Abstract 3019: Targeting transcriptional regulation using CDK7 and CDK9 inhibition as a novel combination therapy in fibrolamellar carcinoma

Abstract Background: Fibrolamellar carcinoma (FLC) is a rare liver cancer with a 5-year survival rate between 35-40%. FLC typically affects a younger patient population, the majority receiving a diagnosis between the ages of 10-35. There are currently no curative systemic therapies and an unmet need for novel and effective therapeutics to improve survival. A unique molecular hallmark of FLC is a fusion event between heat shock protein 40 (DNAJB1) and the catalytic subunit alpha of protein kinase A (PRKACA). This event creates the FLC specific oncoprotein (DNAJ-PKAc) which has been shown to promote FLC tumor formation. Downstream signaling of DNAJ-PKAc remains unknown, however, this fusion protein has been shown to cause enhancer remodeling to upregulate FLC specific drivers of proliferation. Methods: Western blot analysis was performed on patient tumors, normal tissue, and patient derived organoids (PDOs) for active RPB1, including phosphorylation of serine 2, serine 5, and serine 7 (pRBP1-s2, s5, s7). Single agent activity of SY5609 was assessed at 144 hours in PDOs. Synergy was assessed at 144 hours using a dose titration grid. SynergyFinder 3.0 was used to produce Highest Single Agent (HSA) synergy scores. Viability was determined using CellTiterGlo (CTG, 50% v/v). RNA was extracted from PDOs for qPCR to measure relative FLC specific driver gene expression, including SLC16A14 and LINC00473. SY5609 was treated continuously while VIP152 was removed after 24 hours to mimic in vivo pharmacokinetics. Results: Relative to normal tissue, FLC tumors had an increase in pRPB1-s5 (6.38, p<0.05) and pRPB1-s7 (5.82, p<0.05) compared to cholangiocarcinoma (CCA) (2.42, n.s.; 1.10, n.s.) and hepatocellular carcinoma (HCC) (2.61, n.s.; 0.95, n.s.). CDK7 inhibition with SY5609 in FLC PDOs had a 9x greater selectivity than normal liver PDOs and 216x greater than CCA PDOs with IC50=0.39nM, 3.14nM, and 84.22nM, respectively. FLC PDOs treated with physiologic concentrations of SY5609 showed downregulation of FLC specific enhancer driven gene SLC16A14 but not LINC00473 with fold changes of 0.51 and 0.84. SY5609 did not regulate SLC16A14 in CCA PDOs (fold change=2.78) and LINC00473 was undetectable in all samples. The addition of CDK9 inhibition with VIP152 was synergistic in FLC PDOs (HSA=8.79) which was not seen in CCA PDOs (HSA=-1.82). Combination treatment synergistically downregulated FLC specific driver gene LINC00473 (fold change=0.41), and decreased pRBP1-s2, 5, and 7 shown via western blot. Conclusion: Here, we show FLC has an increase in phosphorylated RPB1 specifically at s5 and s7, and a unique sensitivity to CDK7 inhibition with SY5609. The addition of CDK9 inhibition using VIP152 is synergistic at downregulating FLC specific driver genes and decreasing activated RPB1 and viability. Ongoing work is validating this combination in animal models to assess toxicity and in vivo efficacy. Citation Format: Austin Stram, Manabu Nukaya, Patrick Carney, Eleanor Riedl, Jeremy D. Kratz, Sean Ronnekleiv-Kelly. Targeting transcriptional regulation using CDK7 and CDK9 inhibition as a novel combination therapy in fibrolamellar carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3019.