The SRC-family serves as a therapeutic target in triple negative breast cancer with acquired resistance to chemotherapy.

BACKGROUND: Resistance to chemotherapy, combined with heterogeneity among resistant tumors, represents a significant challenge in the clinical management of triple negative breast cancer (TNBC). By dissecting molecular pathways associated with treatment resistance, we sought to define patient sub-groups and actionable targets for next-line treatment. METHODS: Bulk RNA sequencing and reverse phase protein array profiling were performed on isogenic patient-derived xenografts (PDX) representing paclitaxel-sensitive and -resistant tumors. Pathways identified as upregulated in the resistant model were further explored as targets in PDX explants. Their clinical relevance was assessed in two distinct patient cohorts (NeoAva and MET500). RESULTS: Increased activity in signaling pathways involving SRC-family kinases (SFKs)- and MAPK/ERK was found in treatment resistant PDX, with targeted inhibitors being significantly more potent in resistant tumors. Up-regulation of SFKs- and MAPK/ERK-pathways was also detected in a sub-group of chemoresistant patients after neoadjuvant treatment. Furthermore, High SFK expression (of either SRC, FYN and/or YES1) was detected in metastatic lesions of TNBC patients with fast progressing disease (median disease-free interval 27 vs 105 months). CONCLUSIONS: Upregulation of SFK-signaling is found in a subset of chemoresistant tumors and is persistent in metastatic lesions. Based on pre-clinical results, these patients may respond favorably to treatment targeting SFKs.

AXL inhibition improves BRAF-targeted treatment in melanoma.

More than half of metastatic melanoma patients receiving standard therapy fail to achieve a long-term survival due to primary and/or acquired resistance. Tumor cell ability to switch from epithelial to a more aggressive mesenchymal phenotype, attributed with AXLhigh molecular profile in melanoma, has been recently linked to such event, limiting treatment efficacy. In the current study, we investigated the therapeutic potential of the AXL inhibitor (AXLi) BGB324 alone or in combination with the clinically relevant BRAF inhibitor (BRAFi) vemurafenib. Firstly, AXL was shown to be expressed in majority of melanoma lymph node metastases. When treated ex vivo, the largest reduction in cell viability was observed when the two drugs were combined. In addition, a therapeutic benefit of adding AXLi to the BRAF-targeted therapy was observed in pre-clinical AXLhigh melanoma models in vitro and in vivo. When searching for mechanistic insights, AXLi was found to potentiate BRAFi-induced apoptosis, stimulate ferroptosis and inhibit autophagy. Altogether, our findings propose AXLi as a promising treatment in combination with standard therapy to improve therapeutic outcome in metastatic melanoma.

Detection of phenotype-specific therapeutic vulnerabilities in breast cells using a CRISPR loss-of-function screen.

Cellular phenotype plasticity between the epithelial and mesenchymal states has been linked to metastasis and heterogeneous responses to cancer therapy, and remains a challenge for the treatment of triple-negative breast cancer (TNBC). Here, we used isogenic human breast epithelial cell lines, D492 and D492M, representing the epithelial and mesenchymal phenotypes, respectively. We employed a CRISPR-Cas9 loss-of-function screen targeting a 2240-gene 'druggable genome' to identify phenotype-specific vulnerabilities. Cells with the epithelial phenotype were more vulnerable to the loss of genes related to EGFR-RAS-MAPK signaling, while the mesenchymal-like cells had increased sensitivity to knockout of G2 -M cell cycle regulators. Furthermore, we discovered knockouts that sensitize to the mTOR inhibitor everolimus and the chemotherapeutic drug fluorouracil in a phenotype-specific manner. Specifically, loss of EGFR and fatty acid synthase (FASN) increased the effectiveness of the drugs in the epithelial and mesenchymal phenotypes, respectively. These phenotype-associated genetic vulnerabilities were confirmed using targeted inhibitors of EGFR (gefitinib), G2 -M transition (STLC), and FASN (Fasnall). In conclusion, a CRISPR-Cas9 loss-of-function screen enables the identification of phenotype-specific genetic vulnerabilities that can pinpoint actionable targets and promising therapeutic combinations.

EMT-Derived Alterations in Glutamine Metabolism Sensitize Mesenchymal Breast Cells to mTOR Inhibition.

Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention. Here we used 13C carbon analogs of glucose and glutamine to examine differences in their utilization within central carbon and lipid metabolism following EMT in breast epithelial cell lines. We found that there are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of the TCA-cycle, characterized by increased mitochondrial IDH2-mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Using weighted correlation network analysis, we identified cancer drugs whose efficacy against the NCI-60 Human Tumor Cell Line panel is significantly associated with GSH abundance and confirmed these in vitro. We report that EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to mTOR inhibitors. IMPLICATIONS: EMT in breast cells causes an increased demand for glutamine for fatty acid biosynthesis, altering its contribution to glutathione biosynthesis, which sensitizes the cells to mTOR inhibitors.

Inhibition of O-GlcNAc transferase activates tumor-suppressor gene expression in tamoxifen-resistant breast cancer cells.

