Abstract 619: Dynamic protein expression governs cellular PARP inhibitor response and represents a pathway to single cell drug resistance

Abstract

Abstract Protein expression functions as a biomarker for precision drug therapy to more effectively treat cancer or as a measurement of drug resistance. Expression levels are commonly determined in bulk populations of cells at single time points. However, cells often undergo dynamic expression during therapeutic response that may influence drug resistance. Therefore, how changing expression levels dynamically impact cellular response remains largely unknown, limiting determination of bona fide protein biomarkers while leaving mechanisms of therapeutic resistance underappreciated. Drugs inhibiting the DNA repair protein poly(ADP-ribose) polymerase (PARP) have been increasingly used to treat various cancers. PARP inhibitors (PARPi) are especially potent in cancers exhibiting deficient homologous recombination, or BRCAness. Yet, acquired PARPi resistance is common and remains a major area of concern, underscoring the importance of discovering how to overcome resistance mechanisms. To determine the impact of dynamic protein expression on PARPi resistance, we have developed a platform approach that dynamically tracks specific protein expression during drug treatment in single cells. We used CAS9-directed plasmid insertion to precisely couple fluorescent protein expression with translation of PARP1 and, combined with longitudinal (days) single cell tracking, correlated PARP1 expression levels with cellular fate during drug exposure. Remarkably, we found that dynamic expression of PARP1 controls cellular fate - division, death, or senescence. PARPi were effective in breast cancer cells when PARP1 expression remained steady. However, cells with similar initial PARP1 expression that significantly increased expression proved resistant. Furthermore, while all PARPi enzymatically inhibit the target protein, the 5 approved drugs display a differential ability to trap PARP1 onto damaged DNA, producing increased potency. We found dynamic PARP1 expression impacted PARPi resistance differently based on drug trapping ability, suggesting a mechanism to select the most effective PARPi for each patient. Guided by a CRISPR knockout screen, we then determined dynamic expression of other important proteins in PARPi response, finding dynamic expression influence in cellular resistance. These results highlight the role of dynamic DNA damage repair in response to PARPi and present potential combination therapy targets. Complementary to dynamic transcriptional profiling of cell lines under drug treatment, our approach displays significant resistance dependence on dynamic protein expression, but provides increased sensitivity through tracking single cells. Overall, our novel approach to measure dynamic protein expression levels highlights previously unknown avenues of cellular resistance. Citation Format: Bianca Fernandez, J Matthew Dubach. Dynamic protein expression governs cellular PARP inhibitor response and represents a pathway to single cell drug resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 619.