Fig. 4: MALNC decreases during ATRA-induced myeloid differentiation and associates with better OS in AML patients.

A(Top) Relative expression of MALNC determined by RT-qPCR in HL60 and NB4 cells during all-trans retinoic acid-induced differentiation (10 µM ATRA, 96 h). Data are shown as means ± SEM from three biological experiments, and relative to time point 0 h and normalized towards endogenous control TBP. (Bottom) Level of differentiation marker CD11b during ATRA and vehicle (DMSO 0.01%) treated HL60 and NB4 cells. The percentage of CD11 b-positive cells relative to time point 0 h was determined by flow cytometry up to 96 h. Data are shown as means ± SEM from three biological experiments. P-values were determined by Students t-tests: ns- not significant, *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. B Peaks of histone modifications H3K4me3 and H3K27ac and chromatin accessibility (ATAC-seq) around the TSS of MALNC gene (chr14:83,711,285-83,737,170, GRCh37) for ATRA (1 µM, 96 h) and vehicle control (ethanol) treated HL60. Data retrieved from ENCODE (GSE93994 and GSE93993). C Chromatin accessibility (ATAC-seq) in the TSS of MALNC (chr14:83, 253,790-83,261,760 GRCh38) for untreated HL60 as well as ATRA, phorbol 12-myristate-13-acetate (PMA) and Vitamin D3 (VitD3) treated HL60. Data retrieved from ENCODE (GSE79019) D Expression of MALNC among cytogenetic and genetic risk groups in the (top) ClinSeq cohort (n = 304) and (bottom) TCGA-LAML cohort (n = 148). Normalized counts (log2(CPM + 1)) were scaled and shown as box and whisker dot plots with interquartile range (IQR). The sample number is indicated in parentheses (n). P-value determined by Kruskal–Wallis test and then followed by Dunn´s pairwise comparison test. E, F Kaplan-Meier survival curves showing overall survival (OS) of AML patients stratified by MALNC expression (CPM cut-off by median). Data is shown in the ClinSeq-AML cohort of intensively treated patients, including APL (n = 267) or excluding APL patients (n = 257). P-value determined by log-rank test. Data was censored at the time of allogeneic stem cell transplantation (allo-HSCT), if applicable. G Multivariate analysis was performed on non-APL AML patients from ClinSeq cohort (n = 257). FAB French-American-British-AMLclassification, NPM1 Nucleophosmin 1, IDH2 Isocitrate dehydrogenase, wt wild-type, mut mutated, APL acute promyelocytic leukemia.