DNMT3A Limits Myeloid Signaling Responses in Committed T Cells during Normal and Leukemic Development

Abstract

T-cell development and lineage commitment are temporally protracted processes in which the interplay between transcription factors and epigenetic modulators coordinate the sequential exclusion of alternative fates and acquisition of specialized T-cell functions. Alterations during this process can lead to diseases such as T-cell acute lymphoblastic leukemia (T-ALL). While many efforts have focused on the transcription factors which drive normal development and leukemogenesis, the underlying epigenetic constraints remain poorly understood. Further, characterization of the role of particular factors during early stages of human T-cell development is limited by difficulty in obtaining and manipulating primary thymic tissue. Here, we characterize T-cell development as modeled in a chemically defined in vitro differentiation system using primary human CD34+ cord blood (CB) as input material (StemSpan T Cell Generation Kit, Stem Cell Technologies). This culture system yields large numbers of genetically manipulable cells that recapitulate aspects of normal intrathymic development based on immunophenotype, gene expression profiling, T-cell receptor rearrangement, and lineage restriction. DNMT3A is recurrently mutated in human T-ALL with identified variants thought to result in protein loss-of-function (LOF). As well, DNMT3A mutations are enriched in ETP-TALL, a phenotypic subtype with transcriptional signature resembling stem/myeloid-like progenitors. We thus sought to explore the role of DNMT3A in early stages of T cell lineage commitment using the in vitro CB differentiation system in combination with lentiviral shRNA-mediated knockdown (KD) and CRISPR/Cas9-mediated knockout (KO) to mimic DNMT3A LOF mutations. To assess durability of commitment to the T lineage, we transferred cells at the post-commitment CD44- stage from culture conditions supporting T cell differentiation to conditions favoring myeloid differentiation (StemSpan Myeloid Expansion Supplements I and II for granulocytes and monocytes, respectively). We found using clonogenic cell growth assays performed at limiting dilution that T cells with DNMT3A KD/KO were able to grow in myeloid media and expressed an aberrant T/myeloid phenotype at significantly higher frequency as compared to negative controls. Interestingly, this lineage switching activity was nearly exclusively represented in a small subset of cells that expressed myeloid growth factor receptors on the cell surface which were present in both KD and control in vitro derived T cells, as well as in normal thymus at similar frequencies. Minor subsets of myeloid receptor positive cells are also identifiable in established human T-ALL cell lines, and similar to in vitro derived T cells, the receptor positive subset of DNMT3A KD cells showed preferential growth in response to stimulation with their cognate ligands. Phosphoflow assays demonstrated that KD and non-silencing/negative control cells showed similar phosphoprotein signaling responses upon myeloid growth factor stimulation, suggesting that growth responses in KD cells are enacted at the DNA level, presumably by reduced DNA methylation at target gene promoters. These results support a working model in which DNMT3A loss in T-cell progenitors restores their ability to respond positively to myeloid growth factor stimulation both in terms of cell growth and myeloid marker expression. Of note, interrogation of clonal progeny from individual DNMT3A KO cells reveal no evidence of differentiation delay or arrest under T-tropic culture conditions, suggesting that responses to myeloid-tropic conditions are due to reprogramming of T-committed cells rather than increased persistence of uncommitted cells. These processes also appear to be operative in established T-ALL cells, suggesting that DNMT3A LOF mutation may afford T-ALL cells the ability to exploit alternate myeloid factor replete niches in the body.