PROJECT SUMMARY Although the mechanism by which calreticulin (CALR) mutations cause myeloproliferative neoplasms (MPN) has been elucidated, there is currently a fundamental gap in translating this knowledge into innovative therapeutic strategies. The long-term goal is to advance the treatment of CALR-mutant MPN, in particular to develop new therapies with disease-modifying activity and curative potential. The overall objective in this application is to exploit the insights we have gained from understanding the altered biochemical properties and unique molecular dependencies of mutant CALR-driven MPN, to identify novel therapeutic vulnerabilities, including in the context of CALR/ASXL1 co-mutation. The central hypothesis is that CALR-mutant hematopoietic stem cells (HSC) have unique properties, which arise as a consequence of the mechanism of oncogenicity of mutant CALR, which we have previously elucidated. The specific properties, which we hypothesize that CALR-mutant HSC possess, include altered protein homeostasis and a differential dependency on key cellular pathways (e.g. N-glycosylation and protein secretion) for survival. We further hypothesize that co-operating genetic events (e.g. concomitant ASXL1 mutation) alter the chromatin state of CALR-mutant HSC to drive disease progression in MPN. The rationale for the proposed research is that, once we develop novel therapeutic strategies to target the unique properties of CALR-mutant HSC, we will be able to preferentially target CALR-mutant MPN cells in patients. This has the potential to alter the natural history of CALR-mutant MPN, including in the context of ASXL1 co-mutation, which confers a negative prognostic impact on CALR-mutant MPN. Guided by strong preliminary data, the hypothesis will be tested by pursuing three specific aims: 1) Determine protein homeostasis and sensitivity to proteasome inhibition in Calr-mutant HSC; 2) Determine the molecular vulnerabilities of mutant CALR-driven MPN; and 3) Determine the impact of mutant Asxl1 on Calr-mutant MPN in vivo. Under the first aim, a mutant CALR knockin (KI) mouse model that closely recapitulates the features of human CALR-mutant MPN will be employed to measure protein synthesis and proteasome activity in Calr-mutant HSC and to determine if Calr- mutant HSC are differentially sensitive to in vivo proteasome inhibition. Under the second aim, key cellular pathways we have found to be uniquely required for the survival of mutant CALR-expressing hematopoietic cells in an in vitro whole genome CRISPR knockout screen, will be inhibited using functional genetic and pharmacological approaches in mutant CALR KI mice. Under the third aim, the impact of mutant Asxl1 on histone modifications, chromatin state and the transcriptome of Calr-mutant HSC, will be determined using a mutant Asxl1 KI mouse. The approach is innovative through the application of novel murine models, in vitro and in vivo CRISPR/Cas9 gene editing, chemical screening and mass spe...