Biology subjects

Golkowski, M.

Publications and source records attributed to Golkowski, M..

9 recordsLinked to original sources

bioRxiv2026

Response to SMAC mimetics in patient-derived models of triple-negative breast cancer relies on RIPK1 activation

Triple-negative breast cancer (TNBC) is a highly metastatic and aggressive breast cancer subtype with limited targeted treatment options. Second mitochondrial activator of caspases (SMAC) mimetics represent a promising therapeutic class for TNBC patients, although they have yet to be approved clinically. Screening a repository of 69 breast cancer patient-derived xenograft organoid (PDxO) lines revealed that approximately 39% were sensitive to the SMAC mimetic birinapant. To define the basis of resistance in lines that did not respond, we compared birinapant-sensitive (S-) and de novo resistant (R-) TNBC PDxOs. Birinapant caused degradation of inhibitor of apoptosis proteins (IAPs) in both groups but failed to induce cell death in R-PDxOs. We determined that R-PDxOs were susceptible to certain types of apoptosis but were specifically resistant to TNFR-mediated cell death, an integral pathway involved in SMAC mimetic response. Moreover, RIPK1 activation via autophosphorylation, a critical event in TNFR-mediated apoptosis, was absent in both R-PDxOs and PDxOs selected for acquired birinapant resistance. Pharmacologic inhibition or degradation of RIPK1 protected S-PDxOs from birinapant-induced cell death, demonstrating that RIPK1 activation is required for response in TNBC. To investigate how to restore RIPK1 activation in R-PDxOs, we discovered that HOIP, an E3 ubiquitin ligase that inhibits RIPK1 autophosphorylation, was more abundant in R-PDxOs compared to S-PDxOs. Inhibiting HOIP activity restored RIPK1 autophosphorylation and sensitized a subset of R-PDxOs to birinapant. Together, these findings identify impaired RIPK1 autophosphorylation as a mechanism of birinapant resistance in TNBC and suggest that RIPK1 activation state may distinguish SMAC mimetic-sensitive and -resistant tumors. Targeting HOIP may provide a strategy to overcome resistance in selected tumors.

cancer biology↗

bioRxiv2026

Multi-Modal Kinome Profiling Discovers Mesenchymal-Like Polarity Networks that Underly Directed Hepatocellular Carcinoma Cell Migration

Metastasis and associated therapy resistance remain the principal drivers of cancer related death, and there is a pressing need for a deeper mechanistic understanding and anti-metastatic therapies. For patients that suffer from hepatocellular carcinomas (HCCs), which are the most common primary liver cancers, frequent systemic metastasis results in bleak 5-year survival prognoses of only 4%. To metastasize, carcinoma cells must acquire an invasive phenotype, which typically requires switching from an epithelial-like apical-basal polarity to the front-rear polarity of mesenchymal-like cells. Signaling cues that originate in the tumor microenvironment can activate cellular morphogenic programs that drive polarity switching, like the epithelial-mesenchymal transition (EMT). Protein kinases control most cell signaling pathways and are highly actionable drug targets; however, systematic studies determining the kinases that underly the epithelial-mesenchymal polarity switch (EMPS) are lacking. We developed an assay platform that integrates mass spectrometry (MS)-based kinome profiling, broadly capturing kinase network activity, with chemical genetic screening using selective kinase inhibitors and quantitative phase imaging (QPI), serving as the phenotypic readout. Applying this approach that we dubbed morphokin-MS, to epithelial-like HCC cell lines that we induced to undergo EMPS identified a conserved network of 12 kinases that contributed to HCC cell polarity switching and directed cell migration; MS-based kinome profiling of 17 HCC patient tumors showed that these kinase are frequently upregulated in human tumors. morphokin-MS also revealed that death-associated protein kinase 3 (DAPK3) is one of the principal drivers of the EMPS and directed HCC cell migration. Thus, our mechanistic studies revealed that DAPK3 forms a complex with DAPK1 and filamin-A inter-acting protein 1-like (FILIP1L), which act as scaffold proteins that recruit DAPK3 to the centrosome. Pharmaco-logical and genetic inhibition of the DAPK1-DAPK3-FILIP1L complex blocked centrosome repositioning and microtubule polarization toward the leading edge of mesenchymal-like HCC cells, directed cell migration, and invasion. Our morphokin-MS method and comprehensive kinome profiling data will serve as a valuable resource for the cancer research community; our discovery of an inducible mesenchymal-like DAPK1-DAPK3-FILIP1L polarity complex that controls centrosome positioning in motile HCC cells may lead to the development of novel therapeutics for combatting cancer metastasis.

