Shigeki Hirabayashi

Selinexor, a first-in-class exportin1 (XPO1) inhibitor, is an attractive anti-tumor agent because of its unique mechanisms of action; however, its dose-dependent toxicity and lack of biomarkers preclude its wide use in clinical applications. To identify key molecules/pathways regulating selinexor sensitivity, we performed genome-wide CRISPR/Cas9 dropout screens using two B-ALL lines. We identified, for the first time, that paralogous DDX19A and DDX19B RNA helicases modulate selinexor sensitivity by regulating MCL1 mRNA nuclear export. While single depletion of either DDX19A or DDX19B barely altered MCL1 protein levels, depletion of both significantly attenuated MCL1 mRNA nuclear export, reducing MCL1 protein levels. Importantly, combining selinexor treatment with depletion of either DDX19A or DDX19B markedly induced intrinsic apoptosis of leukemia cells, an effect rescued by MCL1 overexpression. Analysis of Depmap datasets indicated that a subset of T-ALL lines expresses minimal DDX19B mRNA levels. Moreover, we found that either selinexor treatment or DDX19A depletion effectively induced apoptosis of T-ALL lines expressing low DDX19B levels. We conclude that XPO1 and DDX19A/B coordinately regulate cellular MCL1 levels and propose that DDX19A/B could serve as biomarkers for selinexor treatment. Moreover, pharmacological targeting of DDX19 paralogs may represent a potential strategy to induce intrinsic apoptosis in leukemia cells.

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5′ single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4 ⁺ T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type–specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type–resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.

Selinexor, a first-in-class exportin1 (XPO1) inhibitor, is an attractive anti-tumor agent because of its unique mechanisms of action; however, its dose-dependent toxicity and lack of biomarkers preclude its wide use in clinical applications. To identify key molecules/pathways regulating Selinexor sensitivity, we performed genome-wide CRISPR/Cas9 dropout screens using two B-ALL lines. We identified, for the first time, that paralogous DDX19A and DDX19B RNA helicases modulate Selinexor sensitivity by regulating MCL1 mRNA nuclear export. While single depletion of either DDX19A or DDX19B barely altered MCL1 protein levels, depletion of both significantly attenuated MCL1 mRNA nuclear export, reducing MCL1 protein levels. Importantly, combining Selinexor treatment with depletion of either DDX19A or DDX19B markedly induced intrinsic apoptosis of leukemia cells, an effect rescued by MCL1 overexpression. Analysis of Depmap datasets indicated that a subset of T-ALL lines expresses minimal DDX19B mRNA levels, and, in fact, either Selinexor treatment or DDX19A depletion effectively induced TP53-independent apoptosis of MOLT-3, a T-ALL line expressing low DDX19B levels. We conclude that XPO1 and DDX19A/B coordinately regulate cellular MCL1 levels and propose that DDX19A/B could serve as biomarkers for Selinexor treatment. Moreover, pharmacological targeting of DDX19 paralogs may represent a potential strategy to induce intrinsic apoptosis in leukemia cells.

Measurable residual disease (MRD) status before transplantation has been shown to be a strong prognostic factor in patients with Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph⁺ ALL). However, the outcomes of unrelated hematopoietic stem cell transplantation based on the MRD status have not been fully investigated. In this retrospective study, we compared the outcomes of 715 consecutive adults with Ph⁺ ALL in complete remission who underwent unrelated cord blood transplantation (UCBT) (single‐unit UCBT, n = 232 [4/6, 5/6, and 6/6 HLA match]), HLA‐matched unrelated bone marrow transplantation (UBMT; n = 292 [8/8 HLA match]), or HLA‐mismatched UBMT (n = 191 [7/8 HLA match]). In the MRD⁺ cohort, adjusted 3‐year leukemia‐free survival rates were 59.8%, 38.3%, and 55.5% after UCBT, HLA‐matched UBMT, and HLA‐mismatched UBMT, respectively. In the MRD⁻ cohort, the corresponding rates were 65.3%, 70.4%, and 69.7%, respectively. The MRD⁺ HLA‐matched UBMT group had a significantly higher risk of relapse than the MRD⁺ HLA‐mismatched UBMT group (hazard ratio [HR] in the MRD⁺ HLA‐mismatched UBMT group, 0.33; 95% confidence interval [CI] 0.15–0.74) and the MRD⁺ UCBT group (HR in the MRD⁺ UCBT group, 0.38; 95% CI 0.18–0.83). Furthermore, HLA‐matched UBMT had a significant effect of MRD on death (HR 1.87; 95% CI 1.19–2.94), relapse or death (HR 2.24; 95% CI 1.50–3.34), and relapse (HR 3.12; 95% CI 1.75–5.57), while UCBT and HLA‐mismatched UBMT did not. In conclusion, our data indicate Ph⁺ ALL patients with positive MRD may benefit from undergoing UCBT or HLA‐mismatched UBMT instead of HLA‐matched UBMT to reduce leukemic relapse.

