Fig 2: Nmnat1 is dispensable for normal hematopoiesis.(A) Quantitative PCR showing the relative expression levels of Nmnat1 in murine hematopoietic cell populations in the BM compared to whole BM (WBM) (n = 3). (B and C) Complete blood cell counts (B) and frequencies of B cell, T cell, and myeloid cell (C) in PB of Nmnat1 WT (Nmnat1+/+), Het (Mx1-Cre; Nmnat1+/fl), and KO (Mx1-Cre; Nmnat1fl/fl) mice at 4 months after poly(I:C) injection (n = 8 to 12). WBC, white blood cells; RBC, red blood cells; HGB, hemoglobin; PLT, platelets. (D) BM cellularity of poly(I:C)-treated Nmnat1 WT, Het, and KO mice (n = 5 to 6). (E) Frequencies of LSK, HSC, MPP, HPC1, and HPC2 in the BM of poly(I:C)-treated Nmnat1 WT, Het, and KO mice (n = 5 to 6). (F) Frequencies of GMP, CMP, and MEP in the BM of poly(I:C)-treated Nmnat1 WT, Het, and KO mice (n = 5 to 6). (G) Competitive BM transplantation with 500,000 Nmnat1 WT, Het, or KO BM cells (CD45.2+), along with 500,000 competitor BM cells (CD45.1+) into lethally irradiated recipient mice. Graphs show the overall percentage of CD45.2+ cells and those in B, T, and myeloid lineages (n = 5 to 6). (H) Model for the role of NMNAT1 in AML and venetoclax resistance. All data represent mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student’s t test unless otherwise noted. See also fig. S5.

Fig 3: NMNAT1 deletion decreases nuclear NAD+ and activates the p53 pathway.(A) Viability of MOLM13 and OCI-AML2 cells treated with FK866 (10 µM) with or without NAD+ precursors (NMN, NA, NR, and NAM; 1 mM). (B) Viability of MOLM13 and OCI-AML2 cells transduced with NMNAT1_sg (+) or negative control sgRNA [NC_sg (-)] with or without NAD+ precursors (NMN, NA, NR, and NAM; 1 mM). (C) Immunofluorescence images of NMNAT1 in MOLM13 cells. (D) Nuclear NAD+ levels in MOLM13 cells after deleting NMNAT1 or a negative control (NC) locus. (E) CRISPR domain screening using sgRNAs targeting different exons of NMNAT1 in Cas9-expressing MOLM13 cells. (F) Relative expansion of MOLM13 cells transduced with sgRNA against NMNAT1 or NC with or without the indicated mouse Nmnat1 constructs. (G) Survival curves of NSG-SGM3 mice transplanted with control or NMNAT1-deleted MOLM13 cells with the indicated murine Nmnat1 constructs. (H and I) Cell cycle analysis by BrdU incorporation (H) and annexin V staining (I) of MOLM13 cells after deleting NMNAT1 or NC. (J) Immunoblotting showing phospho-p53, ?H2AX, and NMNAT1 in MOLM13 cells after lentiviral deleting NMNAT1. (K and L) Annexin V staining (K) or relative expansion (L) of MOLM13 cells after deleting NMNAT1 with or without consecutive targeting of TP53. (M) Relative expression of p53 target genes after deleting NMNAT1. (N) Immunoprecipitation assay showing increased acetylated lysine of p53 in NMNAT1-deleted MOLM13 cells. (O) Relative expansion of NMNAT1-deleted MOLM13 cells expressing WT or K320/373/381R-TP53 constructs. (P) Competitive growth assay after deleting the indicated genes. All data represent mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student’s t test unless otherwise noted. See also fig. S2.

Fig 4: Whole-genome CRISPR screen identifies NAMPT and NMNAT1 as genetic dependencies in AML.(A) A schematic outline of the genome-wide CRISPR-Cas9 screen. (B) A volcano plot showing both positively and negatively selected genes (FDR < 0.25). (C) A schematic showing the three NAD+ biosynthesis pathways, with metabolites and enzymes in normal and italic font, respectively. (D) The read numbers of four sgRNAs against NAMPT (left) and NMNAT1 (right) at day 25 compared to input. (E) Cancer cell line dependency scores (CERES) of genes involved in NAD+ biosynthesis (n = 563). The box plot presents interquartile range, and the whiskers show 95% confidence interval. (F) CERES of NAMPT and NMNAT1 in leukemia (n = 26) and other cancer cell lines (n = 505). Box plots are annotated with the lower quartile, median, and upper quartile for each category’s z scores. (G) Scatter plot showing the correlation between the dependency score of NAMPT and NMNAT1 (n = 563). (H) Immunoblots of NAMPT (left) and NMNAT1 (right) in MOLM13 cells at day 4 after deleting these genes. (I) Competitive growth assay in Cas9-expressing MOLM13 cells that express sgRNAs against negative control (NC) or genes in the NAD+ biosynthesis pathway. The percentages of sgRNA-expressing cells were normalized to those on day 4 after transduction. (J) Measurement of intracellular NAD+ amounts (left) and NAD+/NADH (reduced form of NAD+) ratio (right) in GMP and MLL-AF9–driven L-GMPs (n = 4). (K) Intracellular NAD+ amounts (left) and NAD+/NADH ratio (right) in human cord blood CD34+ HSPCs and PDX-derived AML samples (n = 4). All data represent mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student’s t test unless otherwise noted. See also fig. S1.

Fig 5: NMNAT1 deletion suppresses leukemia progression.(A and B) Frequency of GFP+ leukemia cell in blood (day 25) (A) and survival curve (B) of mice transplanted with MLL-AF9–transduced HSPCs isolated from Nmnat1 WT, Het, and KO mice (n = 8). (C) Representative fluorescence-activated cell sorting plot and quantification of CD34+CD16/32+ L-GMPs (red box) after Nmnat1 deletion (n = 6). (D) Relative nuclear NAD+ levels in GFP+ leukemic cells (n = 4). (E and F) Immunoblotting showing phospho-p53 (Ser15) and ?H2AX (E) and acetylated (lysine) p53 after immunoprecipitation of p53 (F) in L-GMPs. (G) Expression levels of p53 target genes in L-GMPs from Nmnat1 WT, Het, and KO AML mice. (H) Limiting dilution assays performed with GFP+ BM cells from primary recipient mice (n = 6). (I and J) MLL-AF9–transduced HSPCs from Mx1-Cre;Nmnat1+/+ or Mx1-Cre;Nmnat1fl/fl mice before poly(I:C) treatment were transplanted into recipient mice and treated with poly(I:C) (indicated by arrowheads) 5 days after transplantation. Frequency of GFP+ leukemia cell in blood 3 weeks after transplantation (I), and survival curves (J) are shown (n = 10). (K and L) Frequency of GFP+ leukemia cell in PB (K) and survival curves (L) (n = 8) of Nmnat1 WT or Het AML recipient mice treated with vehicle or venetoclax (100 mg/kg, days 5 to 21). (M to P) Survival curves (M and O) and frequency of human CD45+CD34+CD38- LSCs (N and P) in NSG-SGM3 mice transplanted with MLL-AF9 (M and N) or FLT3-ITD (O and P) human AML samples with or without CRISPR-mediated deletion of NMNAT1 (n = 5 to 6). All data represent mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 by unpaired Student’s t test unless otherwise noted. See also fig. S4.