Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.

SUMMARY How specific genetic lesions contribute to transformation of non-malignant myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (sAML) are poorly understood. JARID2 is lost by chromosomal deletions in a proportion of MPN/MDS cases that progress to sAML. In this study, genetic mouse models and patient-derived xenografts demonstrated that JARID2 acts as a tumor suppressor in chronic myeloid disorders. Genetic deletion of Jarid2 either reduced overall survival of animals with MPN or drove transformation to sAML, depending on the timing and context of co-operating mutations. Mechanistically, JARID2 recruits PRC2 to epigenetically repress self-renewal pathways in hematopoietic progenitor cells. These studies establish JARID2 as a bona fide hematopoietic tumor suppressor and highlight potential therapeutic targets.

Background and Aim: The porcine heart bears the best resemblance to the human heart and remains the preferred preclinical model for anatomical, physiological, and medical device studies. In an effort to study phenomena related strictly to ischemia reperfusion and donor preservation protocols, it is essential to avoid the immune responses related to allotransplantation. Orthotopic auto-transplantation is a unique strategy to the field of cardiac transplantation for ex vivo experimentation. Nevertheless, auto-transplantation carries its own technical challenges related to insufficient length of the great vessels that are to be transected and re-anastomosed. Methods: A novel method for orthotopic cardiac auto-transplantation in the porcine model was developed and was described herein. Porcine models were used for ex vivo experimentation of a novel device to study ischemia reperfusion injury. Results: A total of five porcine models were used for ex vivo experimentation of a novel device to mitigate ischemia reperfusion injury and determine effects of donor preservation. Modifications to routine cardiac transplantation protocols to allow for successful auto-transplantation are described. Conclusion: Orthotopic cardiac auto-transplantation in the porcine model is a plausible and technically feasible method for reliable study of ischemia reperfusion injury and donor preservation protocols. Here, we describe methods for both direct orthotopic porcine cardiac auto-transplantations as well as a simplified protocol that can be substituted for full surgical auto-transplantation for the studies of preservation of donor hearts.

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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.