Zinc-dependent multimerization of mutant calreticulin is required for MPL binding and MPN pathogenesis

Calreticulin (CALR) is mutated in the majority of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs). Mutant CALR (CALR(de)(l52)) exerts its effect by binding to the thrombopoietin receptor MPL to cause constitutive activation of JAK-STAT signaling. In this study, we performed an extensive mutagenesis screen of the CALR globular N-domain and revealed 2 motifs critical for CALR(de)(l52) oncogenic activity: (1) the glycan-binding lectin motif and (2) the zinc-binding domain. Further analysis demonstrated that the zinc-binding domain was essential for formation of CALR(de)(l52) multimers, which was a co-requisite for MPL binding. CALR dels2 variants incapable of binding zinc were unable to homomultimerize, form CALR(de)(l52)-MPL heteromeric complexes, or stimulate JAK-STAT signaling. Finally, treatment with zinc chelation disrupted CALR(de)(l52)-MPL complexes in hematopoietic cells in conjunction with preferential eradication of cells expressing CALR(de)(l52) relative to cells expressing other MPN oncogenes. In addition, zinc chelators exhibited a therapeutic effect in preferentially impairing growth of CALR(de)(l52)-mutant erythroblasts relative to unmutated erythroblasts in primary cultures of MPN patients. Together, our data implicate zinc as an essential cofactor for CALR(de)(l52)oncogenic activity by enabling CALR(de)(l52) multimerization and interaction with MPL, and suggests that perturbation of intracellular zinc levels may represent a new approach to abrogate the oncogenic activity of CALR(de)(l52) in the treatment of MPNs.

Automated summary

This study identified key motifs for the oncogenic activity of CALR(de)(l52), highlighting the essential role of the zinc-binding domain in multimerization of CALR(de)(l52) and interaction with MPL. Zinc chelators showed therapeutic potential in preferentially targeting CALR(de)(l52)-mutant cells, suggesting that modulation of intracellular zinc levels could be a novel approach to inhibit the oncogenic activity of CALR(de)(l52) in MPNs treatment.