Biology subjects

Misek, S. A.

Publications and source records attributed to Misek, S. A..

7 recordsLinked to original sources

bioRxiv2023

Inhibition of the Rho/MRTF pathway improves the response of BRAF-resistant melanoma to PD1/PDL1 blockade

Metastatic cutaneous melanoma is a fatal skin cancer. Resistance to targeted and immune therapies limits the benefits of current treatments. Identifying and adding anti-resistance agents to current treatment protocols can potentially improve clinical responses. Myocardin-related transcription factor (MRTF) is a transcriptional coactivator whose activity is indirectly regulated by actin and the Rho family of GTPases. We previously demonstrated that development of BRAF inhibitor (BRAFi) resistance frequently activates the Rho/MRTF pathway in human and mouse BRAFV600E melanomas. In clinical trials, pre-treatment with BRAFi reduces the benefit of immune therapies. We aimed to test the efficacy of concurrent treatment with our MRTF pathway inhibitor CCG-257081 and anti-PD1 in vivo and to examine its effects on the melanoma immune microenvironment. Because MRTF pathway activation upregulates the expression of immune checkpoint inhibitor genes/proteins, we asked whether CCG-257081 can improve the response to immune checkpoint blockade. CCG-257081 reduced the expression of PDL1 in BRAFi-resistant melanoma cells and decreased surface PDL1 levels on both BRAFi- sensitive and -resistant melanoma cells. Using our recently described murine vemurafenib-resistant melanoma model, we found that CCG-257081, in combination with anti-PD1 immune therapy, reduced tumor growth and increased survival. Moreover, anti-PD1/CCG-257081 co-treatment increased infiltration of CD8+ T cells and B cells into the tumor microenvironment and reduced tumor-associated macrophages. Here, we propose CCG-257081 as an anti-resistance and immune therapy-enhancing anti-melanoma agent. Novelty and ImpactWe present a study that provides evidence for a new combined approach for targeting BRAF inhibitor-resistant melanoma. Pharmacological inhibition of the resistance-inducing Rho/MRTF pathway using CCG-257081 enhanced the response to PD1/PDL-1 blockade in vivo. These results indicate a role of the Rho/MRTF pathway in regulating tumor-immune interactions. Thus, CCG-257081 emerges as a potential new anti-resistance agent that can improve the response to immune checkpoint inhibitors in advanced melanoma and, possibly, other cancers.

cancer biology↗

bioRxiv2022

Germline variation contributes to false negatives in CRISPR-based experiments with varying burden across ancestries

Reducing disparities is critical to promote equity of access to precision treatments for all patients with cancer. While socioenvironmental factors are a major driver behind such disparities, biological differences also are likely to contribute. The prioritization of cancer drug targets is foundational for drug discovery, yet whether ancestry-related signals in target discovery pipelines exist has not been systematically explored due to the absence of data at the appropriate scale. Here, we analyzed data from 611 genome-scale CRISPR/Cas9 viability experiments in human cell line models as part of the Cancer Dependency Map to identify ancestry-associated genetic dependencies. Surprisingly, we found that most putative associations between ancestry and dependency arose from artifacts related to germline variants that are present at different frequencies across ancestry groups. In 2-5% of genes profiled in each cellular model, germline variants in sgRNA targeting sequences likely reduced cutting by the CRISPR/Cas9 nuclease. Unfortunately, this bias disproportionately affected cell models derived from individuals of recent African descent because their genomes tended to diverge more from the consensus genome typically used for CRISPR/Cas9 guide design. To help the scientific community begin to resolve this source of bias, we report three complementary methods for ancestry-agnostic CRISPR experiments. This report adds to a growing body of literature describing ways in which ancestry bias impacts cancer research in underappreciated ways.

cancer biology↗

bioRxiv2020

Ibrutinib blocks YAP1 activation and reverses BRAFi resistance in melanoma cells

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 found that ibrutinib 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, another BTK inhibitor 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 in YAP1 target genes and we showed 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.

cancer biology↗

bioRxiv2019

Inhibition of the Myocardin-Related Transcription Factor pathway increases efficacy of Trametinib in NRAS-mutant melanoma cell lines

