Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia

Background: T -ALL is an aggressive hematological tumor that develops as the result of a multi -step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first -line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T -ALL to develop potential targeted therapies. Methods: Pediatric T -ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Thcells. DNA -damage was assessed by immunofluorescence confocal-imaging and single -cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. Results: We discovered that nuclear-WASp is required for suppressing R -loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R -loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, Rloops accumulate in vivo and are processed into DNA -breaks by transcription -coupled -nucleotide -excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R -loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription -linked DNA damage during malignancy and through modification of epigenetic dysregulations. Conclusion: Our findings present a provocative possibility of resetting R -loops as a therapeutic intervention to correct both immune deficiency and malignancy in T -cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T -ALL patients, anticipating WASp ' s requirement for the suppression of T -ALL progression.