Deciphering the Mechanisms of Dedifferentiated Liposarcoma Resistance to CDK4/6-Inhibitor, Palbociclib

Abstract

BACKGROUND: Dedifferentiated liposarcoma (DDLPS) are malignant adipocytic cancers characterized by an amplification of the cell cycle regulatory gene, CDK4. Despite the success of novel CDK4-inhibitors such as palbociclib, innate and acquired resistance to the drugs limits efficacy in patients with DDLPS, so identifying responders and mechanisms that drive resistance is important to maximize the clinical utility of these drugs in sarcoma patients.

Deregulation of the CDKN2A-CCND-CDK4/6-Rb pathway is frequently observed in over 25% of unselected sarcomas and is pathognomonic for specific sarcoma subtypes. Molecular mechanisms and biomarkers of palbociclib resistance have been posited in non-mesenchymal tumors but are poorly understood in DDLPS. Therefore, determining biomarkers of resistant DDLPS cell lines would promote or deter treatment with palbociclib in patients with DDLPS based on their genetic profile. Additionally, combination therapy with CDK4/6 inhibition to target dual genomic derangements is an attractive and yet-to-be-tested approach.

This project serves to determine biomarkers of resistance in dedifferentiated liposarcoma by developing palbociclib-resistant DDLPS cell lines. I hypothesize that downstream cell cycle regulators, CDK2, CDK7, and Cyclin E drive G1/S progression in palbociclib resistant DDLPS cell lines.

AIM 1: To determine the mechanisms and biomarkers that contribute to innate and acquired resistance by comparing the genomic profile of resistant lines to their respective parental line

APPROACH: I will develop palbociclib-resistant DDLPS cell lines by treating the lines at palbociclib's IC50 until the IC50 of resistant lines are approximately 10x the parental lines and then compare protein expression via DNA sequencing as well as SDS PAGE and Western blotting. An alternative approach would use RT-PCR for the genes of interest.

EXPECTED OUTCOME: Resistant lines result in decreased expression of Rb and p16, and an increase in Cyclin E, CDK2, CDK7, and CDK6. No changes are expected in CDK4 or Cyclin D.

AIM 2: To determine the effect palbociclib resistance has on other therapies, I will test a combination of drugs that may prevent resistance in developing tumors or reduce tumor progression in resistant tumors using synergy assays and developing potentially resistant lines.

APPROACH: I will perform a synergy assay on parental and resistant lines with palbociclib and another inhibitor. Additionally, cell lines will be treated with combination therapies that showed synergy to determine how long it takes for the cell lines to gain resistance. Alternative approaches would only test CDK7/9 and mTOR inhibitors which affect upstream and downstream proteins closest to the CDK4/CCND complex and omit continuous treatment.

EXPECTED OUTCOME: CDK7/9, MDM2, mTOR, or PI3K inhibitors will work well combined with CDK4 inhibitors to prevent resistance. CDK2 and other CDK4 inhibitors will work well to combat already resistant tumors. Beneficial combination therapies will show increased cell death compared to palbociclib-alone and will take more time to develop resistance compared to palbociclib-only treatment.