EZH2-Mediated Methylation Regulates JARID2 Protein Stability in Normal and Neoplastic Hematopoiesis

Jarid2 is a co-factor of the polycomb repressive complex 2 (PRC2) that recruits this complex to different sites in the genome to catalyze the repressive epigenetic mark H3K27me3. JARID2 gene deletion was identified as a cooperating mutation that aids in the transformation of MPN to sAML. Previous studies from our lab have demonstrated that Jarid2 functions as a tumor suppressor in Jak2^V617F-mutant MPN by recruiting PRC2 to silence self-renewal gene expression programs in committed hematopoietic progenitor cells. In the present study, we aimed to: (1) further elucidate the molecular mechanism of tumor suppressor function mediated by Jarid2 in Jak2^V617F mutant MPN, and (2) restore JARID2 protein expression in JAK2^V617F-mutant MPN as a novel therapeutic strategy to reverse MPN pathologies and prevent sAML transformation.

Previous studies have shown that Ezh2, the catalytic subunit of PRC2, di- and tri-methylates Jarid2 at its K116 residue. To understand the functional effect of the post-translational modification mediated by Ezh2 on Jarid2 in normal hematopoiesis, we generated a novel Vav-Cre:Jarid2^fl/K116A (Jarid2-K116A) mouse model using CRISPR-Cas9 gene editing. The Jarid2-K116A mutation in the endogenous locus produces a protein that cannot be methylated by Ezh2. The resulting mice express one K116A point mutated copy of Jarid2, while the other copy will be deleted through Vav-Cre recombinase system. We studied the functional effect of the Jarid2-K116A point mutation on normal hematopoiesis through competitive transplantation of long-term hematopoietic stem cells (HSCs). Compared to other Vav-Cre:Jarid2 genotypes (control, Jarid2^fl/+ = Het, and Jarid2^fl/fl = KO), Jarid2-K116A HSCs showed a neomorphic phenotype with Jarid2-K116A recipients showing the highest peripheral blood engraftment (~20% increase) and significant enhancement in HSC self-renewal (~50% increase). This suggests that post-translational modification of Jarid2 by Ezh2 is a critical regulator of HSC function.

Next, we studied the molecular effect of Jarid2-K116A point mutation. Using CRISPR-Cas9 gene editing, we generated Jarid2-K116A 32D cells, and results confirmed that di- and tri-methylation mediated by Ezh2 on K116 residue of Jarid2 was reduced and Jarid2 protein stability was enhanced. This result was also confirmed on whole bone marrow (WBM) cells, where Jarid2-K116A showed significant increase in the protein stability. Knocking out Ezh2 in 32D cells significantly enhanced Jarid2 protein stability. Then, we investigated the effect of K116A point mutation on the protein-protein interactions with Jarid2 by Co-IP / mass spectrometry analysis. Results showed that K116A point mutation significantly decreased the interaction of Jarid2 with the E3 ubiquitin ligase Ppil2. Genetic inhibition of Ppil2 in 32D cells significantly reduced Jarid2 polyubiquitination and enhanced Jarid2 protein stability, similar to the Jarid2-K116A point mutation. These results suggest that Ezh2 mediates di- and tri-methylation of the Jarid2-K116 residue, which act as a signal for the recruitment and interaction of Ppil2 that in turn regulates Jarid2 protein stability by ubiquitination.

To study the effect of enhanced Jarid2 protein stability in MPN, we generated five Mx1-Cre:Jarid2:Jak2^V617F mouse models as follow: Mx1-Cre:Jarid2^+/+, Mx1-Cre:Jarid2^+/+:Jak2^V617F, Mx1-Cre:Jarid2^fl/+:Jak2^V617F, Mx1-Cre:Jarid2^fl/fl:Jak2^V617F, and Mx1-Cre:Jarid2^fl/K116A:Jak2^V617F. Two months post pIpC injection, results showed that enhancing Jarid2 protein stability through K116A point mutation significantly decreased WBC counts, BM and spleen myeloid lineage, and spleen HSCs compared to Jarid2 loss-of-function Jak2^V617F genotypes. Interestingly, western blot analysis using WBM cells showed that Jarid2-K116A significantly decreased phospho-stat3 and phospho-stat5 levels. These results suggest that enhancing Jarid2 protein stability in Jak2^V617F MPN decreases the disease dissemination through inhibition of JAK/STAT signaling. Next, we hypothesized if stabilization of Jarid2 protein by targeting Ppil2 could be applied as a therapeutic approach in MPN. Genetic inhibition of Ppil2 reduced the colony formation capacity and blood engraftment of mouse Jak2^V617F HSPCs. Ongoing studies aim to investigate the effect of PPIL2 inhibition on MF CD34+ patient samples as a precision medicine for treating JAK2^V617F MPN.