Preclinical evaluation of JAK2 specific investigational oligonucleotide for the treatment of MDS/PV.

Abstract

122 Background: Myeloproliferative disorders (MPD) are clonal hematopoietic stem cell malignancies with cytokine independency or hypersensitivity. Polycythemia vera (PV), an acquired MPD characterized by increased blood cell mass and hematocrit and leukocyte count, is associated with incidental myelofibrosis (MF). MDS is characterized by cytopenia and the presence of morphological dysplasia of precursor and mature bone marrow blood cells. PV and MDS leave patients at risk for progression to acute myeloid leukemia. Abnormal cytokine signaling due to an aberrant JAK2-STAT pathway has a vital role in PV and MDS pathogenesis. JAK2 mutations can result in hematologic malignancies, where hyperactive signaling of the JAK2-STAT pathway promotes tumor cell proliferation, invasion, and angiogenesis. Increased JAK2 kinase activity is observed in hematologic malignancies; somatic JAK2 V617F gain-of-function mutations are found in at least 95% of PV patients and is implicated in MDS cases. We hypothesize that preventing JAK2 transcription by antisense oligonucleotide (ASO) mediated exon masking of the JAK2 intron-exon junction will result in reduced JAK2 mRNA, and thus JAK2 protein, by providing nonsense mediated decay (NMD) in the reading frame. This may alleviate disease manifestations of hematologic malignancies like PV and MDS. Methods: This study utilized a HEL cell line harboring the V617F JAK2 gain-of-function mutation. We designed a series 19mer ASO targeting JAK2 exon-intron junctions ( JAK2 intron-exon junction providing NMD in the reading frame, steric, non-RNAse H1) and tested these at a range of concentrations. The ASOs were designed with a phosphorothioate 2’-O-methoxyethyl backbone and prioritized based on in silico binding affinity and limited off-target binding. HEL cells underwent ASO treatment (1µM) and 72-hour incubation. Results: We observed significant JAK2 protein decrease (~50%) in ASO-treated samples compared to untreated samples. JAK2 qPCR results confirmed 40-60% of target transcript. STAT5 phosphorylation status further confirmed this effect, and we report a 35% pSTAT5 reduction. Furthermore, this ASO showed limited off-target effects in silico. siRNA and CRISPR knockout lysate were used as controls. Our preclinical data support this ASO as a highly specific JAK2 agent, affecting direct levels of JAK2 as well as downstream STAT signaling. Conclusions: Around 50-60% of primary MF patients harbor the JAK2 V617F gain-of-function mutation. ASOs offer the capability to directly target JAK2 mutations with high precision and effectively reduce JAK2 protein production, without off-target kinase effects. The ability to reduce JAK2 protein may alleviate the disease burden that patients with hematologic malignancies face, resulting in a higher quality of life through prevention and treatment.