Biology subjects

Kurimchak, A. M.

Publications and source records attributed to Kurimchak, A. M..

9 recordsLinked to original sources

bioRxiv2026

MRCKα represses GEF-H1 mediated RhoA activation to promote ovarian cancer spheroid growth and invasion.

High-grade serous ovarian carcinoma (HGSOC) is characterized by high mortality rates and the frequent development of chemotherapy resistance. A hallmark of HGSOC progression is the formation of multicellular spheroids in malignant ascites that facilitate peritoneal dissemination and metastasis. While the CDC42-regulated kinases MRCK and MRCK{beta} (MRCK) were previously found to be highly expressed in ovarian tumors and to be essential for cell migration and spheroid growth, the underlying molecular mechanisms were poorly defined. Mass spectrometry identified the RhoA-selective guanine nucleotide exchange factor GEF-H1 as a primary interacting partner of MRCK. Functional assays revealed that MRCK inhibition or knockdown led to significantly increased levels of active GEF-H1 and subsequent RhoA activation. MRCK was found to phosphorylate GEF-H1 on Ser174, and pharmacological inhibition of MRCK reduced this phosphorylation and decreased the association of GEF-H1 with -Tubulin. Live-cell imaging and 3D assays demonstrated that MRCK inhibition disrupted cell-cell contacts and impaired the compaction of multicellular structures, ultimately reducing the viability of patient-derived organoids. These findings delineate a novel signaling crosstalk mechanism in which MRCK represses GEF-H1-mediated RhoA activation to facilitate the formation of cell-cell contacts that contribute to the survival and growth of HGSOC cells in 3D multicellular structures. This study highlights MRCK as a potential therapeutic target to inhibit the growth and spread of HGSOC.

cell biology↗

bioRxiv2025

Characterization of MEK1/2 Degraders Uncovers a Kinase-Independent Role for MEK1/2 in the Stabilization and Maturation of CRAF

Altered MAPK signaling frequently occurs in human disease. MEK1 and MEK2 (MEK1/2) are central protein kinases in the MAPK signaling cascade that phosphorylate ERK1/2 promoting cell growth. MEK1/2 degraders offer a strategy to characterize both kinase-dependent and independent functions of MEK1/2. Here, we discovered that MEK1/2 degradation, but not kinase inhibition, caused the subsequent degradation of upstream kinase CRAF via a cell-intrinsic mechanism. MEK1/2 binding to CRAF, but not MEK1/2 catalytic activity, was required for CRAF protein stability and maturation to a functional kinase. In the absence of MEK1/2, a minor pool of newly synthesized immature CRAF that had anti-apoptotic functions remained. Finally, we showed that a stable primed CRAF-MEK1/2 signaling complex existed in cells that required RAS binding to potentiate MEK-ERK phosphorylation. Together, weve discovered a previously unrecognized kinase-independent function of MEK1/2, while contextualizing MEK1/2 as an integral component of the CRAF activation cycle beyond the conventional CRAF-MEK kinase- substrate paradigm.

cancer biology↗

bioRxiv2023

RAPID RESISTANCE TO BET INHIBITORS IS MEDIATED BY FGFR1 IN GLIOBLASTOMA

Bromodomain and extra-terminal domain (BET) proteins are therapeutic targets in several cancers including the most common malignant adult brain tumor glioblastoma (GBM). Multiple small molecule inhibitors of BET proteins have been utilized in preclinical and clinical studies. Unfortunately, BET inhibitors have not shown efficacy in clinical trials enrolling GBM patients. One possible reason for this may stem from resistance mechanisms that arise after prolonged treatment within a clinical setting. However, the mechanisms and timeframe of resistance to BET inhibitors in GBM is not known. To identify the temporal order of resistance mechanisms in GBM we performed quantitative proteomics using multiplex-inhibitor bead mass spectrometry and demonstrated that intrinsic resistance to BET inhibitors in GBM treatment occurs rapidly within hours and involves the fibroblast growth factor receptor 1 (FGFR1) protein. Additionally, small molecule inhibition of BET proteins and FGFR1 simultaneously induces synergy in reducing GBM tumor growth in vitro and in vivo. Further, FGFR1 knockdown synergizes with BET inhibitor mediated reduction of GBM cell proliferation. Collectively, our studies suggest that co-targeting BET and FGFR1 may dampen resistance mechanisms to yield a clinical response in GBM.

cancer biology↗

bioRxiv2023

Unique vulnerability of RAC1-mutant melanoma to combined inhibition of CDK9 and immune checkpoints

RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.

cancer biology↗

bioRxiv2023

MEK1/2-Targeting PROTACs Promote the Collateral Degradation of CRAF in KRAS Mutant Cells

Withdrawal StatementFollowing extensive additional studies, we discovered new findings that suggest that the degradation of CRAF by MS934 is not via collateral degradation through proximity to MEK1/2 but rather via a cell intrinsic mechanism that occurs post MEK1/2 degradation in KRAS mutant cells. We have performed extensive time course experiments exploring MS934-mediated degradation of CRAF, as well as additional MEK1/2 knockdown studies in several cell line models. Data from these studies showed that MEK1/2 proteins are degraded by MS934 several hours before CRAF protein and that genetic depletion of both MEK1 and MEK2 reduces CRAF protein levels equivalent to that observed with MS934. Together, these new findings suggest that CRAF degradation is not due to collateral proximity degradation by MS934 but rather due to a cell intrinsic mechanism post MEK1/2 protein depletion.

