Proteomic analysis of JAK2V617F-induced changes identifies potential new combinatorial therapeutic approaches

Abstract

In excess of 90% of patients with polycythaemia vera (PV) express a mutated form of Janus kinase 2 (JAK2), JAK2V617F. Such aberrant proteins offer great potential for the treatment of these diseases; however, inhibitors to JAK2 have had limited success in the clinic in terms of curing the disease. To understand the effects of this oncogene in haematopoietic cells with the aim of improving treatment strategies, we undertook a systematic evaluation of the effects of JAK2V617F expression using proteomics. The effects of JAK2V617F on over 5000 proteins and 2000 nuclear phosphopeptide sites were relatively quantified using either SILAC or eight-channel iTRAQ mass spectrometry. Pathway analysis of the proteins identified as changing indicated disruption to the p53 and MYC signalling pathways. These changes were confirmed using orthogonal approaches. The insight gained from this proteomic analysis led to the formation of hypothesis-driven analysis on inhibitor-mediated effects on primary cells from patients with a JAK2V617F mutation. Simultaneous inhibition of MYC and upregulation of p53 led to the preferential extinction of JAK2V617F-positive CD34+ cells, illustrating a potential therapeutic benefit from combined targeting of p53 and MYC.

Proteomic Analysis of an Induced Pluripotent Stem Cell Model Reveals Strategies to Treat Juvenile Myelomonocytic Leukemia

“…This implies the possibility of post-translational regulation of protein levels in these cells, as we observed previously in JAK2 mutant-driven polycythemia vera. 25 Given the fact that the perturbation was occurring post-translationally, we undertook a proteomic assessment of the cell lines.…”

“…We have previously demonstrated the efficiency of TP53 activation (Nutlin) in combination with MYC (JQ1) inhibition in the treatment of MPNs. 25 , 28 Nutlin inhibits the interaction between HDM2 and TP53 leading to the stabilization of TP53. 35 JQ1 is a BET bromodomain inhibitor, which reduces transcription by disruption of chromatin-dependent signaling 36 with MYC as a primary target.…”

“…Drugs were used at optimal doses, as previously defined. 25 , 28 Following 14 days under hematopoietic differentiation conditions, embryoid bodies were disrupted and colony-forming assays performed in the presence and absence of the drug. Colony numbers were assessed at 12 days.…”

“…Wild-type, NS and NS/JMML iPSC cells were differentiated toward hematopoietic progenitors over 14 days prior to the enrichment of CD33+ cells using CliniMACS (Miltenyi Biotec). Due to the relatively low yield of these clinically relevant progenitor cells, the three biological replicates for each cell line were pooled prior to being lysed to allow isobaric tagging using 8 channel iTRAQ reagent, nanoflow liquid chromatography, and tandem mass spectrometry, as previously described 25 and detailed in the Supporting Methods . Two 10 μg aliquots of protein from each pool were subject to iTRAQ labeling and MS analysis (as outlined in the Supporting Methods and Supporting Table 1 ).…”

The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34+ Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability

“…We have observed an upregulation of JAK‐STAT pathway and confirmed the increased levels of STAT5 phosphorylation by WES Simple and FC after the incorporation of MSC‐EV in CD34 + cells. Although there are no previous data on the implications of this (probably transient) upregulation of JAK‐STAT on CD34 + cells induced by MSC‐EV incorporation, it is well established that a constitutive upregulation of this signaling pathway (as observed in V617F JAK‐2 mutations in myeloproliferative neoplasms) leads to CD34 + cell proliferation and reduced apoptosis . In addition, an upregulation of JAK/STAT signaling has been shown in CD34 + cells from acute myeloid leukemia patients compared with normal cord blood or peripheral blood stem cells, suggesting that JAK/STAT signaling supports AML cells growth and survival .…”