Introduction: The MPN, polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) are clonal stem cell disorders, which share mutations constitutively activating the physiologic signal transduction pathways for hematopoiesis. Although the MPN have different natural histories, they share in common transformation to myelofibrosis and acute leukemia at differing frequencies and not explainable by a specific mutation. Impaired MPL expression, due to incomplete glycosylation is another common denominator amongst the MPN. Significantly, MPL is the only hematopoietic growth factor receptor expressed in MPN HSC, and we have demonstrated that the PV phenotype in a JAK2 V617F transgenic (V617tg) mouse could be abrogated by elimination of MPL or its ligand, TPO. Impaired MPL expression in the MPN cannot be completely explained by receptor-activated down regulation because not all MPN driver mutations directly activate MPL. However, we have discovered another common denominator in the MPN, variant MPL splicing, eliminating 7 amino acids in the MPL N-terminal domain, a common hotspot for both MPL driver and inactivating mutations, which impairs MPL glycosylation and expression.
Methods: To determine the role of the MPL splice variant (MPL SV) in MPN pathophysiology, we cloned the full length MPLSV cDNA and created a transgenic mouse (MPLSV tg) using the same VAV promoter as the V617Ftg mouse to ensure hematopoietic cell-specific transcription. The MPLSV tg mice were produced by pronuclear injection of purified MPLSV cDNA into B6SJLF1 mice. Founders were crossed into a C57Bl/6 background, and then were crossed with V617Ftg mice in either an MPL knockout or wild type background. Mice were phenotyped by blood counts and necropsy with morphologic and immunophenotyping of bone marrow and tumor masses.
Results: Amongst 19 founder B6SJLF1 mice, 6 expressed the MPLSV transgene with copy numbers ranging from one to 30; single copy number mice had no hematopoietic phenotype but all mice with higher MPLSV tg copy numbers had thrombocytopenia (median platelet count 500,000/µL; range 200- 600,000; wild type, 750,000/µL; 550-950,000). MPLSV tg mice from several different founders bred into a C57Bl/6 background for 6 generations maintained consistent copy numbers, Mendelian ratios and hematopoietic phenotypes with 100% penetrance and an inverse correlation between MPLSV copy number and the platelet count. The number of double transgenic (V617Ftg/MPLSV tg) offspring observed in the MPLSV tg to V617Ftg crosses were slightly lower than expected (20% vs 25%), indicating reduced embryo viability. Four week old V617Ftg/MPLSV tg mice displayed tumorous abnormalities of the head and hips as well as small size and failure to thrive (median weight 8.5 gms; range 8-9; wild type, 13 gms; 10-14), and enlarged spleens (median weight 0.29 gms; range 0.15-0.64; wild type 0.086 gms; 0.05-0.10). Histologically, the V617Ftg/MPLSV tg-associated head and hip abnormalities represented monomorphic myeloid sarcomas extending through the calvarium both extra and intracranially and from the femur into surrounding muscle. Both the marrow and spleen were diffusely infiltrated by large blasts with abundant basophilic cytoplasm, expressing CD34, CD61 and CD117 but lacking CD127, consistent with a myeloid origin. All V617Ftg/MPLSV tg mice either died or required humane sacrifice by 6 weeks. The extramedullary tumor was transplantable in secondary recipients, and flow cytometry-based phenotyping showed that the tumors (both primary and secondary) were CD34, CD117, CD61 and CD71- positive. Penetrance of the leukemia phenotype was 100% in V617Ftg/MPLSV tg from all founder lines with multiple copies of the MPLSV tg, whether in an MPL knockout or wild type background. The leukemia phenotype was never observed with the MPLSV tg alone or with V617Ftg alone despite observation of over 300 V617Ftg mice up to 50 weeks of age.
Conclusion: We identified an MPL SV in human MPN that was functional in mice with a dominant-negative effect with respect to platelet production and at the same time synergized with JAK2 V617F to create a fulminant myeloid malignancy. We have recently shown that knockout of MPL or TPO alone abrogates the PV phenotype in the V617Ftg mouse, whereas the MPLSV uniquely drives a highly penetrant and fulminant leukemia, establishing MPL and TPO as targets for mitigation of malignant transformation in the MPN.
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