydin Iraz T IT Abbate Franco F Rajan Geena Susan GS Badal Brateil B Aifantis Iannis I Desman Garrett G Celebi Julide Tok JT
Pigment cell & melanoma research 20170101 6
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Project description:BRAFV600 -mutated melanoma brain metastases (MBMs) are responsive to BRAF inhibitors, but responses are generally less durable than those of extracranial metastases. We tested the hypothesis that the drug efflux transporters P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) expressed at the blood-brain barrier (BBB) offer MBMs protection from therapy. We intracranially implanted A375 melanoma cells in wild-type (WT) and Abcb1a/b;Abcg2-/- mice, characterized the tumor BBB, analyzed drug levels in plasma and brain lesions after oral vemurafenib administration, and determined the efficacy against brain metastases and subcutaneous lesions. Although contrast-enhanced MRI demonstrated that the integrity of the BBB is disrupted in A375 MBMs, vemurafenib achieved greater antitumor efficacy against MBMs in Abcb1a/b;Abcg2-/- mice compared with WT mice. Concordantly, P-gp and BCRP are expressed in MBM-associated brain endothelium both in patients and in A375 xenografts and expression of these transporters limited vemurafenib penetration into A375 MBMs. Although initially responsive, A375 MBMs rapidly developed therapy resistance, even in Abcb1a/b;Abcg2-/- mice, and this was unrelated to pharmacokinetic or target inhibition issues. Taken together, we demonstrate that both intrinsic and acquired resistance can play a role in MBMs.
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Project description:MEK1/2 and BRAFV600E inhibitors are used to treat BRAFV600E-positive melanoma, with other cancers under evaluation. Genetic perturbation of copper import or pharmacologic reduction of copper with the clinical copper chelator TTM inhibits MEK1/2 kinase activity and reduces BRAFV600E-driven tumorigenesis. In this study, we report that TTM inhibited transformed growth of melanoma cell lines resistant to BRAF or MEK1/2 inhibitors and enhanced the antineoplastic activity of these inhibitors. TTM also provided a survival advantage in a genetically engineered mouse model of melanoma, and when accounting for putative overdosing, trended toward an increase in the survival benefit afforded by BRAF inhibition. This effect was phenocopied by genetically inhibiting copper import in tumors, which was linked to a reduction in MAPK signaling. Thus, TTM reduces copper levels and MAPK signaling, thereby inhibiting BRAFV600E-driven melanoma tumor growth. These observations inform and support clinical evaluation of TTM in melanoma. Cancer Res; 77(22); 6240-52. ©2017 AACR.
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Project description:PurposeDespite unprecedented responses to immune checkpoint inhibitors and targeted therapy in melanoma, a major subset of patients progresses and have few effective salvage options. We have previously demonstrated robust, selective uptake of the peptidomimetic LLP2A labeled with Cu-64 ([64Cu]-LLP2A) for positron emission tomography (PET) imaging in subcutaneous and metastatic models of B16F10 murine melanoma. LLP2A binds with high affinity to very late antigen-4 (VLA-4, integrin α4β1), a transmembrane protein overexpressed in melanoma and other cancers that facilitates tumor growth and metastasis. Yet B16F10 fails to faithfully reflect human melanoma biology, as it lacks certain oncogenic driver mutations, including BRAF mutations found in ≥ 50 % of clinical specimens. Here, we evaluated the PET tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in novel, translational BRAFV600E mutant melanoma models differing in VLA-4 expression-BPR (VLA-4-) and BPRα (VLA-4+).ProceduresBPR cells were transduced with α4 (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [64Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [64Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells was quantified via a plate-based assay. Tracer biodistribution and PET/CT imaging were evaluated in mice bearing subcutaneous BPR and BPRα tumors.Results[64Cu]-LLP2A demonstrated high binding affinity to BPRα (Kd = 1.4 nM) but indeterminate binding to BPR cells. VLA-4+ BPRα and B16F10 displayed comparable time-dependent [64Cu]-LLP2A internalization, whereas BPR internalization was undetectable. PET/CT showed increased tracer uptake in BPRα tumors vs. BPR tumors in vivo, which was validated by significantly greater (p < 0.0001) BPRα tumor uptake in biodistribution analyses.Conclusions[64Cu]-LLP2A discriminates BPRα (VLA-4+) vs. BPR (VLA-4-) melanomas in vivo, supporting translation of these BRAF-mutated melanoma models via prospective imaging and theranostic studies. These results extend the utility of LLP2A to selectively target clinically relevant and therapy-resistant tumor variants toward its use for therapeutic patient care.
