Integrated single-cell transcriptomics and functional genomics reveal RAD51B as an essential gene in near-haploid leukemia

Whole chromosome losses resulting in near-haploid karyotypes are found in a rare subgroup of treatment-refractory acute lymphoblastic leukemia. To systematically dissect the unique physiology and uncover susceptibilities that can be exploited in near-haploid leukemia, we leveraged single-cell transcriptomics and computational inference of cell cycle stages to pinpoint key differences between near-haploid and diploid leukemia cells. Combining cell cycle stage-specific differential expression with gene essentiality metrics obtained from a genome-wide CRISPR-Cas9-mediated knockout screen, we identified the homologous recombination pathway component RAD51B as an essential gene in near-haploid leukemia. DNA damage analyses revealed significantly increased sensitivity of RAD51-mediated repair to RAD51B loss in the G2/M stage of near-haploid cells, suggesting a unique role of RAD51B in the homologous recombination pathway. We observed elevated G2/M checkpoint signaling as well as a RAD51B signature expression program in response to chemotherapy in a xenograft model of human near-haploid B-ALL, as well as overexpression of RAD51B and its associated programs in a large panel of near-haploid B-ALL patients. These data highlight a unique genetic dependency on DNA repair machinery in near-haploid leukemia and demarcate RAD51B as a promising candidate for targeted therapy  in this treatment-resistant disease. KBM7 cell lines (haploid, diploidized), PDX model of near-haploid B-ALL, bone marrow and spleen cells from one mouse each in 6 groups: untreated diploidized B-ALL (WT dip), untreated near-haploid B-ALL (WT hap), vehicle treated diploidized B-ALL (WTVeh dip), vehicle treated near-haploid B-ALL (WTVeh hap), combination chemotherapy treated diploidized B-ALL (WTTX dip), combination chemotherapy treated near-haploid B-ALL (WTTX hap) Please note that the raw data was withrawn on Nov 21, 2022 as it is protected human data that is not consented for public display in an unrestricted database.