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas ([~]50% BRAFV600 mutations and [~]30% NRAS mutations). While targeted therapies have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in NRAS mutant melanomas in part due to their cytostatic effects and primary resistance in this patient population. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. Combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells which have a high level of trametinib resistance. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlights the potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

cancer biology↗

bioRxiv2019

E2F1 Drives Breast Cancer Metastasis by Regulating the Target Gene FGF13 and Altering Cell Migration

In prior work we demonstrated that loss of E2F transcription factors inhibits metastasis. Here we address the mechanisms for this phenotype and identify the E2F regulated genes that coordinate tumor cell metastasis. Transcriptomic profiling of E2F1 knockout tumors identified a role for E2F1 as a master regulator of a suite of pro-metastatic genes, but also uncovered E2F1 target genes with an unknown role in pulmonary metastasis. High expression of one of these genes, Fgf13, is associated with early human breast cancer metastasis in a clinical dataset. Together these data led to the hypothesis that Fgf13 is critical for breast cancer metastasis, and that upregulation of Fgf13 may partially explain how E2F1 promotes breast cancer metastasis. To test this hypothesis we ablated Fgf13 via CRISPR. Deletion of Fgf13 in a MMTV-PyMT breast cancer cell line reduces the frequency of pulmonary metastasis. In addition, loss of Fgf13 reduced in vitro cell migration, suggesting that Fgf13 may be critical for tumor cells to invade out of and escape the primary tumor. The significance of this work is twofold: we have both uncovered genomic features by which E2F1 regulates metastasis and we have identified new pro-metastatic functions for the E2F1 target gene Fgf13.

cancer biology↗

bioRxiv2018

Identification of Pirin as a Molecular Target of the CCG-1423/CCG-203971 Series of Anti-Fibrotic and Anti-Metastatic Compounds

A series of compounds (including CCG-1423 and CCG-203971) discovered through an MRTF/SRF dependent luciferase screen has shown remarkable efficacy in a variety of in vitro and in vivo models, including melanoma metastasis and bleomycin-induced fibrosis. Although these compounds are efficacious, the molecular target is unknown. Here, we describe affinity isolation-based target identification efforts which yielded pirin, an iron-dependent co-transcription factor, as a target of this series of compounds. Using biophysical techniques including isothermal titration calorimetry and X-ray crystallography, we verify that pirin binds these compounds in vitro. We also show with genetic approaches that pirin modulates MRTF-dependent SRE.L Luciferase activation. Finally, using both siRNA and a previously validated pirin inhibitor, we show a role for pirin in TGF-p induced gene expression in primary dermal fibroblasts. A recently developed analog, CCG-257081, which co-crystallizes with pirin, is also effective in the prevention of bleomycin-induced dermal fibrosis.

pharmacology and toxicology↗

bioRxiv2018

Rho-mediated MRTF and YAP1 activation contributes to BRAF inhibitor resistance in Sox9High/Sox10Low melanoma cells.

Over half of cutaneous melanoma tumors have BRAFV600E/K mutations. Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses. In this study we demonstrate that approximately 50-60% of melanoma cell lines with acquired vemurafenib resistance activate the RhoA family signaling pathway. RhoAHigh BRAFi-resistant cells are sensitive to the combination of ROCK inhibitors and vemurafenib. Further, these RhoAHigh cells have >100-fold increase in Sox9 expression and >100-fold decrease in Sox10 expression. Two transcriptional co-activators downstream of RhoA, MRTF and YAP1, are activated in Sox9High/Sox10Low BRAFi-resistant cells. Pharmacological inhibition of these transcriptional mechanisms re-sensitizes the cells to vemurafenib. Analysis of human BRAFi-resistant tumors reveals that many resistant tumors show gene expression signatures consistent with increased RhoA activation or activation of the transcriptional co-activators MRTF and YAP1. A subset of melanoma tumors in the TCGA dataset with low Sox10 expression also have elevated RhoA, MRTF, and YAP1 activation signatures. Taken together, these results support the concept of targeting RhoA-regulated gene transcription pathways as a promising approach for treating or preventing BRAFi-resistance in melanoma.

cancer biology↗