cancer biology↗

bioRxiv2026

Perm1 Gene Therapy Mitigates PRDM16-Associated Cardiomyopathy

BackgroundPathogenic variants in PR domain containing 16 (PRDM16) cause pediatric and adult cardiomyopathies characterized by ventricular dilation, systolic dysfunction, and impaired metabolic maturation. Cardiac deficiency of PRDM16 alters metabolic gene expression and long-chain fatty acid (FA) metabolites. However, the downstream mediators involved are not well characterized. Furthermore, whether improving mitochondrial FA metabolism can prevent PRDM16-associated cardiomyopathy is currently unknown. MethodsIn vivo and in vitro approaches using patient-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and mouse models with Prdm16 deletion/mutation were employed. Transcriptomics and proteomics analyses were conducted, and adeno-associated virus (AAV)-mediated therapy was tested. ResultsHere, we show that a defect in FA metabolism is an early hallmark of PRDM16 cardiac deficiency. We show, for the first time, that PERM1 is a direct downstream target of PRDM16 and is involved in the regulation of FA metabolism through coordinated action with PGC1. Most importantly, neonatal delivery of AAV9-Perm1 in cardiac-specific Prdm16 knockout (Prdm16 cKO) mice markedly improved contractile parameters, reduced left ventricular (LV) dilation, and extended survival. These cardioprotective effects of PERM1 gene therapy occurred independent of restoring FA oxidation. Transcriptional and proteomic analyses of AAV-Perm1-treated Prdm16 cKO mice demonstrated significant improvements in mitochondrial cristae architecture, preservation of sarcomere organization, reduced cardiomyocyte apoptosis, attenuated myocardial fibrosis, and diminished cardiac remodeling. ConclusionsWe identify PERM1 as a direct downstream effector of PRDM16 and uncover a previously unrecognized PRDM16-PGC1-PERM1 axis essential for FA metabolic regulation in the heart. Perm1 gene therapy ameliorated PRDM16-associated cardiomyopathy through post-transcriptional mechanisms involving preservation of mitochondrial and sarcomere integrity. The current study provides preclinical evidence suggesting that Perm1 gene therapy may be a promising therapeutic target to improve the cardiac outcomes of patients affected by pathogenic PRDM16 variants.

