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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 5

Cotargeting CDK4 and MAPK pathways targets different tumor cell subsets.

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Cotargeting CDK4 and MAPK pathways targets different tumor cell subsets....
(A and B) Apoptosis was determined by annexin V and propidium iodide staining after treatment with AZD6244 (5 μM) or palbociclib (5 μM) for 48 hours. Data are presented as the mean ± SD. (C) 344SQ_Z-cad cells were treated with DMSO, mocetinostat (1 μM), AZD6244 (5 μM), or palbociclib (5 μM) for 48 hours followed by fluorescent image acquisition. Scale bar: 50 μm. (D) Images from C were quantified for the percentage of RFP or GFP colored pixels calculated per field of view (FOV). n = 4–6 FOVs. (E) 344SQ_Z-cad cells were treated with DMSO, AZD6244 (5 μM), palbociclib (5 μM), or the combination. NucView 405 Caspase 3 substrate was added to each condition as a readout for apoptosis. Representative fluorescent images were acquired 48 hours after addition of drugs. Scale bar: 25 μm. Arrows indicate apoptotic cells. Images were quantified for total caspase-3+ cells as a percentage of total cells in 4–6 FOVs. (F and G) EVTs were plated in a Matrigel (MG) or a collagen/Matrigel (Coll/MG). After 24 hours, EVTs were treated with DMSO, AZD6244 (5 μM), palbociclib (5 μM), or the combination. Treatment in MG was continued for 9 days and Coll/MG for 5 days, with representative images from last day of the culture shown (scale bar: 200 μm for MG and 100 μm for Coll/MG). Red color indicates epithelial-like cells and green color indicates mesenchymal-like cells. Quantification of percentage RFP and GFP colored pixels in 4–6 FOVs. Data are represented as mean. At the end of the treatment, CellTiter-Glo reagent was added and relative luciferin signal was measured. Treatment groups were compared with DMSO using 1-way ANOVA in all the panels. ****P < 0.0001; ***P < 0.005; **P < 0.001; *P < 0.05.

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