Loss of p300 accelerates MDS-associated leukemogenesis

Abstract

The role that changes in DNA methylation and histone modifications have in human malignancies is poorly understood. p300 and CREB-binding protein (CBP), two distinct but highly homologous lysine acetyltransferases, are mutated in several cancers, suggesting their role as tumor suppressors. In the current study, we found that deletion of p300, but not CBP, markedly accelerated the leukemogenesis ofNup98-HoxD13 (NHD13) transgenic mice, an animal model that phenotypically copies human myelodysplastic syndrome (MDS). p300 deletion restored the ability of NHD13 expressing hematopoietic stem and progenitor cells (HSPCs) to self-renew in vitro, and to expand in vivo, with an increase in stem cell symmetric self-renewal divisions and a decrease in apoptosis. Furthermore, loss of p300, but not CBP, promoted cytokine signaling, including enhanced activation of the MAPK and JAK/STAT pathways in the HSPC compartment. Altogether, our data indicate that p300 has a pivotal role in blocking the transformation of MDS to acute myeloid leukemia, a role distinct from that of CBP.

Figure 1. Lack of p300 accelerates the leukemogenesis of NHD13 BM cells in a transplantation model

(A) BM cells from p300^flox/flox;Mx1Cre+, NHD13;p300^flox/flox;Mx1Cre- (NHD13), NHD13;p300^flox/+;Mx1Cre+, NHD13;p300^flox/flox;Mx1Cre+ and NHD13;CB^Pflox/flox;Mx1Cre+ mice were injected into 8-week old lethally irradiated B6SJL recipient mice (n=12–18). Poly(I:C) administrations were performed 4 weeks post transplantation. Shown is the Kaplan-Meier survival curve of recipient mice of each genotype post poly(I:C) injections. (B) Top panels show BM and spleen cellularity of leukemic NHD13;p300^Δ/Δ mice, compared to that of age-matched NHD13 mice. Bottom panels show WBC, RBC and platelet counts from peripheral blood of leukemic NHD13;p300^Δ/Δ and age-matched NHD13 mice (n=4–5). (C) HSPCs were isolated from recipient mice 2 weeks post poly(I:C) injections. Shown are the representative FACS profiles of Lin- (upper panel), LK and LSK cells (middle panel) of each genotype indicated in the plots. The absolute number of Lin-, LK and LSK BM cells (from 2 femurs and 2 tibias of each mouse) is plotted in the bottom panel.

Figure 2. Increased stem cell self-renewal and reduced cell death are intrinsic to p300 deleted NHD13 BM cells

(A) HSPCs were sorted from the indicated recipient mice 2 weeks post poly(I:C) injection. Serial replating assays were performed and the resulting colonies were counted weekly before being re-plated. Shown is the data from 2 independent experiments. (B) Serial replating assays were performed using HSPCs isolated from the indicated primary mice 2 weeks post poly(I:C) injection. Data plotted here is from 2 separate experiments. (C) Two weeks post poly(I:C) injection, CD34- LSK cells were isolated from each indicate primary mice and sorted individually into 96-well plate for the paired daughter cell assays. Data are presented as mean frequency from one representative assay out of 2 independent experiments in total. (D) Shown are the representative FACS profiles of Annexin-V and 7AAD staining of LK (upper panel) and LSK cells (bottom panel) from the indicated mice.

Figure 3. Differentially expressed genes in p300-null NHD13 HSPCs

HSPCs were isolated from the indicated primary mice and sorted 2 weeks after poly(I:C) injection. Lin- and ckit+ cells were also isolated from NHD13;p300^Δ/Δ mice at leukemia stage. RNA samples were then prepared for RNA-sequencing analyses. (A) Normalized expression profiles of the top 100 genes with the largest standard deviations across all samples, including samples from the leukemic NHD13;p300^Δ/Δ mice. Data represented are variance-stabilizing transformation (VST) counts from the DESeq2 package. The dendrogram shows samples in all groups clustered as expected. (B) Venn diagram shows the overlap of differentially expressed genes in p300 deleted WT vs NHD13 mice. Genes were considered to be differentially expressed if they had a P-value of less than 0.05 and absolute log fold change of more than 1.5. (C) Up- and down-regulated pathways identified using Gene Set Enrichment Analysis (GSEA) after p300 deletion in NHD13 HSPCs. (D) qPCR validation for several differentially expressed genes. Data were normalized to the expression of GAPDH.

Figure 4. Activated MAPK and JAK/STAT signaling in response to cytokine stimulation in NHD13;p300^Δ/Δ HSPCs

BM cells isolated from mice 2 weeks post poly(I:C) injection from wt, NHD13, p300^Δ/Δ, NHD13;p300^Δ/Δ, CBP^Δ/Δ, and NHD13;CBP^Δ/Δ were cultured in DMEM supplemented with 20% FBS. Phosphorylation of (A) ERK1/2, (B) STAT3 and (C) STAT5 in LK and LSK cells were determined by flow cytometry after cells were cytokine staved for 2 hours, and stimulated or un-stimulated with a cytokine cocktail (GM-CSF 200ng/ml, SCF 100ng/ml, IL-3 10ng/ml and IL-6 10ng/ml) for 15 minutes. Left panels show representative FACS profiles. Right panels plot the frequency of pERK1/2, pSTAT3, and pSTAT5 positive cells in LK and LSK population (n=4). 1-Wt; 2-NHD13; 3-p300^Δ/Δ; 4-NHD13;p300^Δ/Δ; 5-CBP^Δ/Δ and 6- NHD13;CBP^Δ/Δ.