Single-cell genomics analysis reveals complex genetic interactions in an in vivo model of acquired BRAF inhibitor resistance.

The evolution of therapeutic resistance is a major obstacle to the success of targeted oncology drugs. While both inter- and intratumoral heterogeneity limit our ability to detect resistant subpopulations that pre-exist or emerge during treatment, our ability to analyze tumors with single-cell resolution is limited. Here, we utilized a cell-based transposon mutagenesis method to identify mechanisms of BRAF inhibitor resistance in a model of cutaneous melanoma. This screen identified overexpression of NEDD4L and VGLL3 as significant drivers of BRAF inhibitor resistance in vivo. In addition, we describe a novel single-cell genomics profiling method to genotype thousands of individual cells within tumors driven by transposon mutagenesis. This approach revealed a surprising genetic diversity among xenograft tumors and identified recurrent co-occurring mutations that emerge within distinct tumor subclones. Taken together, these observations reveal an unappreciated genetic complexity that drives BRAF inhibitor resistance.

Abl kinases can function as suppressors of tumor progression and metastasis.

Introduction: Abl family kinases function as proto-oncogenes in various leukemias, and pro-tumor functions have been discovered for Abl kinases in many solid tumors as well. However, a growing body of evidence indicates that Abl kinases can function to suppress tumor cell proliferation and motility and tumor growth in vivo in some settings. Methods: To investigate the role of Abl kinases in tumor progression, we used RNAi to generate Abl-deficient cells in a model of androgen receptor-indifferent, metastatic prostate cancer. The effect of Abl kinase depletion on tumor progression and metastasis was studied in an in vivo orthotopic model, and tumor cell motility, 3D growth, and signaling was studied in vitro. Results: Reduced Abl family kinase expression resulted in a highly aggressive, metastatic phenotype in vivo that was associated with AKT pathway activation, increased growth on 3D collagen matrix, and enhanced cell motility in vitro. Inhibiting AKT pathway signaling abolished the increased 3D growth of Abl-deficient cells, while treatment with the Abl kinase inhibitor, imatinib, promoted 3D growth of multiple additional tumor cell types. Moreover, Abl kinase inhibition also promoted soft-agar colony formation by pre-malignant fibroblasts. Conclusions: Collectively, our data reveal that Abl family kinases can function to suppress malignant cell phenotypes in vitro, and tumor progression and metastasis in vivo.

Elevated levels of sCD48 are inversely correlated with markers of disease activity in bullous pemphigoid.

sCD48 is elevated in diseases characterized by IgE and eosinophilia. Thus, serum levels sCD48 were evaluated in relation to clinical characteristics of Bullous pemphigoid (BP) patients. sCD48 levels were determined by ELISA in sera from 26 patients with classic BP and 26 healthy controls. Disease severity scores, differential blood counts, and circulating autoantibody levels were obtained. A correlation analysis was performed to establish relationships between sCD48 and clinical and laboratory markers of disease severity. Overall, circulating levels of sCD48 were significantly elevated in BP patients; however, when stratified based on disease severity, patients with mild-moderate disease had higher levels of sCD48 than those with severe disease. A Spearman's correlation analysis identified an inverse relationship between sCD48 and disease activity, serum BP180 IgE and peripheral eosinophil numbers. Further studies are needed to determine the pathologic relevance of these findings.

Functional Genomic Screening Independently Identifies CUL3 as a Mediator of Vemurafenib Resistance via Src-Rac1 Signaling Axis.

