Abstract 1046: Combination of CDK 4/6 inhibitor Palbociclib with Rapamycin synergistically inhibits the growth of hepatocellular carcinoma cells

Abstract

Abstract Introduction: Aberrant cell-cycle regulation is common in cancer, making it an attractive therapeutic target. The cell cycle inhibitor Palbociclib has been shown to have a cytostatic effect on HCC cells via the induction of quiescence and senescence, though it has limited cytocidal effect. We hypothesized that arrested cells upregulate mTOR signaling to stay metabolically active and investigated the effect of combining Palbociclib with the mTOR inhibitor Rapamycin on HCC cell lines in-vitro. Methods: We employed single sample gene set enrichment analysis (ssGSEA) of 10 paired RNA-seq and 15 paired proteomic data sets as well as 371 HCC tumors from TCGA to identify commonly altered molecular markers in HCC, including cell cycle related genes. Subsequently, we studied the effect of Palbociclib and Rapamycin on two HCC cell lines, SNU398 and HuH7. We treated the cell lines with increasing doses of Palbociclib and used cell counting to assess growth inhibition, as well as β-galactosidase activity assay to assess induction of senescence. We then treated both cell lines with Palbociclib for ten days followed by Palbociclib plus Rapamycin for 4 days and performed an MTT assay to assess relative viable cell number. We also did a fixed ratio treatment with Palbociclib and Rapamycin, followed by calculation of combination index to determine the nature of their interaction. Finally, we performed Western Blot analysis for various proteins of the cell cycle and mTOR pathways to elucidate the effects of combination treatment. Results: ssGSEA revealed significant positive enrichment of several cell cycle and mitosis related gene sets. Palbociclib treatment showed a dose-dependent inhibition of growth as well as increase in senescence associated β-galactosidase activity for both SNU398 and HuH7 cell lines. Addition of Rapamycin demonstrated a dose-dependent potentiation of Palbociclib induced growth inhibition, and fixed ratio treatment with the two drugs established a synergistic effect as indicated by a combination index of less than 1. Western blot analysis showed increased levels of intrinsic CDK inhibitors p16, p15, p21 and p27 with Palbociclib treatment. Western blot analysis of proteins of mTOR pathway revealed that Palbociclib treatment caused an increase in levels of p-mTOR as well as its downstream signaling proteins p-4eBP1, p-p70 and p-s6, and addition of Rapamycin suppressed their levels. Conclusion: HCC cells over-express genes and proteins that drive cell cycle progression. Palbociclib induces senescence in hepatocellular carcinoma cell lines along with an associated upregulation in mTOR signaling, and combination with Rapamycin has a synergistic effect on growth inhibition in-vitro possibly by eliminating senescent cells. Citation Format: Ankur Tiwari, Araceli Huerta, Hakim Bouamar, Debodipta Das, Francisco G. Cigarroa, LuZhe Sun. Combination of CDK 4/6 inhibitor Palbociclib with Rapamycin synergistically inhibits the growth of hepatocellular carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1046.