Selective ASO-based JAK2 inhibitor for the treatment of hematological malignancies with JAK2 V617F mutation burden.

e14582 Background: Myeloproliferative disorders are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Due to dysregulation of the JAK-STAT signaling pathway, MPDs display excessive proliferation of megakaryocytic, erythroid, and granulocytic progenitors, causing patients to develop splenomegaly, thrombosis, and bleeding as an overproduction of blood cells occur. The increased kinase activity of JAK2 is present in several hematologic malignancies, and the somatic JAK2 V617F mutation can be found in at least 98% of PV patients. Inhibiting JAK2 acts to suppress hematopoiesis, consequently reducing red blood cell, neutrophil, platelet, and lymphocyte production. We hypothesize that preventing JAK2 transcription by antisense oligonucleotide (ASO)-mediated exon masking of the JAK2 intron-exon junction will selectively reduce JAK2 mRNA and protein by providing NMD in the reading frame. This approach aims to mitigate the clinical manifestations of hematologic malignancies by curtailing JAK2 V617F -driven autonomous cell proliferation. Methods: We designed a series of 19mer ASOs targeting JAK2 exon-intron junctions and tested these at a range of concentrations. A human HEL and SET2 cell line harboring the V617F JAK2 mutation underwent ASO treatment (1µM) and incubation, followed by qPCR and Western blot analyses. Peripheral blood mononuclear cells (PBMCs) obtained from PV patients carrying the V617F JAK2 mutation were plated in methylcellulose, containing erythropoietin, in the presence or absence of ASO (1 µM, 48-hour incubation). Results: Following ASO treatment of HEL cells, JAK2 qPCR results show a ~50% reduction of the target JAK2 transcript, with a dose-response reduction in mRNA observed. Western blot results reveal a significant JAK2 protein decrease (~70%) in ASO-treated HEL cells compared to untreated samples. STAT5 phosphorylation status further confirmed this effect, and we report a 35% pSTAT5 reduction. The ASO was shown to be a selective inhibitor of JAK2 with no significant JAK1, JAK3, or TYK2 inhibition. Evaluation of endogenous erythroid colony (EEC) formation following ASO treatment to PV PBMCs revealed a significant reduction of erythroid colonies. Other non-erythroid colonies were produced in this treatment condition. Conclusions: Our preclinical data supports this ASO as a highly selective JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. ASOs have the potential to target JAK2 mutations with high specificity, effectively decreasing JAK2 protein levels without off-target effects on other kinases. Currently, there is no available therapeutic that selectively inhibits JAK2. We propose that the application of our ASO to selectively reduce JAK2 protein may alleviate the disease burden with decrease in unwanted side effects.