Understanding and Targeting Transcriptional Master Regulators in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) causes nearly 800,000 deaths worldwide and represents one of the fastest- rising tumors in the US. Despite recent FDA approval of combination therapies for HCC, the majority of patients die within two years. A major challenge in this context is the high genetic diversity of HCC and its low degree of actionable mutations. Accordingly, the most effective therapies, such as atezolizumab + bevacizumab, target the immune and tumor microenvironment (TME) but not tumor cells. Tyrosine kinase inhibitors (TKI) such as sorafenib and lenvatinib target growth factor signaling cascades in tumor cells but exert only moderate effects on survival. In addition, the effects of TKI may also be mediated via the TME, and current data suggest that combinations of TKI and immune checkpoint inhibitor (ICI) therapy do not synergize. This suggests that current combination therapies have reached a plateau, leaving direct tumor cell targeting and potentially synergistic combinations of tumor cell plus TME targeting a largely unexplored therapeutic gap. Here, we seek to address this gap by functionally characterizing the transcriptional regulatory landscape of HCC with the goal of uncovering novel tumor cell-intrinsic vulnerabilities. Tumor cells are characterized by aberrant “cancer transcriptomes”, in which specific modules are critical for the maintenance of the cancer state and its hallmarks, such as high proliferation and altered metabolism. Transcriptional master regulators (TMR) represent key nodes within these transcriptomic networks, and about 20-30% of TMR are essential for tumor cells, representing targetable “bottlenecks”. As TMR often operate independently of upstream driver mutations, they are shared by a wider range of patients and represent attractive targets in tumors with diverse mutational profiles such as HCC. Based on promising preliminary data, we seek to computationally establish human HCC TMR from two large patient cohorts, confirm their tumor cell specificity by single-cell RNA-seq-based analyses, and correlate TMR with clinical parameters and outcomes (Aim 1). Next, we seek to validate tumor cell-intrinsic functions of novel activated and repressed TMR, that are shared with our human cohorts and associated with patient survival; for this, we will use CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) in vivo screens and subsequent validation of the top hits by testing their effects on tumor growth via single gene CRISPRi or CRISPRa (Aim 2). Finally, we will therapeutically target tumor cell TMR; and screen and test combination therapy with ICI and VEGF inhibition to evaluate synergies between tumor cell TMR- and TME-targeted therapies (Aim 3). The proposed multidimensional research plan will be realized by a strong multi-disciplinary team (Schwabe, Wangen- steen, Zucman-Rossi, Califano) with a history of collaboration and a synergistic and unique combination of expertise in HCC cohorts, HCC biology, computational biology and in vivo CRISPR. The proposed studies will not only provide deeper understanding of transcriptional regulation in HCC but will also reveal novel tumor-intrinsic candidates for tumor cell-based mono- and combination therapy, and thereby advance therapeutic concepts.