Abstract B01: Genome-scale shRNA screen provides insight into the role of CUL3 in adaptive mechanisms of resistance to mutant BRAF inhibition

Abstract MAPK inhibition with targeted BRAF inhibitors has revolutionized the treatment of BRAF-mutant melanoma. However, in most cases, the effects of mutant BRAF inhibition are transient, owing to the acquisition of drug resistance. To resolve mechanisms that drive resistance to mutant BRAF inhibition, we performed a genome-scale shRNA screen capable of revealing genes whose loss of function confers resistance to vemurafenib (VEM). We validated ten of our top candidates with independent lentiviral shRNA constructs. We found that knockdown of NF1 and CUL3 confers resistance to VEM. This result is consistent with previous genome-scale loss-of-function screens that have also identified NF1 and CUL3 as drivers of resistance to mutant BRAF inhibition. NF1 encodes a protein that hydrolyzes Ras-GTP. Not surprisingly, it has been shown that loss of NF1 results in increased Ras-GTP levels that lead to reactivation of MAPK in the presence of VEM. However, little is known about the mechanistic changes that underlie CUL3-mediated resistance. Here, we show that unlike the classical reactivation of MAPK that is seen with the loss of NF1, loss of CUL3 results in increased signaling through a Rac1-dependent focal-adhesion-like process that is accompanied by sustained MAPK activity in the presence of VEM. Furthermore, we show that this mechanism of resistance can be abrogated by inhibiting the Src kinase family, a nine-member protein family that critically mediates integrin signaling. In relation to other known drivers of resistance to mutant BRAF inhibition, loss of NF1 reactivates MAPK in a manner much like RAS mutations or BRAF alterations/amplification. By contrast, loss of CUL3 leads to resistance reminiscent of a reversible therapy-resistant state that lacks the key genomic alterations in MAPK signaling proteins. Citation Format: Marion Vanneste, Eliot Zhu, Charlotte Feddersen, Afshin Varzavand, Tyler Foley, Lei Zhao, Rob Piper, Christopher Stipp, Adam Dupuy, Michael Henry. Genome-scale shRNA screen provides insight into the role of CUL3 in adaptive mechanisms of resistance to mutant BRAF inhibition [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B01.</jats:p

Abl kinase deficiency promotes AKT pathway activation and prostate cancer progression and metastasis

AbstractAbl family kinases function as proto-oncogenes in various leukemias, and pro-tumor functions have been discovered for Abl kinases in solid tumors as well. However, a growing body of evidence indicates that Abl kinases can function to suppress tumor cell proliferation, motility, and in vivo tumor growth in some settings. To investigate the role of Abl kinases in prostate cancer, we generated Abl-deficient cells in a pre-clinical model of spontaneously metastatic, androgen-indifferent prostate cancer. Loss of 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. Treatment of Abl kinase-expressing cells with the Abl kinase inhibitor imatinib phenocopied the malignant phenotypes observed in Abl-deficient tumor cells. In addition, inhibiting AKT pathway signaling abolished the increased 3D growth of Abl-deficient cells. Our data reveal that Abl family kinases can function as suppressors of prostate cancer progression and metastasis by restraining AKT signaling, a signaling pathway known to be associated with emergence of metastatic castration-resistant prostate cancer.</jats:p

Abstract PR17: Sleeping Beauty mutagenesis reveals a Src-dependent DBL GEF family signaling mechanism driving MAPK inhibitor resistance in BRAF mutant melanoma

Abstract BRAF/MEK inhibition remains an important treatment option for patients with BRAF V600 mutant melanoma who show disease progression on immunotherapy; however, the majority of patients treated with BRAF/MEK (MAPKi) therapy develop MAPKi-resistant disease within two years of initiating treatment. Genomic analysis of drug-resistant melanomas has identified somatic mutations associated with resistance, including BRAF amplification or truncation and RAS mutation. In addition, other forms of acquired resistance involving adaptation and reprogramming have been described, with different studies identifying different drivers in different melanoma cell lines and patient-derived xenografts. The heterogeneity of acquired resistance mechanisms presents a major clinical challenge for identifying novel drug combinations with potential activity in significant subsets of patients. To enable the rapid identification of context-dependent drivers of MAPKi resistance, we adapted our Sleeping Beauty (SB) transposon mutagenesis for use in cell culture. SB mutagenesis in this setting identifies predominantly gain-of-function mechanisms and thus has the ability to identify both genetic mutations (such as expression of truncated proteins) and genes within alternative acquired resistance pathways whose upregulated expression drives MAPKi resistance. Validating our approach, our screen identified N-terminal truncation of BRAF—a known mechanism of vemurafenib resistance. In addition, we identified DBL family guanine exchange factors (GEFs), MCF2 and VAV1, as drivers of MAPKi resistance that we have functionally validated in multiple BRAF V600E mutant melanoma cell lines. Crucially, many DBL family GEFs are well known to be regulated by Src family kinases (SFKs), and our data indicate that GEF-driven MAPKi resistance can be blocked by combination treatment with vemurafenib and saracatinib, a selective SFK inhibitor. Expansion of resistant cells in the presence of vemurafenib or vemurafenib with cobimetinib can be reversed by switching to vemurafenib plus saracatinib. Consistently, we find that adding saracatinib converts MAPKi-mediated cytostasis into cell killing and blocks spontaneous MAPKi resistance in responsive melanoma cell lines. The GEFs signal downstream to a RAC1-PAK kinase module that is required for GEF-driven resistance. Importantly, the potential utility of both SFK inhibitors and PAK inhibitors for overcoming MAPKi resistance has been proposed, but the connection between SFK-dependent and PAK-dependent resistance mechanisms had not been obvious. We show that combined BRAF/SFK inhibition is effective in cell lines in which the GEF-driven mechanism operates, but not in cell lines in which the GEFs do not drive resistance. Our SB mutagenesis system has the ability to rapidly survey the context-dependent landscape of MAPKi resistance in any transfectable cell system, with the potential to reveal connections between seemingly disparate resistance mechanisms. This abstract is also being presented as Poster B04. Citation Format: Charlotte R. Feddersen, Jacob Schillo, Afshin Varzavand, Hayley Vaughn, Andrew Voight, Eliot Zhu, Jesse D Riordan, Christopher S. Stipp, Adam J. Dupuy. Sleeping Beauty mutagenesis reveals a Src-dependent DBL GEF family signaling mechanism driving MAPK inhibitor resistance in BRAF mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR17.</jats:p

