Overexpression of Calreticulin Impairs Self-Renewal Capacity of Hematopoietic Stem Cells

Mutations in JAK2, c-MPL and CALR have all been identified as drivers of MPN pathogenesis. JAK2 and c-MPL are critical for hematopoietic stem cell self-renewal and differentiation, however CALR's physiologic role in hematopoiesis is unknown. To investigate CALR's role in normal and malignant hematopoiesis lethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with CALR^DEL, CALR^WT or empty MSCV-IRES-hCD4 vector retrovirus. As expected, mice transplanted with CALR^DEL developed thrombocytosis, mice transplanted with CALR^WT and empty vector had normal peripheral blood counts. At 6 months post-transplant mice were sacrificed. The percentage of hCD4+ whole bone marrow cells (marks cells expressing gene of interest) was 76.6% for empty vector, 73.1% for CALR^WT, and 8.6% for CALR^DEL. hCD4+ hematopoietic stem/progenitor cells (LKS), common myeloid progenitors (CMP), granulocyte monocyte progenitors (GMP), and megakaryocyte erythroid progenitors (MEP) from empty vector, CALR^WT and CALR^DEL mice were sorted into methylcellulose supplemented with cytokines (mIL-3, hEPO, and mSCF). MEP from CALR^WT mice formed fewer colonies compared to empty vector and CALR^DEL (p>0.004). GMP, CMP, and LKS from empty vector, CALR^WT and CALR^DEL formed equivalent numbers of colonies. To assess self-renewal function colonies were amalgamated and serially replated into methylcellulose at a concentration of 10,000 cells/ml. Upon serial replating CALR^WT LKS cells formed fewer colonies compared to CALR^DEL and empty vector (p>0.001). To more specifically assess the effect of CALR on HSC self renewal potential whole bone marrow from mice transplanted with CALR^DEL, CALR^WT or empty MSCV-IRES-GFP (MIG) vector was serially transplanted into lethally irradiated C57B/6 secondary recipient mice and the percentage of GFP-positive cells in the peripheral blood was measured by flow cytometry over time. Mice transplanted with CALR^WT bone marrow cells quickly lost GFP positive cells in the peripheral blood over time whereas MIG empty vector and CALR^DEL mice retained GFP positive cells for at least 8 months post-transplant.

Because mutant CALR^DEL interacts with and activates the thrombopoietin receptor (c-MPL) to drive MPN and because the defect in hematopoietic stem cell self-renewal that we observed in mice overexpressing CALR^WT is reminiscent of the phenotype of mice lacking c-MPL we hypothesize that CALR^WT interacts with c-MPL and negatively affects its function. Using co- immunoprecipitation of Ba/F3 cells doubly transduced with GFP tagged c-MPL and hCD4 tagged empty vector, CALR^WT or CALR^DEL, we found that CALR^WT and CALR^DEL interacts with c- MPL.

In summary, these data demonstrate that overexpression of CALR^WT negatively affects the self-renewal capacity of hematopoietic stem cells, evidenced by impaired ability to reconstitute secondary hosts and reduced serial re-plating ability in methylcellulose. This may be due to CALR^WT binding to c-MPL and negatively regulating its function.