Biology subjects

Kizhake, S.

Publications and source records attributed to Kizhake, S..

3 recordsLinked to original sources

bioRxiv2026

A Multi-Kinase Inhibitor Screen Identifies mTOR Signaling as a Vulnerability in Advanced Prostate Cancer.

The receptor tyrosine kinase AXL has context-dependent roles in prostate cancer (PCa). Although AXL signaling has been implicated in PCa progression, it can also promote disseminated tumor cell dormancy. Paradoxically, AXL expression has been reported to be lost in metastatic PCa. This raises two critical questions: Is AXL loss associated with aggressive disease, and does it create therapeutically exploitable vulnerabilities? In this study, we comprehensively analyzed AXL expression across pan-cancer and PCa cohorts and found that AXL downregulation was significantly associated with treatment resistance, aggressive PCa phenotypes, and shorter overall survival. To investigate the functional consequences of AXL loss, we used CRISPR-Cas9 based gene editing to generate isogenic AXLKO cells and characterized their proliferative and migratory properties. To identify therapeutically exploitable vulnerabilities associated with AXL loss, we screened these cells against a library of 1,814 kinase inhibitors using the isogenic cell lines. We identified Torin-2 as a candidate therapeutic agent and validated its activity using dose-response assays, which demonstrated an ~ 18-fold lower IC50 in AXLKO cells than in AXLWT cells. In parallel, PRISM/DepMap analysis provided orthogonal evidence of drug dependencies associated with low AXL expression and revealed a selective vulnerability of AXLKO cells to PI3K/AKT/mTOR pathway inhibition. Collectively, our findings link AXL downregulation to aggressive PCa phenotypes and identify mTOR inhibition as a potential therapeutic strategy for AXL-deficient PCa.

cancer biology↗

bioRxiv2026

Beyond Dmax and DC50: A simple tool to evaluate the complex PROTAC-mediated kinetic degradation dynamics

Protein degradation mediated by PROteolysis TArgeting Chimeras (PROTACs) is a complex, dynamic, and multi-step process. The structural diversity of PROTACs, coupled with cellular heterogeneity, introduces considerable variability at each stage. Therefore, a straightforward yet comprehensive approach is essential to effectively dissect these mechanisms. The existing methods for evaluating PROTAC degradation are often time-consuming, labor-intensive, or expensive, and many capture only a partial view of the entire degradation process. Consequently, multiple complementary assays are typically required to obtain the complete picture. Here, we use commercially available Cyclin dependent Kinase 6 (CDK6) targeting PROTACs to demonstrate that a simple, single-step, no-wash, additive-free, GFP-based live-cell imaging approach can rapidly generate dynamic degradation metrics (Dmax, Rmax, TDmax, CDmax, DC50, Km, Tmax, Kdeg, Vmax, DT50, {tau} and TRmax) for comparing and ranking PROTACs within a controlled reporter system. The rapid assay turnaround time, excellent assay qualities and compatibility with high-throughput (HT) formats will facilitate quick hit identification and serve as a guiding assay for hit-to-lead optimization campaigns.

molecular biology↗

bioRxiv2024

Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14{Delta}) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14{Delta} using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14{Delta}-GFP using live cell imaging. We benchmarked out MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was > 15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.

cancer biology↗