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Project description:The presence of colony stimulating factor-1 (CSF1)/CSF1 receptor (CSF1R)-driven tumor-infiltrating macrophages and myeloid-derived suppressor cells (MDSCs) is shown to promote targeted therapy resistance. In this study, we demonstrate the superior effect of a combination of CSF1R inhibitor, PLX3397 and BRAF inhibitor, PLX4720, in suppressing primary and metastatic mouse BRAFV600E melanoma. Using flow cytometry to assess SM1WT1 melanoma-infiltrating leukocytes immediately post therapy, we found that PLX3397 reduced the recruitment of CD11b+ Gr1lo and CD11b+ Gr1int M2-like macrophages, but this was accompanied by an accumulation of CD11b+ Gr1hi cells. PDL1 expression on remaining myeloid cells potentially dampened the antitumor efficacy of PLX3397 and PLX4720 in combination, since PD1/PDL1 axis blockade improved outcome. We also reveal a role for PLX3397 in reducing tumor-infiltrating lymphocytes, and interestingly, this feature was rescued by the co-administration of PLX4720. Our findings, from three different mouse models of BRAF-mutated melanoma, support clinical approaches that co-target BRAF oncogene and CSF1R.
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Project description:Mutations in the promoter of the telomerase reverse transcriptase ( TERT ) gene are the paradigm of a cross-cancer alteration in a non-coding region. TERT promoter mutations (TPMs) are biomarkers of poor prognosis in several tumors, including thyroid cancers. TPMs enhance TERT transcription, which is otherwise silenced in adult tissues, thus reactivating a bona fide oncoprotein. To study TERT deregulation and its downstream consequences, we generated a Tert mutant promoter mouse model via CRISPR/Cas9 engineering of the murine equivalent locus (Tert -123C>T ) and crossed it with thyroid-specific Braf V600E -mutant mice. We also employed an alternative model of Tert overexpression (K5-Tert). Whereas all Braf V600E animals developed well-differentiated papillary thyroid tumors, 29% and 36% of Braf V600E +Tert -123C>T and Braf V600E +K5-Tert mice progressed to poorly differentiated thyroid cancers at week 20, respectively. Braf+Tert tumors showed increased mitosis and necrosis in areas of solid growth, and older animals from these cohorts displayed anaplastic-like features, i.e., spindle cells and macrophage infiltration. Murine Tert promoter mutation increased Tert transcription in vitro and in vivo , but temporal and intra-tumoral heterogeneity was observed. RNA-sequencing of thyroid tumor cells showed that processes other than the canonical Tert-mediated telomere maintenance role operate in these specimens. Pathway analysis showed that MAPK and PI3K/AKT signaling, as well as processes not previously associated with this tumor etiology, involving cytokine and chemokine signaling, were overactivated. Braf+Tert animals remained responsive to MAPK pathway inhibitors. These models constitute useful pre-clinical tools to understand the cell-autonomous and microenvironment-related consequences of Tert-mediated progression in advanced thyroid cancers and other aggressive tumors carrying TPMs.
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Project description:The principal unmet need in BRAFV600E-positive melanoma is lack of an adequate therapeutic strategy capable of overcoming resistance to clinically approved targeted therapies against oncogenic BRAF and/or the downstream MEK1/2 kinases. We previously discovered that copper (Cu) is required for MEK1 and MEK2 activity through a direct Cu-MEK1/2 interaction. Repurposing the clinical Cu chelator tetrathiomolybdate (TTM) is supported by efficacy in BRAFV600E-driven melanoma models, due in part to inhibition of MEK1/2 kinase activity. However, the antineoplastic activity of Cu chelators is cytostatic. Here, we performed high-throughput small-molecule screens to identify bioactive compounds that synergize with TTM in BRAFV600E-driven melanoma cells. Genetic perturbation or pharmacologic inhibition of specific members of the BCL2 family of antiapoptotic proteins (BCL-W, BCL-XL, and MCL1) selectively reduced cell viability when combined with a Cu chelator and induced CASPASE-dependent cell death. Further, in BRAFV600E-positive melanoma cells evolved to be resistant to BRAF and/or MEK1/2 inhibitors, combined treatment with TTM and the clinically evaluated BCL2 inhibitor, ABT-263, restored tumor growth suppression and induced apoptosis. These findings further support Cu chelation as a therapeutic strategy to target oncogene-dependent tumor cell growth and survival by enhancing Cu chelator efficacy with chemical inducers of apoptosis, especially in the context of refractory or relapsed BRAFV600E-driven melanoma. SIGNIFICANCE: This study unveils a novel collateral drug sensitivity elicited by combining copper chelators and BH3 mimetics for treatment of BRAFV600E mutation-positive melanoma.
