Fig. 1: CNOT3 is essential for leukemogenesis.

A CRISPR score rank for essentiality in MOLM13 leukemia cells of subunits of deadenylation complexes—CCR4-NOT complex and PAN2/PAN3 complex. B Immunoblots showing CNOT3 protein in CB-CD34+ cells and primary AML patient cells. C Representative flow plots showing detection of endogenous CNOT3 in healthy donors vs. primary AML patient cells (AML samples). D Quantification of median fluorescent intensity (MFI) of CNOT3 levels in healthy donors (n = 4) vs. primary AML patient cells (n = 16). Data shown as mean ± s.e.m, p value calculated by Mann–Whitney test. E–I MOLM-13 expressing either a scramble (control) shRNA or CNOT3-targeting shRNAs (KD33 and KD-37). E Immunoblots showing efficient knock down of CNOT3. F Cell proliferation. G Quantitative summary of flow cytometry analysis of myeloid markers CD11b and CD14. H Representative H&E images. I Percentage of Annexin-V positive cells by flow cytometry. J, K MOLM-13 cells expressing empty vector (control) or cDNA expressing CNOT3 (CNOT3-OV). J Representative immunoblots confirming CNOT3 overexpression. K Cell proliferation. Graphs (F, G, I, K) showing data as mean ± s.e.m, n = 3 independent experiments, p values calculated by two-tailed student’s t test. L Percentage of human CD45 positive cells in peripheral blood (PB) of NSG recipient mice 4 weeks post transplantation. Control n = 5; KD-33 n = 4; KD-37 n = 4. mean ± s.e.m, p < 0.001, Mann–Whitney test. M Kaplan–Meier curves. Control n = 10; KD-33 n = 10; KD-37 n = 10. p < 0.001, Log-rank test. N Immunoblots showing knockdown of CNOT3 n = 1 sample. O In vitro culture n = 6 primary samples and P Colony formation. n = 5 primary samples. Data shown mean ±  s.e.m., p < 0.001, two-tailed Student’s t test. Q Experimental scheme to assess leukemia in vivo. R–T Percentage of human leukemia CD45+ cells in recipient animals in three independent PDX models. N = 5 per group. mean ± s.e.m, p values calculated by Mann–Whitney test. U, V High expression of CNOT3 mRNA correlates with poor prognosis in AML patients. Kaplan–Meier curves showing outcomes of AML patients in U TCGA-AML high n = 117 vs. low n = 46; V Beat AML high n = 271 vs. low n = 141. p values calculated using Log-rank test. Source data are provided as Source Data files for figures (B, D–G, I–T).