Increased phosphorylation of Rb and protein expression of cyclin E confers resistance to CDK4/6-inhibitors in the CDK4-amplified cancer, dedifferentiated liposarcoma
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Date
2022-04
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Abstract
Background:
Dedifferentiated liposarcomas (DDLPS) are malignant adipocytic cancers characterized by an amplification of the cell cycle regulatory gene, CDK4. Palbociclib, a CDK4/6-inhibitor, is a standard of care treatment; however, innate and acquired resistance to the drug limits efficacy in patients with DDLPS. To this end, we developed palbociclib-resistant DDLPS cell lines derived from patient samples to better characterize their phenotypic and genomic differences.
Methods:
Three human CDK4 amplified DDLPS cell lines (224A, 246, 863) and one control cell line without CDK4 amplification and RB loss (LiSa2) were brought into culture and treated with palbociclib at their respective IC25 for 6 months to develop resistance. Parental lines were grown as controls in tandem with the developing resistant lines to compare molecular changes in response to palbociclib. Cell viability was measured by XTT, cell cycle analysis was measured with Flow cytometry using a PI stain, and protein expression was measured using whole cell lysates for Western blotting.
Results:
The four cell lines had IC50s to palbociclib ranging from 15.0 to 11.4 µM. Long-term treated strains demonstrated consistent reductions in the measured IC50s (8.99 to 7.08 µM). After treatment with 10 µM of palbociclib for 48 hours, two parental cell lines showed a greater increase in cells in the G1 fraction than long-term treated cells and two cell lines demonstrated the opposite where parental cell lines had fewer cells in G1 after treatment compared to long-term treated cell. Protein expression by Western indicated that the initial response to palbociclib in parental cell lines decreased pRb protein levels and increased Cyclin E and CDK6. After 6 months of treatment for all cell lines, a statistically significant increase in protein levels of pRb and Cyclin E were observed in the long-term treated cell lines. No changes were seen in CDK4, Cyclin D, or p16.
Conclusion:
Here we present the development of two novel, palbociclib resistant, DDLPS cell lines. DDLPS resistant cell lines able to bypass the G1/S-checkpoint in the presence of CDK4/6-inhibitors demonstrated increased phosphorylation of Rb and increased total Cyclin E. Confirmation via next generation sequencing of RNA expression in parental and resistant lines are underway.
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Keywords
cancer, sarcoma, biomarkers, drug resistance