Supplementary Information from α3β1 Integrin Suppresses Prostate Cancer Metastasis via Regulation of the Hippo Pathway

&lt;p&gt;RNAi targeting sequences, Supplemental Figure Legends, &amp; Supplemental Figures S1-S11&lt;/p&gt;</jats:p

Data from α3β1 Integrin Suppresses Prostate Cancer Metastasis via Regulation of the Hippo Pathway

&lt;div&gt;Abstract&lt;p&gt;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 &lt;i&gt;in vivo&lt;/i&gt; model of prostate cancer metastasis via depletion of α3β1 integrin, a correlation observed in a significant proportion of prostate cancers. Our data describe a mechanism whereby α3β1 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 α3β1-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. &lt;i&gt;Cancer Res; 76(22); 6577–87. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;</jats:p

Abstract B08: Identification and characterization of Rho family GTPases as drivers of drug resistance in <i>BRAFV600</i> mutant melanoma

Abstract The serine/threonine protein kinase BRAF is mutated in approximately 50% of cutaneous melanomas, leading to hyperactivation of the MAPK/ERK pathway. The most common mutations, BRAFV600, can be targeted by selective kinase inhibitors, such as vemurafenib. Although initial clinical response to BRAF inhibition (BRAFi) is encouraging, 90% of patients develop drug resistance within a few months. Drug resistance can be delayed, but not prevented, by combining BRAFi with an MEK inhibitor (MEKi), such as cobimetinib. While some resistance mechanisms are known, disease progression on drug cannot be explained in all patients. We performed a gain-of-function mutagenesis screen utilizing the Sleeping Beauty transposon system to identify novel drivers of resistance in BRAFV600E mutant melanoma cells sensitive to current therapies. We chose four of the top candidates from our screen and validated the ability to drive resistance to both vemurafenib and vemurafenib-cobimetinib combination treatment in multiple melanoma cell lines. In an effort to determine the broader role of candidate vemurafenib-resistance drivers, we conducted an additional in vivo mutagenesis screen, of which genetic analysis is ongoing. Our initial cell-based screen identified two members of the Dbl family of guanine nucleotide exchange factors (GEFs), VAV1 and MCF2, as candidate drivers of vemurafenib resistance. A375 melanoma cells overexpressing VAV1 or MCF2 maintain significant growth under vemurafenib treatment, while control cells do not. Functional tests of VAV1 and MCF2 identified that the active form of two Rho family members, RAC1 and CDC42, increases following treatment with vemurafenib, suggesting a PAK-mediated pathway of resistance. In addition, all candidates that were tested elevated ERK signaling in the presence of vemurafenib. Many of the extracellular signaling pathways known to drive increased vemurafenib resistance activate Rho signaling. Our results suggest that Dbl family members may play an important role in this process. Understanding how Rho activation occurs and its consequences for drug resistance in melanoma will provide critical insights into the design and validation of future targeted therapies. Citation Format: Jacob L. Schillo, Charlotte R. Feddersen, Afshin Varzavand, Hayley R. Vaughn, Lexy S. Wadsworth, Andrew P. Voigt, Eliot Y. Zhu, Jesse D. Riordan, Christopher S. Stipp, Adam J. Dupuy. Identification and characterization of Rho family GTPases as drivers of drug resistance in BRAFV600 mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B08.</jats:p

Supplementary Information from α3β1 Integrin Suppresses Prostate Cancer Metastasis via Regulation of the Hippo Pathway

RNAi targeting sequences, Supplemental Figure Legends, & Supplemental Figures S1-S11</p

Table_1_Abl kinases can function as suppressors of tumor progression and metastasis.xlsx

IntroductionAbl 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.MethodsTo 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.ResultsReduced 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.ConclusionsCollectively, our data reveal that Abl family kinases can function to suppress malignant cell phenotypes in vitro, and tumor progression and metastasis in vivo.</p