cancer biology↗

bioRxiv2023

FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55/PP2A phosphatase

The Ser/Thr protein phosphatase 2A (PP2A) is a highly conserved collection of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). PP2A/B55 substrate recruitment was thought to be mediated by charge-charge interactions between the surface of B55 and its substrates. Challenging this view, we recently discovered a conserved SLiM [RK]-V-x-x-[VI]-R in a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowle et al. eLife 2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55/PP2A. This conserved SLiM is necessary for FAM122A binding to B55 in vitro and in cells. Computational structure prediction with AlphaFold2 predicts an interaction consistent with the mutational and biochemical data and supports a mechanism whereby FAM122A uses the SLiM in the form of a short -helix to dock to the B55 top groove. In this model, FAM122A spatially constrains substrate access by occluding the catalytic subunit with a second -helix immediately adjacent to helix 1. Consistently, FAM122A functions as a competitive inhibitor as it prevents binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55/PP2A in cell lysates. Ablation of FAM122A in human cell lines reduces the rate of proliferation, progression through cell cycle transitions and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a SLiM-dependent, substrate-competitive inhibitor of B55/PP2A that suppresses multiple functions of B55 in the DNA damage response and in timely progression through the cell cycle interphase.

molecular biology↗

bioRxiv2021

MDR1 Drug Efflux Pump Promotes Intrinsic and Acquired Resistance to PROTACs in Cancer Cells

PROTACs (Proteolysis-Targeting Chimeras) represent a promising new class of drugs that selectively degrade proteins of interest from cells. PROTACs targeting oncogenes are avidly being explored for cancer therapies, with several currently in clinical trials. Drug resistance represents a significant challenge in cancer therapies, and the mechanism by which cancer cells acquire resistance to PROTACs remains poorly understood. Using proteomics, we discovered acquired and intrinsic resistance to PROTACs in cancer cells can be mediated by upregulation of the drug efflux pump MDR1. PROTAC-resistant cells could be re-sensitized to PROTACs through co-administering MDR1 inhibitors. Notably, co-treatment of MDR1-overexpressing colorectal cancer cells with MEK1/2 or KRASG12C degraders and the dual ErbB receptor/MDR1 inhibitor lapatinib exhibited potent drug synergy due to simultaneous blockade of MDR1 and ErbB receptor activity. Together, our findings suggest that concurrent blockade of MDR1 will likely be required in combination with PROTACs to achieve durable protein degradation and therapeutic response in cancer.

cancer biology↗

bioRxiv2020

Multiplex-inhibitor bead mass spectrometry reveals differential kinome signatures in newly diagnosed and recurrent glioblastoma

Glioblastoma (GBM) is the most common and aggressive adult brain tumor. Despite years of research, clinical trials have not improved the outcome for GBM. Standard of care for newly diagnosed GBM includes surgical resection, followed by radiation and chemotherapy. Tumor recurrence is inevitable and since most patients are not candidates for a second surgical resection, there is an urgent need to identify resistance mechanisms that arise in recurrent GBM. We postulated that examining the differences of activated kinases between newly diagnosed and recurrent GBM may provide insight to resistance mechanisms. To map the kinome landscape of newly diagnosed (nGBM) and recurrent GBM (rGBM) patient derived xenograft tumors, we used Multiplexed Inhibitor Beads and Mass Spectrometry (MIB-MS). We performed pathway analysis of kinases that differed in MIB-binding between nGBM and rGBM to identify kinase-driven signaling pathways. We also analyzed transcriptional profiles to determine the overlap in signaling pathways seen using proteomics or transcriptomics. Using MIB-MS kinome profiling, we found key differences in kinase-driven signaling pathways that may account for the increase in aggressive behavior seen in recurrent GBM. This included a shift in pathways driving cell invasion and proliferation, as well as upregulation of signaling pathways that drive GBM stem-cell like cell differentiation. Analysis of RNA-sequencing showed no statistically significant differences between enriched gene ontologies in nGBM and rGBM, demonstrating the importance of MIB-MS kinome profiling. Collectively, these studies suggest that kinome profiling may inform future clinical trials for kinase inhibitors in GBM.

cancer biology↗

bioRxiv2020

Kinome Profiling of Primary Endometrial Tumors Using Multiplexed Inhibitor Beads and Mass Spectrometry Identifies SRPK1 As Candidate Therapeutic Target

Protein kinases (collectively, termed the kinome) represent one of the most tractable drug targets in the pursuit of new and effective cancer treatments. However, less than 20% of the kinome is currently being explored as primary targets for cancer therapy, leaving the majority of the kinome untargeted for drug therapy. Chemical proteomics approaches such as Multiplexed Inhibitor Beads and Mass Spectrometry (MIB-MS) have been developed that measure the abundance of a significant portion of the kinome, providing a strategy to interrogate kinome landscapes and dynamics. Kinome profiling of cancer cell lines using MIB-MS has been extensively characterized, however, application of this method to measure tissue kinome(s) has not been thoroughly explored. Here, we present a quantitative proteomics workflow specifically designed for kinome profiling of tissues that pairs MIB-MS with a newly designed super-SILAC kinome standard. Using this workflow, we mapped the kinome landscape of endometrial carcinoma (EC) tumors and normal endometrial (NE) tissues and identified several kinases overexpressed in EC tumors, including Serine/Arginine-Rich Splicing Factor kinase, (SRPK1). Immunohistochemical (IHC) analysis of EC tumor TMAs confirmed MIB-MS findings and showed SRPK1 protein levels were highly expressed in endometrioid and uterine serous cancer (USC) histological subtypes. Querying large-scale genomics studies of EC tumors revealed high expression of SRPK1 correlated with poor survival. Inhibition of SRPK1 in USC cells altered mRNA splicing, downregulating several oncogenes including MYC and Survivin resulting in apoptosis. Taken together, we present a SILAC-based MIB-MS kinome profiling platform for measuring kinase abundance in tumor tissues, and demonstrate its application to identify SRPK1 as a plausible kinase drug target for the treatment of EC.

cancer biology↗