MPL W515L expression induces TGFβ secretion and leads to an increase in chemokinesis via phosphorylation of THOC5

Abstract

The thrombopoietin receptor (MPL) has been shown to be mutated (MPL W515L) in myelofibrosis and thrombocytosis yet new approaches to treat this disorder are still required. We have previously shown that transcriptome and proteomic effects do not correlate well in oncogene-mediated leukemogenesis. We therefore investigated the effects of MPL W515L using proteomics. The consequences of MPL W515L expression on over 3300 nuclear and 3500 cytoplasmic proteins were assessed using relative quantification mass spectrometry. We demonstrate that MPL W515L expression markedly modulates the CXCL12/CXCR4/CD45 pathway associated with stem and progenitor cell chemotactic movement. We also demonstrated that MPL W515L expressing cells displayed increased chemokinesis which required the MPL W515L-mediated dysregulation of MYC expression via phosphorylation of the RNA transport protein THOC5 on tyrosine 225. In addition MPL W515L expression induced TGFβ secretion which is linked to sphingosine 1-phosphate production and the increased chemokinesis. These studies identify several pathways which offer potential targets for therapeutic intervention in the treatment of MPL W515L-driven malignancy. We validate our approach by showing that CD34+ cells from MPL W515L positive patients display increased chemokinesis and that treatment with a combination of MYC and sphingosine kinase inhibitors leads to the preferential killing of MPL W515L expressing cells.

AXL Inhibition Extinguishes Primitive JAK2 Mutated Myeloproliferative Neoplasm Progenitor Cells

“…The role of AXL in drug resistance in CML has been linked to increases in LYN kinase activity 18,20,21 . Given these facts and our previous work demonstrating the similarity between the proteomic perturbations observed in CML and PV 11,12 and our observation that LYN kinase is upregulated by JAK2 V617F 12 and MPL W515L 22 we evaluated AXL as a candidate drug target in MPN.…”

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

“…The data derived from the proteomic analyses presented a phenotype of NS/JMML cells that were different in the expression of motility proteins, which translated into a distinct difference in motility itself in response to CXCL12. The role of this pathway (CXCL12/CD45/Src protein kinases/spliceosome complex proteins/MYC) in leukemogenesis has been elucidated by us in the past. ,, Previous transcriptional analysis highlighted MYC as a potential regulator of the changes observed, while computational analysis of the global protein changes suggested NF-κb and TP53 as hubs differentially affected in NS and NS/JMML cells. TP 53 is rarely mutated in JMML, , which argues for translational or post-translational regulation of this nodal point in transformation processes in JMML.…”

“…Chemotaxis assays were performed using a Boyden chamber assay in a 96-well plate format (Neuroprobe), as previously described. 26 The number of CD33+ cells migrating in response to 200 ng/mL of CXCL12 was assessed over 6 h.…”

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

“…PTPRC and TGFβ were chosen because we have previously reported a reduction in PTPRC expression 18 and upregulation of TGFβ expression 15 induced by a range of other leukaemogenic oncogenes. 19 In addition, TGFβ plays an important role in the pathogenesis of CML 20 and myelofibrosis. 21 Western blot analysis of PTPRC expression in the presence of activated JAK2 mutants ( Figure 1a ) demonstrated decreased levels compared with control cells.…”