There is a fundamental gap in understanding how mutations in calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, cause myeloproliferative neoplasms (MPN). Continued existence of this gap represents an important problem because despite the high frequency of CALR mutations in MPN (40%), there are currently no treatment strategies to specifically target CALR-mutant cells in MPN. Furthermore, although the recently FDA-approved JAK2 inhibitors can provide palliative benefit to MPN patients, including those harboring CALR mutations, JAK2 inhibition does not preferentially target the MPN clone and therefore does not have curative potential in these diseases. The long-term goal is to understand the mechanisms by which mutant CALR induces MPN in order to exploit these insights for therapeutic gain. The overall objective in this application is to determine how mutant CALR transforms hematopoietic cells to engender MPN. The central hypothesis is that mutant CALR, by virtue of its mutant-specific C-terminus and altered sub-cellular localization develops novel protein binding interactions, including with the thrombopoietin receptor, MPL that induce JAK2-STAT signaling pathway activation, to drive the development of clonal hematopoiesis and the MPN phenotype. The rationale for the proposed research is that, once we understand how mutant CALR induces MPN, it will be possible to identify CALR-specific molecular dependencies that can be exploited therapeutically to develop novel treatment approaches. Guided by strong preliminary data, the hypothesis will be tested by pursuing three specific aims: 1) Dissect the requirement for MPL and the mutant CALR C- terminus in oncogenic transformation; 2) Determine the effects of mutant CALR expression on hematopoiesis in vivo; and 3) Determine the key proteins that differentially bind mutant CALR. Under the first aim, a combination of mutagenesis-based structure-function analyses and sub-cellular localization studies (already confirmed as feasible in the applicants' hands), will be applied to several well-established in vitro model systems, to further dissect the interaction between MPL and mutant CALR and define its precise role in inducing MPN. Under the second aim, the mechanisms that allow CALR-mutant hematopoietic stem cells (HSC) to become clonally dominant and induce MPN in vivo will be determined using mouse models of mutant CALR-driven MPN, that are already on hand. Under the third aim, in collaboration with a leader-in-the-field in proteomics, the novel protein binding interactions of mutant CALR will be determined and those required for its transforming capacity will be validated using functional genomics. The approach is innovative through the application of novel murine models, in vitro and in vivo CRISPR/Cas9 genome editing and mass spectrometry (MS)-based quantitative proteomics. The proposed research is significant because it will uncover the mechanisms underlying the pathogenesis of mutant CALR-driven MPN. Ultimately, such knowledge has the potential to inform the development of curative treatment strategies for this disease.

Public Health Relevance

The proposed research is relevant to public health because it will determine the mechanisms by which mutations in the gene, calreticulin (CALR), cause a group of blood disorders called myeloproliferative neoplasms (MPN). CALR mutations are present in 40% MPN patients and give rise to blood disorders characterized by excess blood cell production, an increased risk of transforming into leukemia and no curative treatment options apart from stem cell transplantation. Thus, the proposed research is relevant to the part of the NIH's mission that pertains to understanding mechanisms of disease with the goal of identifying better, more specific treatment options, thus reducing the burden of human disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Molecular and Cellular Hematology Study Section (MCH)
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El Kassar, Nahed
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Brigham and Women's Hospital
United States
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Elf, Shannon; Abdelfattah, Nouran S; Baral, April J et al. (2018) Defining the requirements for the pathogenic interaction between mutant calreticulin and MPL in MPN. Blood 131:782-786
Ciboddo, Michele; Mullally, Ann (2018) JAK2 (and other genes) be nimble with MPN diagnosis, prognosis, and therapy. Hematology Am Soc Hematol Educ Program 2018:110-117
Abdelfattah, Nouran S; Mullally, Ann (2018) Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells. J Vis Exp :
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