In this study, we probed the importance of O-GlcNAc transferase (OGT) activity for the survival of tamoxifen-sensitive (TamS) and tamoxifen-resistant (TamR) breast cancer cells. Tamoxifen is an antagonist of estrogen receptor (ERα), a transcription factor expressed in over 50% of breast cancers. ERα-positive breast cancers are successfully treated with tamoxifen; however, a significant number of patients develop tamoxifen-resistant disease. We show that in vitro development of tamoxifen-resistance is associated with increased sensitivity to the OGT small molecule inhibitor OSMI-1. Global transcriptome profiling revealed that TamS cells adapt to OSMI-1 treatment by increasing the expression of histone genes. This is known to mediate chromatin compaction. In contrast, TamR cells respond to OGT inhibition by activating the unfolded protein response and by significantly increasing ERRFI1 expression. ERRFI1 is an endogenous inhibitor of ERBB-signaling, which is a known driver of tamoxifen-resistance. We show that ERRFI1 is selectively downregulated in ERα-positive breast cancers and breast cancers driven by ERBB2. This likely occurs via promoter methylation. Finally, we show that increased ERRFI1 expression is associated with extended survival in patients with ERα-positive tumors (p = 9.2e-8). In summary, we show that tamoxifen-resistance is associated with sensitivity to OSMI-1, and propose that this is explained in part through an epigenetic activation of the tumor-suppressor ERRFI1 in response to OSMI-1 treatment.

O-GlcNAc Transferase Inhibition Differentially Affects Breast Cancer Subtypes.

Post-translational modification of intracellular proteins with a single N-acetylglucosamine sugar (O-GlcNAcylation) regulates signaling, proliferation, metabolism and protein stability. In breast cancer, expression of the enzyme that catalyzes O-GlcNAcylation - O-GlcNAc-transferase (OGT), and the extent of protein O-GlcNAcylation, are upregulated in tumor tissue, and correlate with cancer progression. Here we compare the significance of O-GlcNAcylation in a panel of breast cancer cells of different phenotypes. We find a greater dependency on OGT among triple-negative breast cancer (TNBC) cell lines, which respond to OGT inhibition by undergoing cell cycle arrest and apoptosis. Searching for the cause of this response, we evaluate the changes in the proteome that occur after OGT inhibition or knock-down, employing a reverse-phase protein array (RPPA). We identify transcriptional repressor - hairy and enhancer of split-1 (HES1) - as a mediator of the OGT inhibition response in the TNBC cells. Inhibition of OGT as well as the loss of HES1 results in potent cytotoxicity and apoptosis. The study raises a possibility of using OGT inhibition to potentiate DNA damage in the TNBC cells.

Stroma-induced phenotypic plasticity offers phenotype-specific targeting to improve melanoma treatment.

Cancer cells' phenotypic plasticity, promoted by stromal cells, contributes to intra-tumoral heterogeneity and affects response to therapy. We have disclosed an association between fibroblast-stimulated phenotype switching and resistance to the clinically used BRAF inhibitor (BRAFi) vemurafenib in malignant melanoma, revealing a challenge in targeting the fibroblast-induced phenotype. Here we compared molecular features and drug sensitivity in melanoma cells grown as co-cultures with fibroblasts versus mono-cultures. In the presence of fibroblasts, melanoma cells switched to the dedifferentiated, mesenchymal-like, inflammatory phenotype that showed reduced sensitivity to the most of 275 tested cancer drugs. Fibroblasts, however, sensitized melanoma cells to PI3K inhibitors (PI3Ki) and particularly the inhibitor of GSK3, AR-A014418 (GSK3i), that showed superior efficacy in co-cultures. The proteome changes induced by the BRAFi + GSK3i combination mimicked changes induced by BRAFi in mono-cultures, and GSK3i in co-cultures. This suggests that the single drug drives the response to the combination treatment, depending on fibroblast presence or absence, consequently, phenotype. We propose that the BRAFi and GSK3i (or PI3Ki) combination exemplifies phenotype-specific combinatorial treatment that should be beneficial in phenotypically heterogeneous tumors rich in stromal interactions.

Fibroblast-induced switching to the mesenchymal-like phenotype and PI3K/mTOR signaling protects melanoma cells from BRAF inhibitors.

The knowledge on how tumor-associated stroma influences efficacy of anti-cancer therapy just started to emerge. Here we show that lung fibroblasts reduce melanoma sensitivity to the BRAF inhibitor (BRAFi) vemurafenib only if the two cell types are in close proximity. In the presence of fibroblasts, the adjacent melanoma cells acquire de-differentiated mesenchymal-like phenotype. Upon treatment with BRAFi, such melanoma cells maintain high levels of phospho ribosomal protein S6 (pS6), i.e. active mTOR signaling, which is suppressed in the BRAFi sensitive cells without stromal contacts. Inhibitors of PI3K/mTOR in combination with BRAFi eradicate pS6high cell subpopulations and potentiate anti-cancer effects in melanoma protected by the fibroblasts. mTOR and BRAF co-inhibition also delayed the development of early-stage lung metastases in vivo. In conclusion, we demonstrate that upon influence from fibroblasts, melanoma cells undergo a phenotype switch to the mesenchymal state, which can support PI3K/mTOR signaling. The lost sensitivity to BRAFi in such cells can be overcome by co-targeting PI3K/mTOR. This knowledge could be explored for designing BRAFi combination therapies aiming to eliminate both stroma-protected and non-protected counterparts of metastases.