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Project description:In the present study, the anti-tumor effects of combination treatment with an siRNA targeting B-Raf proto-oncogene serine/threonine kinase (BRAF)V600E and phosphoinositide 3-kinase (PI3K) signaling pathway inhibitors was investigated in melanoma cell lines harboring BRAFV600E. Human melanoma A375 and WM115 cells were treated with siRNA targeting to BRAF or BRAFV600E, combined with treatment with PI3K signaling pathway inhibitors. CCK-8 and EdU proliferation assays were performed to assess cell viability and proliferation, respectively, following treatment. In addition, flow cytometry analysis was performed to determine cell cycle distribution, and western blot analysis was performed to analyze the activity of the extracellular signal-regulated kinase (ERK) and PI3Ksignaling pathways following treatment. Targeting BRAFV600E using small interfering (si)RNA significantly decreased cell viability and DNA replication in tumor cell lines that harbor oncogenic BRAFV600E. Inhibition of BRAFV600E by siRNA combined with treatment with PI3K or mammalian target of rapamycin signaling pathway inhibitors significantly decreased cell viability and proliferation compared with siRNA or inhibitor treatment alone. Concomitant BRAFV600E and PI3K inhibition led to G1/S phase arrest in melanoma cells. However, melanoma cells in which oncogenic BRAFV600E is not highly expressed (WM115 cells) were not sensitive to BRAFV600E targeted therapy. The PI3K signaling pathway inhibitors were more effective in this cell line. The results from the present study provide an insight into the potential effectiveness of combination therapy and personalized cancer treatments.
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Project description:Phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and protein kinase C (PKC), and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, owing to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI3K). Selective inactivation of Pdk1 in the melanocytes of Braf(V600E)::Pten(-/-) or Braf(V600E)::Cdkn2a(-/-)::Pten(-/-) mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-hydroxytamoxifen. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in Braf(V600E)::Pten(-/-) mice. Pdk1(-/-) melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten wild-type (WT) genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes, and inhibition of FOXO3a restored proliferation and colony formation of Pdk1(-/-) melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.
| S-EPMC3955742 | biostudies-literature
Project description:To optimally integrate targeted kinase inhibitors and immunotherapies in the treatment of melanoma, it will be critical to understand how BRAFV600E mutational status and BRAFV600E inhibition influence the expression of genes that govern antitumor immune responses. Because major histocompatibility complex (MHC) molecules are critical for interactions between tumor cells and lymphocytes, we investigated the impact of BRAFV600E-selective inhibitors on the expression of MHC molecules. We found that the treatment of A375 melanoma cells with vemurafenib enhances the induction of MHC Class I and Class II molecules by interferon γ and IFNα2b. Consistent with these findings, we observed that the forced overexpression of BRAFV600E has the opposite effect and can repress the baseline expression of MHC Class I molecules in A375 cells. Further studies utilizing eight other melanoma cell lines revealed that the vemurafenib-mediated enhancement of MHC induction by IFNγ only occurs in the context of homozygous, but not heterozygous, BRAFV600E mutation. These findings suggest that BRAFV600E activity directly influences the expression of MHC molecules and the response to Type I and Type II IFNs. Furthermore, our data suggest that the effect of vemurafenib on the expression of immune system-relevant genes may depend on the zygosity of the BRAFV600E mutation, which is not routinely assessed in melanoma patients.
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Project description:BackgroundDisseminated BRAFV600E melanoma responds to BRAF inhibitors (BRAFi) but easily develops resistance with poor prognosis. Secretome plays a pivotal role during tumour progression causing profound effects on therapeutic efficacy. Secreted M-CSF is involved in both cytotoxicity suppression and tumour progression in melanoma. We aimed to analyse the M-CSF contribution in resistant metastatic melanoma to BRAF-targeted therapies.MethodsConditioned media from melanoma cells were analysed by citoarray. Viability and migration/invasion assays were performed with paired melanoma cells and tumour growth in xenografted SCID mice. We evaluated the impact of M-CSF plasma levels with clinical prognosis from 35 metastatic BRAFV600E-mutant melanoma patients.ResultsBRAFi-resistant melanoma cells secretome is rich in pro-tumour cytokines. M-CSF secretion is essential to induce a Vemurafenib-resistant phenotype in melanoma cells. Further, we demonstrated that M-CSF mAb in combination with Vemurafenib and autophagy blockers synergistically induce apoptosis, impair migration and reduce tumour growth in BRAFi-resistant melanoma cells. Interestingly, lower M-CSF plasma levels are associated with better prognosis in metastatic melanoma patients.ConclusionsSecreted M-CSF induces a BRAFi-resistant phenotype and means worse prognosis in BRAFV600E metastatic melanoma patients. These results identify secreted M-CSF as a promising therapeutic target toward BRAFi-resistant melanomas.
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