Heterodimeric JAK-STAT Activation as a Mechanism of Persistence to JAK2 Inhibitor Therapy

The identification of somatic activating mutations in JAK21–4 and in the thrombopoietin receptor (MPL)5 in the majority of myeloproliferative neoplasm (MPN) patients led to the clinical development of JAK2 kinase inhibitors6,7. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms, but does not significantly reduce or eliminate the MPN clone in most MPN patients. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic JAK2 inhibition. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible, such that JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, murine models, and patients treated with JAK2 inhibitors. RNA interference and pharmacologic studies demonstrate that JAK2 inhibitor persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors

Heterodimeric JAK-STAT Activation as a Mechanism of Persistence to JAK2 Inhibitor Therapy

The identification of somatic activating mutations in JAK21–4 and in the thrombopoietin receptor (MPL)5 in the majority of myeloproliferative neoplasm (MPN) patients led to the clinical development of JAK2 kinase inhibitors6,7. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms, but does not significantly reduce or eliminate the MPN clone in most MPN patients. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic JAK2 inhibition. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible, such that JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, murine models, and patients treated with JAK2 inhibitors. RNA interference and pharmacologic studies demonstrate that JAK2 inhibitor persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors

Targeting megakaryocytic induced fibrosis by AURKA inhibition in the myeloproliferative neoplasms

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleen of patients are full of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines including TGF-β. Although the JAK inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or significantly reverse fibrosis. Here we show through pharmacologic and genetic studies that, apart from JAK2, Aurora kinase A (AURKA) is a novel therapeutic target in PMF. MLN8237, a selective AURKA inhibitor promoted polyploidization and differentiation of PMF megakaryocytes and displayed potent anti-fibrotic and anti-tumor activity in vivo. We also reveal that loss of one allele of AURKA is sufficient to ameliorate fibrosis and other PMF phenotypes in vivo. Our data suggest that megakaryocytes are drivers of fibrosis and that targeting them with AURKA inhibitors will provide therapeutic benefit in PMF

A simple method for extraction of high molecular weight genomic DNA from buccal cells in mouthwash

477-481With tremendous advances made in the field of molecular biology techniques, the analysis of genomic DNA has become a matter of routine. However, there is a growing need for better, cost-effective, non-invasive methods for sample collection and DNA extraction. A simple protocol for sample collection and extraction of high molecular weight genomic DNA from buccal cells in mouthwash has been standardized. The method does not require elaborate instrumentation and can be applied to large population based studies. It has been observed that good quality high molecular weight genomic DNA can be obtained from exfoliated buccal cells in the early morning mouthwash samples, and that the DNA yield from similar samples decreases during the day, with very low yields obtained in the late evening. The amount of DNA obtained is enough for approximately 300 PCR amplifications using 100 ng DNA as template. DNA so obtained was used successfully for numerous amplifications by PCR with human gene specific primers and can be used for Restriction Fragment Length Polymorphism (RFLP) studies as well as for other molecular biology applications

Glycosaminoglycan measured from synovial fluid serves as a useful indicator for progression of Osteoarthritis and complements Kellgren–Lawrence Score

Background: Plain radiography is the first choice for diagnosis and monitoring of knee-osteoarthritis (OA) while, Kellgren–Lawrence score (KL) is most widely used to grade OA severity. However, incompetency for reproducibility of joint space measurement in longitudinal assessment and non-linearity of KL-score system, limits radiography-based early diagnosis of the disease. Glycosaminoglycan (GAG) is direct cartilage-degradation product, which can be measured biochemically. We strived to correlate KL-score and GAG from OA patients to compliment KL-system. Methods: We obtained 34 synovial-fluid (SF) samples from 28 OA patients (few bilateral) with different disease severity using arthrocetesis. All patients were categorised using radiographic KL-score-system. SFs were further analysed for GAG estimation using 1,2-dimethylmethylene blue (DMMB) assay. Results: A substantial increase in GAG was noted in KL-grade-II and III, comparing grade-I patients, indicating amplified cartilage-degradation. KL-grade-IV patients revealed further rise in GAG reflecting more cartilage-loss. Another category of grade-IV patients with lower GAG were also detected, indicating close to total cartilage-loss. Conclusions: Accurate diagnosis of cartilage-loss remains a challenge with OA due to limitations of KL-system; thus no target intervention is available to arrest active cartilage-loss. We propose, GAG-estimation in OA patients, characterizes accurate biochemical depiction of cartilage degeneration. General Significance: Radiology often fails to reveal an accurate cartilage loss, associated with OA. GAG levels from the SFs of OA patients' serve as a useful marker, which parallels cartilage degeneration and strengthen radiographic grading system, ultimatel

