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Pre-leukemic Loss of Tet2 Perturbs Emergency Myelopoiesis Pathway Activation
James W. Swann DVM PhD1, Oakley C. Olson PhD1, Emmanuelle Passegué PhD1
1Columbia Stem Cell Initiative, Columbia University, 650 W 168th Street, New York, NY 10032
Acute myeloid leukemia (AML) is driven by acquisition of somatic mutations in hematopoietic stem and progenitor cells (HSPC), with initial mutations in epigenetic regulators such as TET2 creating a pre-leukemic state with enhanced self-renewal and impaired differentiation. Subsequent mutations in downstream progenitors co-operate to cause transformation, resulting in accumulation of malignant blast cells. However, disease progression occurs at different rates among individuals, suggesting environmental factors could contribute by expanding pre-leukemic cells at the expense of wild type (WT) HSPCs to facilitate clonal dominance. In regenerative or inflammatory conditions, emergency myelopoiesis (EM) pathways orchestrate expansion of myeloid cells through molecular, metabolic, and cellular remodelling of HSPCs. However, the role of EM pathways in driving clonal expansion of pre-leukemic HSPCs remains unknown.
To determine how Tet2 loss impacts EM pathway activation, we injected the myeloablative drug 5-fluorouracil (5FU) in WT mice or mice with Vav1-iCre-mediated conditional deletion of Tet2 in hematopoietic cells (Tet2cKO). 5FU caused greater expansion of HSPC populations, including hematopoietic stem cells (HSC) and myeloid-biased multipotent progenitor 3 (MPP3) compared to WT mice. However, this did not translate to greater outburst of neutrophils in vivo, and HSCs from 5FU-injected Tet2cKO mice were less likely to form mature myeloid cells ex vivo than WT HSCs,suggesting EM pathway activation and Tet2 loss cooperate to impair HSPC differentiation. Importantly, this effect was not caused by 5FU-induced DNA damage because a similar effect was observed with injection of recombinant granulocyte-colony stimulating factor (G-CSF), which is a key driver of myeloid cell expansion produced in response to 5FU-induced depletion of myeloid cells. Expanded HSCs from 5FU-injected Tet2cKO mice were more quiescent and had a lower apoptotic rate than WT HSCs. Accordingly, 5FU did not impair reconstitution of Tet2cKO HSCs upon transplantation as in WT HSCs. Collectively, these data suggested that Tet2cKO HSPCs resisted conventional EM pathway activation, resulting in accumulation of HSCs with pre-leukemic features.
To understand the mechanism for these effects, we conducted single cell RNA sequencing followed by SCENIC inference of transcription factor (TF) regulons on 5FU activated HSCs. We found that while WT HSC activation was associated with expected signatures Myc and p53 induction, Tet2cKO HSCs had signatures of altered AP-1 activity. We confirmed these findings using Tet2cKO mice with a c-Myc-GFP reporter, in which HSPCs did not upregulate Myc upon 5FU injection, as in WT mice. Conversely, while 5FU injection decreased Fos expression in WT HSCs by RT-qPCR, consistent with its role in quiescence exit, expression of Fos and Jun was increased in Tet2cKO HSCs. These results reveal differential engagement of AP-1 factors in response to EM activation in pre-leukemic HSCs that might contribute to their persisting self-renewal capacity.
To establish whether aberrant EM responses extended to inflammation, we injected Tet2cKO mice with IL-1β. Akin to 5FU, Tet2cKO HSPCs exhibited reduced activation, with lower CD41 expression on HSCs and lesser expansion of MPP3s. To determine whether this effect was caused by altered activation of the master myeloid TF PU.1, we crossed Tet2cKO mice to PU.1-eYFP reporter mice. We showed that the difference in acute IL-1β response was associated with lesser upregulation of PU.1 in Tet2cKO MPP3 compared to WT HSPCs, demonstrating attenuated EM pathway engagement upon inflammation.
Our data demonstrate that loss of Tet2 limits activation of HSPCs upon inflammatory stimulation and EM pathway engagement, which might endow Tet2cKO HSCs with a selective persistence advantage over WT HSCs that are induced to differentiate. This phenomenon is associated with differences in TF usage and a key role for AP-1. We are now working to understand how these mechanisms might interact to drive clonal expansion and promote the emergence of fully transformed leukemic stem cells in AML.