physiology↗

bioRxiv2024

diaPASEF-Powered Chemoproteomics Enables Deep Kinome Interaction Profiling

Protein-protein interactions (PPIs) underlie most biological functions. Devastating human conditions like cancers, neurological disorders, and infections, hijack PPI networks to initiate disease, and to drive disease progression. Understanding precisely how diseases remodel PPI networks can, therefore, help clarify disease mechanisms and identify therapeutic targets. Protein kinases control most cellular processes through protein phosphorylation. The 518 human kinases, known as the kinome, are frequently dysregulated in disease and highly druggable with ATP-competitive inhibitors. Kinase activity, localization, and substrate recognition are regulated by dynamic PPI networks composed of scaffolding and adapter proteins, other signaling enzymes like small GTPases and E3 ligases, and phospho-substrates. Accordingly, mapping kinase PPI networks can help determine kinome activation states, and, in turn, cellular activation states; this information can be used for studying kinase-mediated cell signaling, and for prioritizing kinases for drug discovery. Previously, we have developed a high-throughput method for kinome PPI mapping based on mass spectrometry (MS)-based chemoproteomics that we named kinobead competition and correlation analysis (kiCCA). Here, we introduce 2nd generation (gen) kiCCA which utilizes data-independent acquisition (dia) with parallel accumulation serial fragmentation (PASEF) MS and a re-designed CCA algorithm with improved selection criteria and the ability to predict multiple kinase interaction partners of the same proteins. Using neuroblastoma cell line models of the noradrenergic-mesenchymal transition (NMT), we demonstrate that 2nd gen kiCCA (1) identified 6.1-times more kinase PPIs in native cell extracts compared to our 1st gen approach, (2) determined kinase-mediated signaling pathways that underly the neuroblastoma NMT, and (3) accurately predicted pharmacological targets for manipulating NMT states. Our 2nd gen kiCCA method is broadly useful for cell signaling research and kinase drug discovery.

biochemistry↗

bioRxiv2024

Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

Raf kinases are crucial effectors in the Ras-Raf-Mek-Erk signaling pathway, making them important targets for the development of cancer therapeutics. This study investigates the variable potency of DFG-out-stabilizing Raf inhibitors in mutant KRas-expressing cell lines. We demonstrate that inhibitor potency correlates with basal Raf activity, with more active Raf being more susceptible to inhibition. We further show that DFG-out-stabilizing inhibitors disrupt high-affinity Raf-Mek interactions, promoting the formation of inhibited Raf dimers. Furthermore, we identify cobimetinib as a Mek inhibitor that uniquely sensitizes Raf to DFG-out inhibitors by disrupting autoinhibited Raf-Mek complexes. Building on this insight, we developed cobimetinib analogs with enhanced sensitization properties. Our findings provide a mechanistic framework for understanding the cellular determinants of DFG-out-stabilizing inhibitor sensitivity and offer strategies for optimizing synergistic Raf-Mek inhibitor combinations.

cell biology↗

bioRxiv2024

HAF Prevents Hepatocyte Apoptosis and Hepatocellular Carcinoma through Transcriptional Regulation of the NF-κB pathway.

BackgroundHepatocellular carcinoma (HCC) incidence is increasing worldwide due to the obesity epidemic, which drives metabolic dysfunction-associated steatohepatitis (MASH) that can lead to HCC. However, the molecular pathways that lead to MASH-HCC are poorly understood. We have previously reported that male mice with global haploinsufficiency of hypoxia-associated factor, HAF (SART1+/-) spontaneously develop MASH/HCC. However, the cell type(s) responsible for HCC associated with HAF loss are unclear. ResultsSART1-floxed mice were crossed with mice expressing Cre-recombinase within hepatocytes (Alb-Cre; hepS-/-) or macrophages (LysM-Cre, macS-/-). Only hepS-/- mice (both male and female) developed HCC suggesting that HAF protects against HCC primarily within hepatocytes. HAF-deficient macrophages showed decreased P-p65 and P-p50 and in many major components of the NF-{kappa}B pathway, which was recapitulated using HAF siRNA in vitro. HAF depletion increased apoptosis both in vitro and in vivo, suggesting that HAF mediates a tumor suppressor role by suppressing hepatocyte apoptosis. We show that HAF regulates NF-{kappa}B activity by controlling transcription of TRADD and RIPK1. Mice fed a high-fat diet (HFD) showed marked suppression of HAF, P-p65 and TRADD within their livers after 26 weeks, but manifest profound upregulation of HAF, P-65 and TRADD within their livers after 40 weeks of HFD, implicating deregulation of the HAF-NF-{kappa}B axis in the progression to MASH. In humans, HAF was significantly decreased in livers with simple steatosis but significantly increased in HCC compared to normal liver. ConclusionsHAF is novel transcriptional regulator of the NF-{kappa}B pathway that protects against hepatocyte apoptosis and is a key determinant of cell fate during progression to MASH and MASH-HCC.