JAK2V617F-Mediated Phosphorylation of PRMT5 Down-Regulates Its Methyltransferase Activity and Promotes Myeloproliferation

Abstract Abstract 794 Background: The cytoplasmic, non-receptor JAK2 tyrosine kinase is mutated at amino acid residue 617 (from valine to phenylalanine) in most patients with myeloproliferative neoplasms (MPNs), resulting in a constitutively activated kinase that phosphorylates STAT proteins in the absence of upstream signals. Overexpression of JAK2V617F leads to cytokine-independent growth of Ba/F3 cells and the JAK2V617F transgenic and knockin mice develop a disease phenotype resembling human polycythemia vera. Results: We hypothesized that the JAK2V617F occurs so consistently in MPNs because it gains some functional property. The type II arginine methyltransferase PRMT5 was initially identified because of its interaction with JAK2 in a yeast two hybrid screen. We examined the interaction between JAK2 and PRMT5 and found that JAK2V617F and JAK2K539L (another active JAK2 kinase) bound PRMT5 more strongly than did wild-type JAK2. PRMT5 mediates the symmetrical dimethylation of arginine residues within histones H2A and H4 and methylates other cellular proteins as well, such as p53. The oncogenic forms of JAK2 acquire the ability to phosphorylate PRMT5, which greatly impaired its methyltransferase activity. We have shown the in vivo importance of this post-translation modification as treating JAK2V617F-positive cells (but not the wild-type JAK2-harboring cells) with different JAK2 inhibitors significantly increased histone arginine methylation levels. To define the effect of inhibiting PRMT5 activity on hematopoiesis, we knocked down PRMT5 in human cord blood derived CD34+ cells using shRNA and observed increased colony formation and erythroid differentiation; In contrast, PRMT5 overexpression in these cells led to reduced colony formation and inhibition of erythroid differentiation. Furthermore, overexpression of PRMT5, especially a phosphorylation site mutant form of PRMT5 (PRMT5M6), diminishes the proliferative and erythroid generating capacity of JAK2V617F+ CD34+ cells isolated from MPN patients to a greater degree than normal cord blood CD34+ cells. Importantly, we found marked increase in PRMT5 phosphorylation in JAK2V617F-positive MPN patents relative to normal cord blood CD34+ cells, suggesting that this phosphorylation is important for the myeloproliferation phenotype. Conclusion: we show that the oncogenic mutant forms of JAK2 kinase, such as JAK2V617F and JAK2K539L, are not simply constitutively active forms of wild-type JAK2, rather they have specific gains-of-function that allow them to phosphorylate PRMT5 and down-regulate its enzymatic activity. Inhibition of PRMT5 contributes to the myeloproliferation and erythroid differentiation promoting effects of JAK2V617F. This gain-of –function mutation results in cross-talk between oncogenic kinases and histone arginine methylation. Taken together, we demonstrate a novel link between the mutant JAK2 kinases and PRMT5 methyltransferase activity, which contributes to MPN pathogenesis. Further insights about the shared gene expression profile of JAK2 inhibition vs. PRMT5 knockdown will be presented to understand the basics for the behavior change in hematopoietic stem/progenitor cells brought about by these two interventions. Disclosures: No relevant conflicts of interest to declare