Biology subjects

Barekatain, Y.

Publications and source records attributed to Barekatain, Y..

7 recordsLinked to original sources

bioRxiv2025

CDK8 remodels the tumor microenvironment to resist the therapeutic efficacy of targeted KRASG12D inhibition in pancreatic ductal adenocarcinoma

Mutations in KRAS are a dominant driver of pancreatic ductal adenocarcinoma (PDAC), with over 40% of PDAC patients presenting with KRASG12D mutations. The recent development of small molecule inhibitors targeting KRASG12D has enabled targeting of mutant KRAS signaling and suppression of PDAC; however, the contribution of the tumor microenvironment (TME) to the sustained therapeutic efficacy of KRASG12D inhibition and mechanism/s of resistance to KRASG12D suppression remain to be elucidated. Here, we employed spatial transcriptomics, single cell RNA sequencing, and CODEX-based spatial proteomics to evaluate cancer cell intrinsic and extrinsic responses to KRASG12D inhibition with MRTX1133. While KRASG12D inhibition initially increases CD11c+ cells with impactful T cell infiltration within proximity to cancer cells, long-term treatment with MRTX1133 resulted in reversal of the immune responses leading to KRASG12D therapy resistance promoted by CDK8, a multiprotein mediator complex associated kinase. CDK8 imparts resistance in part through induction of downstream CXCL2 chemokine secretion, inhibition of FAS expression, and remodeling of the TME to promote immune evasion. Targeting CDK8 by itself and in combination with CTLA-4 immunotherapy overcomes resistance to KRASG12D inhibition with prolonged survival with translational implications.

cancer biology↗

bioRxiv2023

Oncogenic KrasG12D specific non-covalent small molecule inhibitor reprograms tumor microenvironment and reverses early precursor lesions and in combination with immunotherapy regresses advanced PDAC in a CD8+ T cells dependent manner

Pancreatic ductal adenocarcinoma (PDAC) is associated with mutations in Kras, a known oncogenic driver of PDAC; and the KRASG12D mutation is present in nearly half of PDAC patients. Recently, a non-covalent small molecule inhibitor (MRTX1133) was identified with specificity to the KrasG12D mutant protein. Here we explore the impact of KrasG12D inhibition by MRTX1133 on advanced PDAC and its influence on the tumor microenvironment. Employing different orthotopic xenograft and syngeneic tumor models, eight different PDXs, and two different autochthonous genetic models, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8+ effector T cells, decreases myeloid infiltration, and reprograms cancer associated fibroblasts. Autochthonous genetic mouse models treated with MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8+ T cells and immune checkpoint blockade therapy (iCBT) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of mutant Kras in advanced PDAC and human patient derived organoids (PDOs) induces Fas expression in cancer cells and facilitates CD8+T cell mediated death. These results demonstrate the efficacy of MRTX1133 in different mouse models of PDAC associated with reprogramming of stromal fibroblasts and a dependency on CD8+ T cell mediated tumor clearance. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with iCBT in clinical trials.

cancer biology↗

bioRxiv2022

Quantification of phosphonate drugs by 1H-31P HSQC shows that rats are better models of primate drug exposure than mice

The phosphonate group is a key pharmacophore in many anti-viral, anti-microbial, and anti-neoplastic drugs. Due to its high polarity and short retention time, detecting and quantifying such phosphonate-containing drugs with LC/MS-based methods is challenging and requires derivatization with hazardous reagents. Given the emerging importance of phosphonate-containing drugs, developing a practical, accessible, and safe method for their quantitation in pharmacokinetics (PK) studies is desirable. NMR-based methods are often employed in drug discovery but are seldom used for compound quantitation in PK studies. Here, we show that proton-phosphorous (1H-31P) heteronuclear single quantum correlation (HSQC) NMR allows for quantitation of the phosphonate-containing enolase inhibitor HEX in plasma and tissue at micromolar concentrations. Although mice were shown to rapidly clear HEX from circulation (over 95% in <1 hr), the plasma half-life of HEX was more than 1hr in rats and nonhuman primates. This slower clearance rate affords a significantly higher exposure of HEX in rat models compared to mouse models while maintaining a favorable safety profile. Similar results were observed for the phosphonate-containing antibiotic, fosfomycin. Our study demonstrates the applicability of the 1H-31P HSQC method to quantify phosphonate-containing drugs in complex biological samples and illustrates an important limitation of mice as preclinical model species for phosphonate-containing drugs.

pharmacology and toxicology↗

bioRxiv2020

Structure-guided microbial targeting of antistaphylococcal prodrugs

Carboxy ester prodrugs have been widely employed as a means to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can successfully mask problematic chemical features that prevent cellular uptake and can be used to target delivery of compounds to specific tissues. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, curbing their potential therapeutic applications. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes due to a paucity of information about the selectivity of microbial esterases. Here we identify the bacterial esterases, GloB and FrmB, that are required for carboxy ester prodrug activation in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, which revealed several promoieties likely to be serum esterase-resistant while still being microbially labile. These studies lay the groundwork for structure-guided design of anti-staphyloccal promoieties and expand the range of molecules to target staphyloccal pathogens.

