Epigenetic Plasticity in ATP-Dependent Chromatin Remodeling Complexes in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the second most common leukemia worldwide with a median age of ~65 years at diagnosis. Myelodysplastic syndromes (MDS) are hematopoietic stem/progenitor cell (HSPC)-initiated clonal disorders of aged individuals that may transform into secondary AML. Cancer genome sequencing studies have identified somatic mutations in genes that encode chromatin regulators. However, mutational profiling alone may not help identify tumor associated transcriptional plasticity, one important hallmark in tumorigenesis. Nucleosome remodeler and deacetylase (NuRD) is an ATP-dependent chromatin remodeling complex, regulating cell-fate commitment and transcriptional architecture. Loss of Mi-2β (CHD4, ATPase subunit of NuRD) causes erythroid leukemia in mice. However, recent report suggests that CHD4 depletion inhibits AML development (Sperlazza J, et al., Blood 2015). Therefore, NuRD involvement in MDS/AML remains incompletely understood.

We have analyzed gene expression of NuRD subunits in a cohort of elderly (median age 60 yrs) patients presenting with MDS. Expression of MBD3 , an integral scaffold of NuRD, but not other NuRD subunits, was significantly downregulated (2.5-fold; P = 0.004) in MDS (n=24) bone marrow (BM) CD34⁺ HSPCs compared to age-matched normal (n=6) BM CD34⁺ cells. Clonal hematopoiesis emerges as an integral component of hematopoietic aging and myeloid malignancies. We asked whether loss of MBD3 expression was associated with HSPC-aging. MBD3 expression was similar ( P = 0.194) in CD34⁺ HSPCs derived from umbilical cord blood (n=6) and aged normal BM. Next, we extended our RT-qPCR analysis in a cohort of de novo AML (n=82) patients, which also suggested significant loss (3.5-fold; P = 0.003) of MBD3 in AML BM nuclear cells (BMNCs) compared to normal BM HSPCs. In addition, there were loss of expression of MTA1, MTA2, MTA3 , RBBP7 subunits ( P < 0.001) in AML. Immunoblot analysis suggested dramatic loss of MBD3 in AML BMNCs, and co immunoprecipitation (Co-IP) analysis using antibody against intact CHD4, demonstrated association of residual NuRD subunits in primary AML cells. Sucrose density gradient centrifugation analysis further confirmed nucleation of residual NuRD (hereafter known as NuRD^Δ) in AML BMNCs. Essentially these data identify HSPC-autonomous loss of MBD3, and indicate NuRD^Δ nucleation as a general phenomenon in AML.

We sought to determine chromatin occupancy of NuRD^Δ in primary AML cells using ChIP-sequencing. CHD4 ChIP-seq identified ~ 9,000 target genes on average in AML (n=3) BMNCs. Interestingly, RNA-seq analysis in matched AML cohort compared to normal hematopoietic cells, and functional annotation clustering using Gene Ontology (GO)-terms on the CHD4 ChIP-seq identified gene set suggested an enrichment of transcripts that are involved in regulating small GTPase signaling, cytoskeletal organization and cell trafficking. Small GTPases play pivotal role in regulating myeloid leukemia cell engraftment and survival (Sengupta A, et al., Blood 2010; Thomas EK, Cancelas JA et al., Cancer Cell 2007). Lentivirus-mediated loss of function experiments and biochemical studies suggested NuRD^Δ contribution in myeloid leukemia cell-microenvironment interaction. We further tested the hypothesis whether NuRD^Δ function involves MBD2/MeCP1-mediated transcriptional dysregulation. Importantly, MBD3 antagonistically regulated H3K27me3, and consistent with this observation a subset of CHD4 ChIP-seq occupied genes in AML significantly overlapped with H3K27me3 binding. Pathway analysis suggested that those genes are involved in RNA transport and metabolism. Pharmacological inhibition of H3K27me3-methyl transferase activity along with small GTPase inhibition attenuated AML cell survival. SWI/SNF, another ATP-dependent chromatin remodeler, represents one of the most commonly mutated (~ 20%) chromatin modulators in human cancer. Co-IP experiments demonstrated interaction between NuRD^Δ and leukemic SWI/SNF (having altered stoichiometry) in AML, and ChIP-seq analysis further identified leukemic SWI/SNF and NuRD^Δ co-occupancy in AML BMNCs. In summary, we illustrate that in human primary AML cells, there is absence of specific subunits of NuRD complex; NuRD^Δ, crosstalks with leukemic SWI/SNF and effects leukemia cell survival and transcriptional dysregulation.