Abstract
Background. Activating mutations in the thrombopoietin receptor (MPL), its proximal signaling partner Janus Kinase 2 (JAK2) or its endoplasmic reticulum chaperone, calreticulin (CALR), can lead to myeloproliferative neoplasms (MPNs). These chronic hematopoietic clonal neoplasms are associated with abnormal myeloid cell proliferation, cytokine production, as well as bone marrow fibrosis. The precise pathogenic mechanism leading to essential thrombocythemia (ET) and primary myelofibrosis (PMF), two closely linked MPNs, and the functional impact of MPL and CALR mutations on Mpl signaling and intracellular trafficking are yet to be determined. MPNs can evolve into acute myeloid leukemia (AML); however, the mechanisms of this transformation are mostly unknown. Here we report a unique case of post-ET MF with AML transformation, presenting with previously unreported double mutations: MPL W515R and a CALR 52-base pair deletion (Type I).
Case Report. A 69 yo female was diagnosed with ET in 1996, and subsequently progressed to post-ET MF in 2008. Molecular studies in 2008 were negative for JAK2 V617F and the BCR / ABL1 fusion. At this time, the patient was treated with hydroxyurea and erythropoietin. In 2010, the patient participated in the phase III COMFORT-I trial of ruxolitinib versus placebo and was randomized to ruxolitinib. Ruxolitinib was discontinued in 2014 due to severe anemia and transfusion dependence. The patient also showed no response after 3 cycles of the phase I/II trial of PRI-724, a β-catenin/wnt pathway inhibitor. She resumed ruxolitinib in 2015, but developed worsening leukocytosis and myeloid immaturity, progressing to AML in January 2016. She stopped ruxolitinib and received azacytidine for one cycle. However, the patient developed tumor lysis syndrome and acute on chronic kidney injury and expired. Molecular studies at the time of leukemic transformation revealed CALR type I and MPL W515R mutations. A repeat test for JAK2 V617F was negative.
Aim.This study focuses on the functional characterization of the MPL / CALR double mutations, their impact on Mpl intracellular trafficking and signaling, and their correlation with disease progression.
Methods. Cryopreserved PBMCs and/or granulocytes obtained at diagnosis and upon transformation to AML were used as source of primary cells to determine the mutational status of the patient. Sequencing, single colony assay (SCA), proximity ligation assay, flow cytometry, biotinylation assays, as well as western blotting analysis, performed on primary cells, were used to elucidate the function and trafficking of Mpl mutants in the context of mutant calreticulin. Multiplex cytokine profiling at different stages of disease progression was also evaluated. UT-7, megakaryoblastic cells, were subjected to CRISPR/Cas9-based genome engineering to model Mpl and calreticulin mutations and further investigate the complex interplay between these players in MPNs pathogenesis.
Results. At diagnosis, Sanger sequencing revealed the presence of a CALR Type I mutation while JAK2 and MPL were negative. gDNA sequencing of samples collected between diagnosis and AML transformation showed the presence of consistent heterozygous CALR Type I mutation but expanding MPL W515R mutation. Analysis of ~300 single colonies at transformation, grown in the presence (200 colonies) or in the absence (100 colonies) of cytokines, confirmed the presence of heterozygous CALR mutations in 100% of tested colonies. Notably, 22.4% were WT, 75.3% were heterozygous and 2.3% were homozygous for MPL W515R. Additionally, a myeloid gene panel analysis at AML transformation showed the presence of an additional ASXL1 mutation. Proximity ligation/microscopy assay performed on CD34+ cells showed that Mpl interacts with mutant calreticulin in a different cellular compartment, outside the ER and near the plasma membrane, than with wildtype calreticulin, indicating that receptor trafficking and signaling are closely related in MPN pathogenesis.
Conclusion. We report a unique case of CALR mutation associated with post-ET MF. Subsequent acquisition of MPL W515R by the MPN clone occurred temporally with AML transformation. Mechanistic studies using gene-edited UT-7 cell models are ongoing. This study will provide important insight in the understanding of MPNs progression and the relative contribution of CALR and MPL mutations to neoplastic transformation.
Gotlib: CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Pharmacyclics: Research Funding; Promedior: Research Funding; Deciphera: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Promedior: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Research Funding; Deciphera: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Author notes
Asterisk with author names denotes non-ASH members.
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