Abstract PR005: Forced accumulation of R-loops through inhibiton of <i>RNase H</i> impairs the proliferation of <i>TET</i>-deficient cancer cells
Authors: Isaac F López-Moyado, Otokani Ortega de la Tejera, Lot M Hernández-Espinosa, Arthur X Cheng, Roberta Nowak, Zhen Dong, Caitlin Brown, André Nussenzweig, Anjana Rao
Journal: Cancer Research (2025)

Abstract Among the many features of cancer cells, they commonly display aberrant accumulation of RNA-DNA hybrids known as R-loops. R-loops are formed in healthy cells during many cellular processes, including transcription and DNA replication, and are crucial for proper maintenance of telomeres and centromeres, as well as for adequate mitochondrial function. However, aberrant accumulation of R-loops can lead to increases in DNA damage and inflammation. R-loop accumulation has been documented in different types of cancer, including BRCA1-mutant breast cancer, diffuse large B cell lymphoma, and myelodysplastic syndrome (MDS). Indeed, accumulation of R-loops has been proposed as a unifying mechanism underlying MDS and other myeloid malignancies. Ten-eleven translocation enzymes (TET1, TET2, TET3) are 5-methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers. We have shown that mice with profound, acute TET deficiency induced by deletion of all Tet genes (hereafter referred to as Tet iTKO mice) show rapid onset of an uncontrolled malignant myeloid expansion. Expanded myeloid cells from Tet iTKO mice show increased levels of R-loops, together with increased DNA damage and inflammation, compared to control myeloid cells. Using Tet iTKO hematopoietic stem and progenitor cells (HSPC), we performed an in vivo CRISPR knockout screen of genes encoding RNA and DNA sensors, as well as R-loop regulators, to find how loss of genes related to nucleic acid sensing and innate immune response would affect the proliferation of TET-deficient myeloid cells. We observed that loss of enzymes that prevent the accumulation of R-loops impaired the uncontrolled malignant myeloid expansion of Tet iTKO cells. CRISPR-mediated depletion of the gene encoding RNase H1, an enzyme that specifically degrades the RNA strand in R-loops, further increased R-loop levels and DNA damage, and decreased proliferation and in vivo expansion of TET-deficient myeloid cancer cells, compared to Tet iTKO cells treated with non-targeting sgRNAs. Moreover, the accumulation of DNA breaks in Tet iTKO cells upon Rnaseh1 loss was not observed in Tet wild-type cells similarly depleted for RNase H1, suggesting a role for TET enzymes and potentially TET-oxidized 5-methylcytosines in DNA repair, which will be discussed in this presentation. Overall, these findings indicate that further increasing R-loops in TET-deficient cancer cells blocks their proliferation, and suggest that RNase H depletion or inhibition could be a new therapeutic strategy for cancer treatment in cancers that display augmented levels of R-loops, independent of the presence of TET mutations. Citation Format: Isaac F López-Moyado, Otokani Ortega de la Tejera, Lot M Hernández-Espinosa, Arthur X Cheng, Roberta Nowak, Zhen Dong, Caitlin Brown, André Nussenzweig, Anjana Rao. Forced accumulation of R-loops through inhibiton of RNase H impairs the proliferation of TET-deficient cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr PR005.