Zinc-Dependent Homomultimerization of Mutant Calreticulin Is Required for MPN Pathogenesis
Authors: Rivera Jeanne F , Baral April Joy , Smyth Rachael , Nadat Fatima , Burman Emma , Alameer Ghadah , Boxall Sally A , Jackson Brian R , Mullally Ann , Chen Edwin 
Published: February 2020

Recurrent mutations in the ER chaperone calreticulin (CALR) are found in ~30% of MPNs. All known CALR mutations result in a +1-frameshift of the CALR reading frame that retains the N-domain and P-arm of wild-type CALR and generates a novel mutant-specific C-terminal domain encoded by an alternative reading frame. Multiple groups have previously demonstrated that mutant CALR (CALRMUT) physically binds to and activates the thrombopoietin receptor MPL and causes inappropriate receptor activation and downstream JAK-STAT signaling in a ligand-independent manner. These oncogenic activities of CALRMUT have been shown to be dependent on a threshold of basic amino acid residues in the mutant-specific C-terminus and on the lectin motif within the N-domain.
In this study, we extended the analysis of the role of the N-domain in CALRMUT activity by performing a comprehensive alanine mutagenesis screen of all 177 non-alanine residues within the N-domain and assessed the ability of alanine-mutants to confer cytokine independent growth in Ba/F3-MPL cells. We identified two functional groups of residues as being important for CALRMUT activity: (i) Residues associated with CALR lectin motif. Consistent with previous work which had shown that Asp135 and Asp317 was essential for CALRMUT activity, we observed that loss of any of 4 residues within the lectin motif (Cys105, Lys111, Gly133 and Asp135) led to total impairment in the ability of CALRMUT to confer cytokine independence, bind MPL or elicit JAK-STAT signaling. Surprisingly, other residues implicated in lectin activity (Tyr109, Tyr128, Met131) had either only a partial effect or no effect on CALRMUT oncogenic activity, suggesting that different regions within the lectin motif of CALR have differential effects in facilitating MPL binding. (ii) Residues associated with zinc binding. We also identified a group of zinc-binding histidine residues (His99, His145 and His170) as being important for modulating CALRMUT activity. We observed that loss of any single histidine residue (1xHis) led to partial abrogation of cytokine independence in Ba/F3-MPL cells. Strikingly, combined loss of two (or more) histidines (2xHis) led to complete impairment in the capacity to confer cytokine independent growth or elicit JAK-STAT signaling, in conjunction with impaired ability to physically associate with Zn2+ by affinity chromatography. In addition, 2xHis-CALRMUT failed to bind MPL in pulldown assays or colocalize with MPL in FRET-based immunocytochemical analysis.
We next investigated in greater detail how the zinc binding capacity of CALRMUT regulates its oncogenic activity. Previous studies have shown that CALRMUT forms homomultimers (Araki, Leukemia, 33:122), and that zinc is able to increase homomultimerization of recombinant wild-type CALR in vitro (Li, Biochemistry, 40:11193). We therefore tested the multimerization capacity of lectin-deficient or zinc binding-deficient CALRMUT by assessing the ability of heterologously-expressed FLAG-tagged CALRMUT species to complex and co-immunoprecipitate with a V5-tagged CALRMUT species in 293T cells. We observed that lectin-deficient CALRMUT and 1xHis-CALRMUT retained the capacity to form homomultimers, but the 2xHis- CALRMUT could not, suggesting that zinc binding was required for homomultimer formation. Treatment with ZnCl2 which increases free intracellular Zn2+ resulted in increased CALRMUT homomultimerization in a dose-dependent manner, as well as increased binding to MPL within heteromeric complexes. Finally, treatment with the zinc chelator TPEN led to decreased CALRMUT homomultimer formation, decreased CALRMUT-MPL heteromultimeric complexes and attenuated JAK-STAT signaling. Cumulatively, our data suggest that zinc is required for homomultimerization of CALRMUT, and is a necessary prerequisite event to facilitate binding to and activation of MPL in CALR-mutated MPNs.

Disclosures
Mullally: Janssen: Research Funding.