Research Projects

Calreticulin (CALR) is an endoplasmic reticulum (ER) chaperone protein that is essential for protein homeostasis. In 2013, +1 frame shift mutations were discovered in the last exon (exon 9) of calr in patients with myeloproliferative neoplasm (MPN). The patients presented with high platelet counts with/without associated thrombosis and fibrosis of the marrow. MPN patients progressed into secondary acute myeloid leukemia with poor prognosis. The frameshift mutations in CALR altered the negatively charged amino acid into methionine-rich and positively charged amino acids along with deletion of the ER retrieval sequence (KDEL). This induced secretion of the mutant CALR as well as lowered its Ca+2 buffering activity in the ER. Further, mutant CALRs interacted specifically with the thrombopoietin receptor and co-localized with the receptor on the surface of hematopoietic stem-progenitor cells, megakaryocytes and platelets. This activated the receptor leading to JAK2-STAT1/3/5 dependent signalling and cytokine independent growth and myeloproliferation. However, key differences were observed in MPN driven by mutant CALR and MPN driven by constitutively active JAK2 V617F. It was observed that CALR mutations induced an early clonal dominance and was seen in younger patients when compared to JAK2 V617F mutation. This indicated that mutant CALR driven MPN pathophysiology cannot be entirely explained by TpoR-JAK2 dependent signalling. We hypothesized that the mutant CALR tail that is unique to the human proteome may have acquired novel interactors or may have lost CALR WT interactors leading to changes in cellular physiology. Our preliminary analysis of the CALR mutant and WT C-terminus specific interactome revealed two proteins that are crucial to lysosomal biogenesis and distribution. Recent articles have indicated that quiescent hematopoietic stem cells (HSCs) maintain enlarged lysosomes possibly due to reduced lysosomal activity. Exit from quiescence was characterized by increased oxidative phosphorylation, increased mTORC1 signalling and reduced lysosomal size. Therefore, in this project we hypothesize that the CALR mutants alter the lysosome distribution and numbers in the cells resulting in altered cellular metabolism and proliferation. We will use both transient and retroviral expression systems to analyse the effects of the mutant CALR on lysosomal biogenesis, size and subcellular distribution. We will use state of the art confocal microscopy, super resolution microscopy, flow cytometry and biochemistry to address these questions. Finally, using mutant CALR interactor specific knock-down/knock-out we will examine the effects of the interactor on cellular metabolism and proliferation. Our results are expected to shed light on the pathophysiology of myeloproliferation.