Purpose:The anti-leukemic activity of allogeneic hematopoietic cell transplantation (HCT) depends on both the intensity of conditioning regimen and the strength of the graft-versus-leukemia (GVL) effect. However, it is unclear whether the sensitivity of the GVL effects differs between donor type and graft source.Experimental Design:We retrospectively evaluated the effect of acute and chronic graft-versus-host disease (GVHD) on transplant outcomes for adults with acute leukemia (n = 6,548) between 2007 and 2017 using a Japanese database. In all analyses, we separately evaluated three distinct cohorts based on donor type [(8/8 allele-matched sibling donor, 8/8 allele-matched unrelated donor, and unrelated single-cord blood (UCB)].Results:The multivariate analysis, in which the development of GVHD was treated as a time-dependent covariate, showed a reductive effect of grade I–II acute GVHD on treatment failure (defined as 1-leukemia-free survival; P < 0.001), overall mortality (OM; P < 0.001), relapse (P < 0.001), and non-relapse mortality (NRM; P < 0.001) in patients receiving from UCB. A reductive effect of limited chronic GVHD on treatment failure (P < 0.001), OM (P < 0.001), and NRM (P < 0.001) was also shown in patients receiving from UCB. However, these effects were not always shown in patients receiving from other donors. The beneficial effects of mild acute and chronic GVHD after UCB transplantation on treatment failure were noted relatively in subgroups of patients with acute myelogenous leukemia and a non-remission status.Conclusions:These data suggested that the development of mild GVHD could improve survival after UCB transplantation for acute leukemia.

Purpose:The anti-leukemic activity of allogeneic hematopoietic cell transplantation (HCT) depends on both the intensity of conditioning regimen and the strength of the graft-versus-leukemia (GVL) effect. However, it is unclear whether the sensitivity of the GVL effects differs between donor type and graft source.Experimental Design:We retrospectively evaluated the effect of acute and chronic graft-versus-host disease (GVHD) on transplant outcomes for adults with acute leukemia (n = 6,548) between 2007 and 2017 using a Japanese database. In all analyses, we separately evaluated three distinct cohorts based on donor type [(8/8 allele-matched sibling donor, 8/8 allele-matched unrelated donor, and unrelated single-cord blood (UCB)].Results:The multivariate analysis, in which the development of GVHD was treated as a time-dependent covariate, showed a reductive effect of grade I–II acute GVHD on treatment failure (defined as 1-leukemia-free survival; P < 0.001), overall mortality (OM; P < 0.001), relapse (P < 0.001), and non-relapse mortality (NRM; P < 0.001) in patients receiving from UCB. A reductive effect of limited chronic GVHD on treatment failure (P < 0.001), OM (P < 0.001), and NRM (P < 0.001) was also shown in patients receiving from UCB. However, these effects were not always shown in patients receiving from other donors. The beneficial effects of mild acute and chronic GVHD after UCB transplantation on treatment failure were noted relatively in subgroups of patients with acute myelogenous leukemia and a non-remission status.Conclusions:These data suggested that the development of mild GVHD could improve survival after UCB transplantation for acute leukemia.

To identify molecules/pathways governing Venetoclax (VEN) sensitivity, we performed genome-wide CRISPR/Cas9 screens using a mouse AML line insensitive to VEN-induced mitochondrial apoptosis. Levels of sgRNAs targeting March5, Ube2j2 or Ube2k significantly decreased upon VEN treatment, suggesting synthetic lethal interaction. Depletion of either Ube2j2 or Ube2k sensitized AML cells to VEN only in the presence of March5, suggesting coordinate function of the E2s Ube2j2 and Ube2k with the E3 ligase March5. We next performed CRISPR screens using March5 knockout cells and identified Noxa as a key March5 substrate. Mechanistically, Bax released from Bcl2 upon VEN treatment was entrapped by Mcl1 and Bcl-XL and failed to induce apoptosis in March5 intact AML cells. By contrast, in March5 knockout cells, liberated Bax did not bind to Mcl1, as Noxa likely occupied Mcl1 BH3-binding grooves and efficiently induced mitochondrial apoptosis. We reveal molecular mechanisms underlying AML cell-intrinsic VEN resistance and suggest a novel means to sensitize AML cells to VEN.