cancer biology↗

bioRxiv2021

Displacement of PKA catalytic subunit from AKAP signaling islands drives pathology in Cushing's syndrome

Mutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushings syndrome. Here we define mechanisms of action for the PKAc-L205R and W196R variants. Both Cushings mutants are excluded from A kinase anchoring protein (AKAP) signaling islands and consequently diffuse throughout the cell. Kinase-dead experiments show that PKA activity is required for cortisol hypersecretion. However, kinase activation is not sufficient, as only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate mutant PKAc into AKAP signaling islands abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Phosphoproteomics show that PKAc-L205R and W196R engage different mitogenic signaling pathways. ERK activity is elevated in adrenal-specific PKAc-W196R knock-in mice. Conversely, PKAc-L205R attenuates Hippo signaling, thereby upregulating the YAP/TAZ transcriptional co-activators. Thus, aberrant localization of each Cushings variant promotes the transmission of a distinct downstream pathogenic signal.

molecular biology↗

bioRxiv2021

Multiplexed profiling of kinase interactomes quantifies cellular network plasticity

Cells utilize protein-protein interaction (PPI) networks to receive, transduce, and respond to stimuli. Interaction network rewiring drives devastating diseases like cancers, making PPIs attractive targets for pharmacological intervention. Kinases are druggable nodes in PPI networks but high-throughput proteomics approaches to quantify disease-associated kinome PPI rewiring are lacking. We introduce kinobead competition and correlation analysis (Ki-CCA), a chemoproteomics approach to simultaneously map hundreds of endogenous kinase PPIs. We identified 2,305 PPIs of 300 kinases across 18 diverse cancer lines, quantifying the high plasticity of interaction networks between cancer types, signaling, and phenotypic states; this database of dynamic kinome PPIs provides deep insights into cancer cell signaling. We discovered an AAK1 complex promoting epithelial-mesenchymal transition and drug resistance, and depleting its components sensitized cells to targeted therapy. Ki-CCA enables rapid and highly multiplexed mapping of kinome PPIs in native cell and tissue lysates, without epitope tagged baits, protein labeling, or antibodies.

biochemistry↗

bioRxiv2019

Kinome-centric pharmacoproteomics identifies signaling pathways underlying cellular responses to targeted cancer drugs

ABSTRACTKinase-dependent signaling networks are frequently dysregulated in cancer, driving disease progression. While kinase inhibition has become an important therapeutic approach many cancers resist drug treatment. Therefore, we need both reliable biomarkers that predict drug responses and new targets to overcome drug resistance. Determining the kinase(s) that control cancer progression in individual cancers can pose a significant challenge. Genomics has identified important, yet limited numbers of kinase driver mutations. Transcriptomics can quantify aberrant gene expression, but it cannot measure the protein phosphorylation that regulates kinase-dependent signaling network activity. Proteomics measures protein expression and phosphorylation and, therefore, quantifies aberrant signaling network activity directly. We developed a kinome-centric pharmacoproteomics platform to study signaling pathways that determine cancer drug response. Using hepatocellular carcinoma (HCC) as our model, we determined kinome activity with kinobead/LC-MS profiling, and screened 299 kinase inhibitors for growth inhibition. Integrating kinome activity with drug responses, we obtained a comprehensive database of predictive biomarkers, and kinase targets that promote drug sensitivity and resistance. Our dataset specified pathway-based biomarkers for the clinical HCC drugs sorafenib, regorafenib and lenvatinib, and we found these biomarkers enriched in human HCC specimens. Strikingly, our database also revealed signaling pathways that promote HCC cell epithelial-mesenchymal transition (EMT) and drug resistance, and that NUAK1 and NUAK2 regulate these pathways. Inhibition of these kinases reversed the EMT and sensitized HCC cells to kinase inhibition. These results demonstrate that our kinome pharmacoproteomics platform discovers both predictive biomarkers for personalized oncology and novel cancer drug targets.

cancer biology↗