RNase H2 is a novel therapeutic target in triple negative breast cancer

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Project Summary Due to their hyperproliferative nature and intrinsic genomic instability, triple-negative breast cancer (TNBC) cells exhibit high levels of replication stress, which occurs when the DNA replication machinery encounters obstacles that impede the replication process. How TNBC cells adapt to these high levels of replication stress remains poorly understood. These adaptive mechanisms, if identified, would reveal specific targets in TNBC and provide an effective strategy for TNBC treatment. To this end, we generated innovative cell models and discovered that one major mechanism required for TNBC cells to survive high replication stress is an increase in the enzyme RNase H2. RNase H2 acts to remove ribonucleotides that have been improperly incorporated into the genome, a key driver of replication stress. Subsequent bioinformatic analysis revealed that RNASEH2A, the catalytic subunit of RNase H2, is overexpressed in 89% of TNBC tumors and all the TNBC cell lines that we tested. More importantly, we found that RNase H2 inhibition, by genetic depletion or by the chemical inhibitor R14, specifically kills TNBC cells both in vitro and in vivo with minimal effects on nontumorigenic mammary epithelial cells. These important findings indicate that RNase H2 inhibition may be a promising therapeutic strategy for TNBC treatment. Intriguingly, we also found that RNase H2 inhibition activated the stimulator of interferon genes (STING) pathway and increased expression of key T-cell-attracting cytokines in TNBC cells and sensitized mouse TNBC tumors to anti-PD-1 immunotherapy, suggesting that the therapeutic effects of RNase H2 inhibition may be potentiated by anti-PD-1 therapy. All of these exciting findings support the hypotheses that RNase H2 inhibition offers a promising therapeutic strategy to treat TNBC and that it may be enhanced by anti-PD-1 immunotherapy. These hypotheses will be tested via 3 specific aims: (1) To identify the underlying mechanisms of the therapeutic efficacy of RNase H2 inhibition in TNBC. We will determine if limiting levels of dNTPs leads to increased misincorporation of ribonucleotides into the genomes of TNBC cells, and if inhibition of RNase H2 in TNBC prevents removal of these misincorporated ribonucleotides, consequently leading to unsustainably high replication stress and cell death. We will also evaluate the potential mechanisms mediating the escape of TNBC from RNase H2 inhibition and strategies to overcome resistance. (2) To determine the therapeutic potential of R14 for TNBC treatment. We will determine the in vivo tolerability of R14 in mice to determine the maximum tolerated dose as well as any potential toxicities. We will then assess the efficacy of R14 treatment in 10 TNBC patient-derived xenograft models representative of 5 TNBC subtypes. (3) To determine the therapeutic efficacy of the combination of RNase H2 inhibition with PD-1 immunotherapy in TNBC. We will evaluate the therapeutic efficacy of the R14/PD-1 immunotherapy combination in TNBC using 5 syngeneic TNBC mouse models. We will also assess if and how R14-mediated therapies affect the tumor immune microenvironment.