Patients with malignant melanoma have a 5-year survival rate of only 15-20% once the tumor has metastasized to distant tissues. While MAP kinase pathway inhibitors (MAPKi) are initially effective for the majority of patients with melanoma harboring BRAFV600E mutation, over 90% of patients relapse within 2 years. Thus, there is a critical need for understanding MAPKi resistance mechanisms. In this manuscript, we performed a forward genetic screen using a whole genome shRNA library to identify negative regulators of vemurafenib resistance. We identified loss of NF1 and CUL3 as drivers of vemurafenib resistance. NF1 is a known driver of vemurafenib resistance in melanoma through its action as a negative regulator of RAS. However, the mechanism by which CUL3, a key protein in E3 ubiquitin ligase complexes, is involved in vemurafenib resistance was unknown. We found that loss of CUL3 was associated with an increase in RAC1 activity and MEKS298 phosphorylation. However, the addition of the Src family inhibitor saracatinib prevented resistance to vemurafenib in CUL3KD cells and reversed RAC1 activation. This finding suggests that inhibition of the Src family suppresses MAPKi resistance in CUL3KD cells by inactivation of RAC1. Our results also indicated that the loss of CUL3 does not promote the activation of RAC1 through stabilization, suggesting that CUL3 is involved in the stability of upstream regulators of RAC1. Collectively, our study identifies the loss of CUL3 as a driver of MAPKi resistance through activation of RAC1 and demonstrates that inhibition of the Src family can suppress the MAPKi resistance phenotype in CUL3KD cells by inactivating RAC1 protein.

Src-Dependent DBL Family Members Drive Resistance to Vemurafenib in Human Melanoma.

The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a BRAFV600E mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/RAC1/PAK signaling axis capable of driving resistance to both current and next-generation BRAFis. However, we show that the SRC inhibitor, saracatinib, can block the DBL-driven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance. SIGNIFICANCE: A simple, rapid, and flexible genetic screening approach identifies genes that drive resistance to MAPK inhibitors when overexpressed in human melanoma cells.

α3ß1 Integrin Suppresses Prostate Cancer Metastasis via Regulation of the Hippo Pathway.

Existing anticancer strategies focused on disrupting integrin functions in tumor cells or tumor-involved endothelial cells have met limited success. An alternative strategy is to augment integrin-mediated pathways that suppress tumor progression, but how integrins can signal to restrain malignant behavior remains unclear. To address this issue, we generated an in vivo model of prostate cancer metastasis via depletion of α3ß1 integrin, a correlation observed in a significant proportion of prostate cancers. Our data describe a mechanism whereby α3ß1 signals through Abl family kinases to restrain Rho GTPase activity, support Hippo pathway suppressor functions, and restrain prostate cancer migration, invasion, and anchorage-independent growth. This α3ß1-Abl kinase-Hippo suppressor pathway identified α3 integrin-deficient prostate cancers as potential candidates for Hippo-targeted therapies currently under development, suggesting new strategies for targeting metastatic prostate cancer based on integrin expression. Our data also revealed paradoxical tumor suppressor functions for Abl kinases in prostate cancer that may help to explain the failure of Abl kinase inhibitor imatinib in prostate cancer clinical trials. Cancer Res; 76(22); 6577-87. ©2016 AACR.

Integrin α3ß1 regulates tumor cell responses to stromal cells and can function to suppress prostate cancer metastatic colonization.

Integrin α3ß1 promotes tumor cell adhesion, migration, and invasion on laminin isoforms, and several clinical studies have indicated a correlation between increased tumoral α3ß1 integrin expression and tumor progression, metastasis, and poor patient outcomes. However, several other clinical and experimental studies have suggested that α3ß1 can possess anti-metastatic activity in certain settings. To help define the range of α3ß1 functions in tumor cells in vivo, we used RNAi to silence the α3 integrin subunit in an aggressive, in vivo-passaged subline of PC-3 prostate carcinoma cells. Loss of α3 integrin impaired adhesion and proliferation on the α3ß1 integrin ligand, laminin-332 in vitro. Despite these deficits in vitro, the α3-silenced cells were significantly more aggressive in a lung colonization model in vivo, with a substantially increased rate of tumor growth that significantly reduced survival. In contrast, silencing the related α6 integrin subunit delayed metastatic growth in vivo. The increased colonization of α3-silenced tumor cells in vivo was recapitulated in 3D collagen co-cultures with lung fibroblasts or pre-osteoblast-like cells, where α3-silenced cells showed dramatically enhanced growth. The increased response of α3-silenced tumor cells to stromal cells in co-culture could be reproduced by fibroblast conditioned medium, which contains one or more heparin-binding factors that selectively favor the growth of α3-silenced cells. Our new data suggest a scenario in which α3ß1 regulates tumor-host interactions within the metastatic tumor microenvironment to limit growth, providing some of the first direct evidence that specific loss of α3 function in tumor cells can have pro-metastatic consequences in vivo.

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5.

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.