The Role of Jarid2 in Leukemic Transformation of Chronic Myeloid Neoplasms
Authors: Celik Hamza , Martens Andrew , Mallaney Cates , Eultgen Elizabeth , Kothari Alok , Fishberger Gregory , Challen Grant A. 
Published: October 2019

Abstract
Despite the increasing use of targeted therapies, a subset of patients with myeloproliferative neoplasms (MPN) transform to secondary acute myeloid leukemia (sAML). MPN patients who develop sAML have a dismal outcome, with a median survival of six-months. The mechanisms and pathways that contribute to transformation from MPN to sAML have not been well delineated. The most commonly mutated genes in MPN include JAK2, MPL and CALR and are likely responsible for initiation of the disease. Although these mutations have potential roles in the pathogenesis and for some cases progression to sAML, their role in sustaining the sAML clone is challenged by the finding that some patients with post-MPN sAML who harbor these mutations in their primary MPNs have no evidence of the same mutation in the leukemic blasts. Recent genome sequencing studies identified deletions of JARID2, associated with Polycomb Repressive Complex 2 (PRC2) involved in implementing global H3K27me3, only in leukemic phase of the disease, but not in chronic phase MPNs. This data suggests that JARID2 deletion could be a sAML-specific transforming event by acting as a tumor suppressor in HSCs.
We show in 32D cells, Jarid2 pull-down is able to co-immunoprecipitate core PRC2 proteins, Ezh2 and Suz12, and Jarid2 depletion using shRNAs leads to reduction in global H3K27 methylation. These data suggest Jarid2 acts in concert with PRC2 in hematopoietic cells to mediate H3K27 methylation. To examine the function of Jarid2 in vivo, we generated a Jarid2 knockout mouse model (Mx1-CRE:Jarid2fl/fl; Jarid2-KO) in which Jarid2 is conditionally deleted in HSCs. Hematopoiesis in these mice was compromised with a 3-fold reduction in hematopoietic stem cell (HSC) number, defective B-cell generation in the bone marrow (BM), a differentiation block in T-cell development in thymus, and a significant reduction in peripheral blood counts.
A competitive transplantation strategy was then employed to assess the potential of Jarid2-KO HSCs. One-hundred phenotypically defined Jarid2-KO HSCs (Lineage- Sca-1+ c-Kit+ CD48- CD150+) from 8-week old mice were transplanted into lethally irradiated recipient mice along with 250,000 whole bone marrow cells from genetically distinguishable wild-type mice. Preliminary analysis of these mice show that the loss of Jarid2 is deleterious for HSC function, leading to reduced lymphoid and enhanced myeloid output and failure to maintain HSC population compared to control HSCs. To further dissect the role of Jarid2 in HSC self-renewal, 18-weeks post-transplant, 100 HSCs were re-purified from the bone marrow of primary recipient mice and transplanted into the secondary recipients along with 250,000 fresh wild-type competitor cells. In this transplant setting, Jarid2-KO HSCs failed to contribute to any PB lineages (myeloid, B-cell and T-cell). Together, these data suggest that Jarid2 is essential for HSC maintenance and is required for HSC self-renewal.
To study the tumor suppressor role of Jarid2 we are using mouse models of the MPN mutation FLT3ITD in combination with Jarid2 deletion to assess the function of Jarid2 as a sAML tumor suppresser. We have established a mouse model by crossing Mx1-CRE:Jarid2fl/fl mice with FLT3ITD/+ mice to generate a Mx1-CRE:Jarid2fl/fl FLT3ITD/+ strain. These mice express the germline ITD mutation under control of the endogenous murine FLT3 promoter and develop MPN with a median survival of 10 months. To mimic the genetic progression of chronic stage MPN to sAML, the genetic deletion of Jarid2 is induced in these mice by pIpC injections once MPN is established at 3-months of age. Blood counts of these mice (2 months after Jarid2 deletion, aged 5 months old) started showing the signs of worsening MPN in the absence of Jarid2 such as, high WBC counts and increased neutrophil differentials compared to control (Mx1-CRE: FLT3ITD/+).
Our ultimate goal is to understand the genetic processes associated with progression of MPN to sAML, which could eventually improve treatment outcomes for patients who can be identified as at increased risk for undergoing sAML transformation.

Disclosures
No relevant conflicts of interest to declare.