biochemistry↗

bioRxiv2020

Accelerated erythrocyte senescence causes dose-limiting anemia of antimalarial enolase inhibitors

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase - a critical enzyme in glycolysis - and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.

pharmacology and toxicology↗

bioRxiv2019

Robust detection of oncometabolic aberrations by 1H-13C heteronuclear single quantum correlation in live cells and intact tumors ex-vivo

Extensive efforts have been made to use non-invasive 1H magnetic resonance (MR) spectroscopy to quantify metabolites that are diagnostic of specific disease states. Within the realm of precision oncology, these efforts have largely centered on quantifying 2-hydroxyglutarate (2-HG) in tumors harboring isocitrate dehydrogenase 1 (IDH1) mutations. As many metabolites have similar chemical shifts, the resulting 1H spectra of intact biological material are highly convoluted, limiting the application of 1H MR to high abundance metabolites. Hydrogen-Carbon Heteronuclear single quantum correlation 1H-13C HSQC is routinely employed in organic synthesis to resolve complex spectra but has received limited attention for biological studies. Here, we show that 1H-13C HSQC offers a dramatic improvement in sensitivity compared to one-dimensional (1D) 13C NMR and dramatic signal deconvolution compared to 1D 1H spectra in an intact biological setting. Using a standard NMR spectroscope without specialized signal enhancements features such as magic angle spinning, metabolite extractions or 13C-isotopic enrichment, we obtain well-resolved 2D 1H-13C HSQC spectra in live cancer cells, in ex-vivo freshly dissected xenografted tumors and resected primary tumors. We demonstrate that this method can readily identify tumors with specific genetic-driven oncometabolite alterations such as IDH mutations with elevation of 2-HG as well as PGD-homozygously deleted tumors with elevation of gluconate. These data support the potential of 1H-13C HSQC as a non-invasive diagnostic tool for metabolic precision oncology.

biophysics↗

bioRxiv2019

Methylthioadenosine is Not Dramatically Elevated in MTAP-Homozygous Deleted Primary Glioblastomas

In BriefThe co-deletion of MTAP in the CDKN2A locus is a frequent event in diverse cancers including glioblastoma. Recent publications report that significant accumulations of the MTAP substrate, methylthioadenosine (MTA), can sensitize MTAP-deleted cancer cells to novel inhibitors of PRMT5 and MAT2A for targeted therapy against tumors with this particular genetic alteration. In this work, using comprehensive metabolomic profiling, we show that MTA is primarily secreted, resulting in exceedingly high levels of extracellular MTA in vitro. We further show that primary human glioblastoma tumors minimally accumulate MTA in vivo, which is likely explained by the metabolism of MTA by MTAP-competent stromal cells. Together, these data challenge whether the metabolic conditions required for therapies to exploit vulnerabilities associated MTAP deletions are present in primary human tumors, questioning their translational efficacy in the clinic.\n\nHighlightsO_LIMethylthioadenosine (MTA) is elevated in MTAP-deleted cancer cells in vitro, which provides a selective vulnerability to PRMT5 and MAT2A inhibitors\nC_LIO_LIAccumulation of MTA in MTAP-deleted cancer cells is predominately extracellular, suggesting active secretion of MTA.\nC_LIO_LIMTAP-deleted primary human glioblastoma tumors show minimal intratumoral elevations of MTA, which is likely explained by secretion and metabolism by MTAP-competent stromal cells.\nC_LI\n\nSUMMARYHomozygous deletion of the CDK2NA locus frequently results in co-deletion of methylthioadenosine phosphorylase (MTAP) in many fatal cancers such as glioblastoma multiforme (GBM), resulting in elevations of the substrate metabolite, methylthioadenosine (MTA). To capitalize on such accumulations, therapeutic targeting of protein arginine methyltransferase 5 (PRMT5) and methionine adenosyl transferase (MAT2A) are ongoing. While extensively corroborated in vitro, the clinical efficacy of these strategies ultimately relies on equally significant accumulations of MTA in human tumors. Here, we show that in vitro accumulation of MTA is a predominately extracellular phenomenon, indicating secretion of MTA from MTAP-deleted cells. In primary human GBMs, we find that MTA levels are not significantly higher in MTAP-deleted compared to MTAP-intact tumors or normal brain tissue. Together, these findings highlight the metabolic discrepancies between in vitro models and primary human tumors and should thus be carefully considered in the development of the precision therapies targeting MTAP-homozygous deleted GBM.

cancer biology↗