Biology subjects

Shokat, K. M.

Publications and source records attributed to Shokat, K. M..

8 recordsLinked to original sources

bioRxiv2025

Genome-wide CRISPR screens identify PTGES3 as a novel AR modulator

The androgen receptor (AR) is a critical driver of prostate cancer (PCa). To study regulators of AR protein levels and oncogenic activity, we created the first live cell quantitative endogenous AR fluorescent reporters. Leveraging this novel AR reporter, we performed genome-scale CRISPRi flow cytometry sorting screens to systematically identify genes that modulate AR protein levels. We identified and validated known AR protein regulators including HOXB13 and GATA2 and also unexpected top hits including PTGES3, a poorly characterized gene in PCa. PTGES3 repression resulted in loss of AR protein, cell cycle arrest, and cell death in AR-driven PCa models. PTGES3 is not a commonly essential gene, and our data nominate it as a prime PCa therapeutic target. Clinically, analysis of PCa data demonstrate that PTGES3 expression is associated with AR-directed therapy resistance. Mechanistically, we show PTGES3 binds directly to AR, forms a protein complex with AR in the nucleus, regulates AR protein stability in vitro and in vivo and modulates AR function in the nucleus at AR target genes. PTGES3 represents a novel therapeutic target for overcoming known mechanisms of resistance to existing AR-directed therapies in PCa.

cancer biology↗

bioRxiv2025

Deazaguanylation is a nucleobase-protein conjugation required for type IV CBASS immunity

7-deazapurines are nucleobase analogs essential for nucleic acid modifications in nearly all cellular life. Here, we discover a role for 7-deazapurines in protein modification within type IV CBASS anti-phage defense and define functions for CBASS ancillary proteins Cap9 and Cap10 in nucleobase-protein conjugation. A structure of Cap10 reveals a tRNA transglycosylase-family enzyme remodeled to bind the modified N-terminus of a partner cGAS/DncV-like nucleotidyltransferase linked to a 7-amido-7-deazaguanine (NDG) nucleobase. The structure of Cap9 explains how this QueC-like enzyme co-opts a 7-deazapurine biosynthetic reaction mechanism for NDG conjugation. We demonstrate that Cap9, Cap10, and NDG conjugation are essential for host defense against phage infection. Our results define a previously unknown 7-deazapurine protein modification and explain how nucleobase biosynthetic machinery has been repurposed for antiviral immunity.

microbiology↗

bioRxiv2024

Multiplexed mosaic tumor models reveal natural phenotypic variations in drug response within and between populations

Many agents that show promise in preclinical cancer models lack efficacy in patients due to patient heterogeneity that is not captured in traditional assays. To address this problem, we have developed GENEVA, a platform that measures the molecular and phenotypic consequences of drug perturbations within diverse populations of cancer cells at single-cell resolution, both in vitro and in vivo. Here, we apply GENEVA to study the KRAS G12C inhibitors, recapitulating known properties of these drugs and uncovering a previously unknown role for mitochondrial activation in cell death induced by KRAS inhibition. We demonstrate that this finding can be leveraged for the development of combination therapies with greater efficacy. Finally, we show that the application of GENEVA with in vivo mouse models revealed epithelial to mesenchymal transition (EMT) as a key mechanism for resistance to KRAS G12C inhibition.

cancer biology↗

bioRxiv2022

Probing the KRas Switch II Groove by Fluorine NMR Spectroscopy

While there has been recent success in the development of KRasG12C inhibitors, unmet needs for selective inhibitors and tool compounds targeting the remaining oncogenic KRas proteins remain. Here, we applied trifluoromethyl-containing ligands of KRas proteins as competitive probe ligands to assay the occupancy of the switch II pocket by 19F NMR spectroscopy. Structure-activity-relationship studies of probe ligands increased the sensitivity of the assay and identified structures that differentially detected each nucleotide state of KRasG12D. These differences in selectivity, combined with the high resolution of 19F NMR spectroscopy, enabled this method to be expanded to assay both nucleotide states of the protein simultaneously. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/500267v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@2fe99corg.highwire.dtl.DTLVardef@19223bdorg.highwire.dtl.DTLVardef@1686090org.highwire.dtl.DTLVardef@19b9d05_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Tissue-Restricted Inhibition of mTOR Using Chemical Genetics

mTOR is a highly conserved eukaryotic protein kinase that coordinates cell growth and metabolism and plays a critical role in cancer, immunity, and aging. It remains unclear how mTOR signaling in individual tissues contributes to whole-organism processes because mTOR inhibitors, like the natural product Rapamycin, are administered systemically and target multiple tissues simultaneously. We developed a chemical-genetic system, termed selecTOR, that restricts the activity of a Rapamycin analog to specific cell populations through targeted expression of a mutant FKBP12 protein. This analog has reduced affinity for its obligate binding partner FKBP12, which reduces its ability to inhibit mTOR in wild-type cells and tissues. Expression of the mutant FKBP12, which contains an expanded binding pocket, rescues the activity of this Rapamycin analog. Using this system, we show that selective mTOR inhibition can be achieved in S. cerevisiae and human cells, and we validate the utility of our system in an intact metazoan model organism by identifying the tissues responsible for a Rapamycin-induced developmental delay in Drosophila. Significance StatementmTOR plays a number of critical organismal roles, including in cell growth, development, immunity and aging, but dissecting the tissue-specific influences of mTOR has proven challenging. This work describes a simple system for identifying the specific tissues and cells responsible for the diverse functions of mTOR, and we show that our system can be used in organisms ranging from yeast to humans.

biochemistry↗

bioRxiv2021

KRAS is vulnerable to reversible switch-II pocket engagement in cells

Current small molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket, exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the switch-II pocket is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a novel cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the switch-II pockets of many GTP hydrolysis-deficient KRAS hotspot (G12, G13, Q61) mutants are accessible using non-covalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the switch-II pocket as a privileged drug binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.

cancer biology↗

bioRxiv2020

A GTP-state specific cyclic peptide inhibitor of the GTPase Gαs

The G protein-coupled receptor (GPCR) cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable receptors and enzymes with the exception of the G subunit, Gs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 1012 cyclic peptides in order to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gs, respectively. Both macrocyclic peptides fine-tune Gs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/3 pocket and block effector interactions. The Gs inhibitors showed strong activity in cellular contexts through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gs provides a path for further development of state-dependent GTPase inhibitors.

biochemistry↗

bioRxiv2017

Kinome rewiring reveals AURKA is a molecular barrier to the efficacy of PI3K/mTOR-pathway inhibitors in breast cancer

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest. We hypothesized that dynamic changes in signaling, including adaptation and feedback, limit drug efficacy. Using a quantitative chemoproteomics approach we mapped dynamic changes in the kinome in response to various agents and identified signaling changes that correlate with drug sensitivity. Measurement of dynamics across a panel of breast cancer cell lines identified that maintenance of CDK4 and AURKA activity was associated with drug resistance. We tested whether incomplete inhibition of CDK4 or AURKA was a source of therapy failure and found that inhibition of either was sufficient to sensitize most breast cancer cells to PI3K, AKT, and mTOR inhibitors. In particular, drug combinations including the AURKA inhibitor MLN8237 were highly synergistic and induced apoptosis through enhanced suppression of mTOR signaling to S6 and 4E-BP1 leading to tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of target therapy and indicates that Aurora kinase co-inhibition could unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer.

cancer biology↗