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Targeting CDK4 overcomes EMT-mediated tumor heterogeneity and therapeutic resistance in KRAS-mutant lung cancer
Aparna Padhye, … , Christopher A. Bristow, Don L. Gibbons
Aparna Padhye, … , Christopher A. Bristow, Don L. Gibbons
Published July 26, 2021
Citation Information: JCI Insight. 2021;6(17):e148392. https://doi.org/10.1172/jci.insight.148392.
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Research Article Oncology

Targeting CDK4 overcomes EMT-mediated tumor heterogeneity and therapeutic resistance in KRAS-mutant lung cancer

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Abstract

Lack of sustained response to therapeutic agents in patients with KRAS-mutant lung cancer poses a major challenge and arises partly due to intratumor heterogeneity that defines phenotypically distinct tumor subpopulations. To attain better therapeutic outcomes, it is important to understand the differential therapeutic sensitivities of tumor cell subsets. Epithelial-mesenchymal transition is a biological phenomenon that can alter the state of cells along a phenotypic spectrum and cause transcriptional rewiring to produce distinct tumor cell subpopulations. We utilized functional shRNA screens, in in vitro and in vivo models, to identify and validate an increased dependence of mesenchymal tumor cells on cyclin-dependent kinase 4 (CDK4) for survival, as well as a mechanism of resistance to MEK inhibitors. High zinc finger E-box binding homeobox 1 levels in mesenchymal tumor cells repressed p21, leading to perturbed CDK4 pathway activity. Increased dependence on CDK4 rendered mesenchymal cancer cells particularly vulnerable to selective CDK4 inhibitors. Coadministration of CDK4 and MEK inhibitors in heterogeneous tumors effectively targeted different tumor subpopulations, subverting the resistance to either single-agent treatment.

Authors

Aparna Padhye, Jessica M. Konen, B. Leticia Rodriguez, Jared J. Fradette, Joshua K. Ochieng, Lixia Diao, Jing Wang, Wei Lu, Luisa S. Solis, Harsh Batra, Maria G. Raso, Michael D. Peoples, Rosalba Minelli, Alessandro Carugo, Christopher A. Bristow, Don L. Gibbons

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Figure 1

Mesenchymal lung cancer cells exhibit increased dependency on CDK4 for growth.

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Mesenchymal lung cancer cells exhibit increased dependency on CDK4 for g...
(A) Schematic illustration of the workflow of the shRNA dropout screens. A library of lentiviral particles expressing 10 different barcoded shRNAs was transduced into murine KP-mutant lung cancer cells. The cells were cultured in vitro or implanted in nude or syngeneic 129/Sv mice and later sequenced for barcoded shRNAs and compared with reference cells. (B) Results from Kinome and FDAome shRNA dropout screens in 393P and 344P cell lines and tumors compared based on the redundant shRNA activity (RSA). Top differential hits are labeled on the graphs, most important being CDK4. Venn diagram shows comparisons across different conditions and top hits identified. (C) Cluster plot analysis of correlation between EMT score and CDK4 mRNA expression of 118 human NSCLC cell lines. (D) Western blot analysis of CDK4 pathway after 6 days of CDK4 knockdown. (E) In vitro cell viability after 48-hour palbociclib treatment in a panel of epithelial and mesenchymal murine (393P, 344SQ) and human (H358, H1299) lung cancer cell lines. n = 8 per drug concentration. The curve was generated using a nonlinear regression fit model. Vertical error bars shown. **P < 0.001, 2-tailed Student’s t test. (F) 344SQ and 393P cells were treated for 24, 48, and 72 hours with 1 and 5 μM palbociclib, and Western blot analysis was utilized to demonstrate drug efficacy over a dose range. Cleaved caspase-3 was used as an apoptotic marker.

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ISSN 2379-3708

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