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Ibrutinib blocks YAP1 activation and reverses BRAFi resistance in melanoma cells

ABSTRACT: Most BRAF-mutant melanoma tumors respond initially to BRAFi/MEKi therapy, although few patients have durable long-term responses to these agents. The goal of this study was to utilize an unbiased computational approach to identify inhibitors which reverse an experimentally derived BRAFi resistance gene expression signature. Using this approach, we predicted that ibrutinib, a BTK inhibitor, effectively reverses this signature, and we demonstrate experimentally that ibrutinib re-sensitizes a subset of BRAFi-resistant melanoma cells to vemurafenib. Ibrutinib is used clinically as a BTK inhibitor; however, neither BTK deletion nor treatment with acalabrutinib, an analog of ibrutinib with reduced off-target activity, re-sensitized cells to vemurafenib. These data suggest that ibrutinib acts through a BTK-independent mechanism in vemurafenib re-sensitization. To better understand this mechanism, we analyzed the transcriptional profile of ibrutinib-treated BRAFi-resistant melanoma cells and found that the transcriptional profile of ibrutinib was highly similar to that of multiple SRC kinase inhibitors. Since ibrutinib, but not acalabrutinib, has significant off-target activity against multiple SRC family kinases, it suggests that ibrutinib may be acting through this mechanism. Furthermore, genes either upregulated or downregulated by ibrutinib treatment are enriched with YAP1 target genes and we experimentally demonstrate that ibrutinib, but not acalabrutinib, reduces YAP1 activity in BRAFi-resistant melanoma cells. Taken together, these data suggest that ibrutinib, or other SRC family kinase inhibitors, may be useful for treating some BRAFi/MEKi-refractory melanoma tumors.

ORGANISM(S): Homo sapiens

PROVIDER: GSE145990 | GEO | 2020/02/27

REPOSITORIES: GEO

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Project description:Nearly 50% of cutaneous melanomas carry activating mutations on the BRAF oncogene, and the combination of BRAF- and MEK-inhibitors (BRAF/MEKi) is frequently used for their clinical management. One major drawback of BRAF/MEKi targeted therapy is the rapid development of therapeutic resistance, which can occur via multiple mechanisms, including the metabolic rewiring of cancer cells that often involves the upregulation of mitochondrial bionergetics and NAD+ biosynthetic pathways. mTORC2 is a signaling complex that requires the presence of its essential RICTOR subunit to play its regulatory functions in cell growth and metabolism. mTORC2 is believed to play mostly pro-oncogenic roles in several tumor types, including melanoma. However, bioinformatics analysis of TCGA melanoma patients’ database revealed that low RICTOR levels in tumors correlate with an overall worse clinical outcome. GSEA analysis of low-RICTOR tumors evidenced also a gene expression signature suggestive of activation of mitochondrial energy producing pathways. On these bases, we have hypothesized that inhibition of mTORC2 activity may render BRAFV600E melanoma cells resistant to BRAFi/MEKi. We show here that RICTOR/mTORC2-deficient cells are intrinsically tolerant to BRAFi/MEKi, and anticipate the onset of resistance to BRAFi after sustained drug exposure both in vitro and in vivo, indicating that mTORC2 activity normally opposes the acquisition of targeted therapy resistance in BRAFV600E melanomas. Mechanistically, RICTOR-deficient cells show an enhanced mitochondrial respiratory potential and increased expression of nicotinamide phosphoribosyltransferase (NAMPT) protein, the rate-limiting enzyme of NAD+ salvage pathway, and pharmacological inhibition of these processes in RICTOR-deficient cells is sufficient to restore sensitivity to BRAFi. Thus, our work identifies a novel role for mTORC2 in favoring the responses of BRAF-mutated melanoma cells to targeted therapy, and suggest that the evaluation of the intratumor level of RICTOR may help to predict the responses of melanoma patients to these treatments.

Project description:Myeloid-derived suppressor cell (MDSC) levels are elevated in cancer patients and contribute to reduced efficacy of immune checkpoint therapy. MDSC express Bruton’s Tyrosine Kinase (BTK) and BTK inhibition with ibrutinib, an FDA-approved irreversible inhibitor of BTK, leads to reduced MDSC expansion/function in mice and significantly improves the anti-tumor activity of anti-PD-1 antibody treatments. Single-cell RNA sequencing (scRNA-seq) was used to characterize the effect of ibrutinib on gene expression of FACS-enriched MDSC from patients with different cancer types (breast, melanoma, head and neck squamous cell cancer - HNSCC). Melanoma patient MDSC were treated in vitro for 4h with 5 µM ibrutinib or DMSO, processed for scRNA-seq using the Chromium 10x Genomics platform, and analyzed via the Seurat v4 standard integrative workflow. Baseline gene expression of MDSC from breast and HNSCC cancer patients revealed similarities among the top expressed genes. In vitro ibrutinib treatment of MDSC from melanoma patients resulted in significant changes in gene expression. GBP1, IL 1β and CXCL8 were among the top downregulated genes while RGS2 and ABHD5 were among the top upregulated genes (p<0.001). double positive CD14+CD15+ MDSC and PMN-MDSC responded similarly to BTK inhibition and exhibited more pronounced gene changes when compared to early MDSC and M-MDSC. Pathway analysis revealed significantly downregulated pathways including TREM1, nitric oxide signaling, and IL 6 signaling (p<0.004). ScRNA-seq revealed characteristic gene expression patterns for MDSC from different cancer patients. BTK inhibition led to the downregulation of multiple genes and pathways important to MDSC function and migration. Myeloid-derived suppressor cell (MDSC) levels are elevated in cancer patients and contribute to reduced efficacy of immune checkpoint therapy. MDSC express Bruton’s Tyrosine Kinase (BTK) and BTK inhibition with ibrutinib, an FDA-approved irreversible inhibitor of BTK, leads to reduced MDSC expansion/function in mice and significantly improves the anti-tumor activity of anti-PD-1 antibody treatments. Single-cell RNA sequencing (scRNA-seq) was used to characterize the effect of ibrutinib on gene expression of FACS-enriched MDSC from patients with different cancer types (breast, melanoma, head and neck squamous cell cancer - HNSCC). Melanoma patient MDSC were treated in vitro for 4h with 5 µM ibrutinib or DMSO, processed for scRNA-seq using the Chromium 10x Genomics platform, and analyzed via the Seurat v4 standard integrative workflow. Baseline gene expression of MDSC from breast and HNSCC cancer patients revealed similarities among the top expressed genes. In vitro ibrutinib treatment of MDSC from melanoma patients resulted in significant changes in gene expression. GBP1, IL 1β and CXCL8 were among the top downregulated genes while RGS2 and ABHD5 were among the top upregulated genes (p<0.001). double positive CD14+CD15+ MDSC and PMN-MDSC responded similarly to BTK inhibition and exhibited more pronounced gene changes when compared to early MDSC and M-MDSC. Pathway analysis revealed significantly downregulated pathways including TREM1, nitric oxide signaling, and IL 6 signaling (p<0.004). ScRNA-seq revealed characteristic gene expression patterns for MDSC from different cancer patients. BTK inhibition led to the downregulation of multiple genes and pathways important to MDSC function and migration.

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Ibrutinib blocks YAP1 activation and reverses BRAFi resistance in melanoma cells

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