Fig. 6: PA2G4 mediates the effect of HDACis on EVI1 and MYC.

A Circle plot showing chromatin-associated proteins immunoprecipitated with an anti-EVI1 antibody by RIME. The bar length indicates the mean of mass spectrometry spectral count (SPEC) of uniquely identified proteins of two biological replicates per condition. Bar colors indicate protein ontology of the EVI1 interactors. B Histograms show the normalized gene expression levels of SERBP1, RPL14, RPL18, RPS2, PA2G4, RPS10, FBL, PABPC1, RACK1, RSL1D1, and DDX21 following 16 hr of 0.5 µM AR-42, 2 µM entinostat in UCSD/AML1, or 0.8 µM AR-42 and 4 µM entinostat in MOLM1 compared to vehicle. Statistical significance was determined using DESeq2. Whiskers show median values (central black lines) and 25^th and 75^th percentiles (bottom and top bounds), respectively. The bars represent values that exceed 1.5 times the interquartile range (IQR) from the edge of each box: (vehicle n = 6, treated n = 12). C Tracks showing EVI1 binding and H3K27ac enrichment across the DDX21, FBL, and PA2G4 locus in HNT34 cells treated with vehicle (DMSO), AR-42 and entinostat. The bottom bar represents the genes (hg19), and the y-axis represents normalized read density scaled to 1 million. D PA2G4, EVI1, ∆EVI1, and MYC expression in TF1, HNT34, and UCSD/AML1 cells 6 days after shRNA transduction (n = 3 biological replicates). E Effect of PA2G4 overexpression on HNT34 cells treated with HDACis. Western blot analysis showing PA2G4, EVI1, ∆EVI1, and MYC in wild-type or PA2G4-overexpressing HNT34 cells after 24 h of treatment with HDACi at the indicated doses (n = 2 biological replicates). (F) Histogram indicates the percentage of viable (white), dead (black), or rescued (red) HNT34 cells (n = 20.000) by the overexpression of PA2G4 on the basis of positivity for annexin V/PI staining after 72 h of HDACis treatment (n = 2 biological replicates). G Percentage of PA2G4, EVI1, and MYC mRNA relative to the control gene ACTB (ΔΔCT) in HNT34 and UCSD/AML1 cells 6 days after shRNA transduction. Data are presented as mean ± SD (n = 3). Statistical significance was determined by a two-tailed unpaired, parametric t-test. H Proteasome inhibition rescues WS6-induced EVI1 and MYC protein degradation. EVI1, ∆EVI1, MYC expression, and PA2G4 in HNT34 and UCSD/AML1 cell lines after treatment with DMSO and WS6 (24 hr) or MG132 (4 hr) at the indicated concentrations (n = 2 biological replicates). NT = non-targeting, sh#65 = shRNA directed against PA2G4 in C and F. Source data are provided as a Source Data file. See also Supplementary Fig. 9-10 and Supplementary Data 4.