He, Fan

Abstract Myeloproliferative neoplasms (MPNs) are typically ascribed to hyperactive JAK-STAT signaling via mutations in JAK2, CALR, or MPL. Here, we characterize how activating mutations in the calcium sensor STIM1, previously established as the cause of Stormorken Syndrome (SS), a rare congenital disorder, are sufficient to drive development of myelofibrosis (MF). We further uncover distinct interactions between STIM1 activity and JAK2 versus CALR driven MPNs. We initially identified two SS patients with activating mutations in STIM1 (R304W and S88G) and features of MF, including marked reticulin fibrosis with megakaryocytic hyperplasia and atypia. This led us to hypothesize that altered calcium signaling may play a role in the development of MF in both SS and MPNs. Single cell RNA sequencing performed on samples from both patients demonstrated elevated NFκB inflammatory signaling in monocyte populations, suggesting a link between altered STIM1 and store operated calcium entry (SOCE) activity and aberrant inflammation, thereby contributing to MF development. To corroborate these findings from our SS patients, we generated a knock-in mouse model of the heterozygous R304W mutation. Induction of hematopoietic-specific expression of mutant Stim1 with Vav-Cre recapitulated the hematological features of SS, including elevated SOCE activity and thrombocytopenia. We also recapitulated MF features including the development of bone marrow fibrosis and severe osteosclerosis, with the mice developing splenomegaly as young as 9 weeks old. In ex vivo megakaryocyte differentiation assays with our Stim1 R304W knock-in mice, we also observed aberrant cytoskeletal organization and elevated TGFβ mRNA expression. When induction was driven universally through CMV-Cre, we also validated that mice recapitulated multiple SS features, including thrombocytopenia, tubular aggregate myopathy, growth deficiency, and the development of bone marrow fibrosis as young as 10 weeks. We additionally identified a separate cohort of 9 related individuals with SS and STIM1 mutations (L92V) as well as a separate patient with the R304W mutation. In ex vivo megakaryocytic differentiation assays, cells from SS patients demonstrated defective thrombopoiesis with significantly decreased proplatelet formation, and displayed aberrant cytoskeletal formation within the pro-platelet termini, in conjunction with increased expression of TGFβ. Further supporting a role for STIM1 in MPNs, we identified upregulation of STIM1 expression in MPN patient CD34+ hematopoietic stem/progenitors' cells (HSPCs), we well as megakaryocyte progenitors and platelets, including those with CALR or JAK2 mutations. To evaluate the functional role of STIM1 in MPNs, we generated patient-derived xenograft (PDX) models with CALR and JAK2-mutantpatient samples subjected to CRISPR ablation of STIM1. In an initial CALR-mutant PDX experiment, abrogation of STIM1 led to decreased human CD45+ cell engraftment in conjunction with prolonged survival. In a second CALR-mutant PDX experiment, STIM1 targeting did not overtly impact engraftment or survival. In contrast, CRISPR ablation of STIM1 in JAK2-mutant PDX models led to exacerbated disease phenotypes, including increased human CD45+ cell engraftment, increased splenomegaly, and early lethality. Altogether, this study represents the first demonstration of MF development in patients with Stormorken Syndrome and reveals a previously unrecognized role of aberrant SOCE underlying SS and MPNs. We recapitulated these findings in a novel conditional mouse model. These findings also illustrate unique interactions between STIM1 and JAK2 and CALR driven MPNs. These observations have important implications for the development of targeted therapeutic approaches for these disorders.

Activating mutations in the calcium sensor STIM1 underlie Stormorken Syndrome (SS), a rare congenital disorder that causes thrombocytopenia and tubular aggregate myopathy. We recently identified two patients with SS and confirmed mutations in STIM1 (R304W and S88G), who were found to have features consistent with myelofibrosis (MF), as indicated by bone marrow biopsies demonstrating megakaryocyte hyperplasia and atypia in conjunction with marked reticulin fibrosis. The unusual MF findings in these two patients raised the hypothesis that altered calcium signaling represents a shared hallmark of congenital platelet disorders such as Stormorken Syndrome and myeloproliferative neoplasms (MPNs) including MF. Analyzing gene expression data from public datasets, we found increased expression of STIM1 in megakaryocyte progenitors and platelets, as well as CD34+ hematopoietic stem/progenitor cells (HSPCs), from MPN patients (including those with CALR or JAK2 mutations) vs healthy controls. These findings led us to hypothesize that dysregulation of STIM1 and store operated calcium entry (SOCE) signaling may contribute to altered megakaryopoiesis and development of fibrosis in both SS and MPNs. To further delineate these processes, we analyzed samples from our two SS patients, demonstrating constitutive activation of SOCE causing increased calcium flux, as well as decreased aggregation in mature platelets. We also performed transmission electron microscopy (TEM) and identified platelets with abnormal granularity in dense and alpha granules. We also performed single cell RNA-seq on SS patient peripheral blood mononuclear cells, which revealed enhanced NFkB inflammatory signaling, suggesting that altered signaling driven by STIM1 mutations may drive aberrant inflammation to contribute to MF development. To corroborate these initial observations, we identified a separate family cohort of 9 individuals in Italy with SS and confirmed STIM1 mutation (L92V). In ex vivo megakaryocytic differentiation assays, cells derived from these patients demonstrated a defect in thrombopoiesis with significantly decreased pro-platelet formation and adhesion to extracellular matrix. To further characterize the effects of the gain of function STIM1 mutations seen in our patients, we developed a novel, conditional knock-in mouse model of the heterozygous R304W mutation in the coiled coil (CC) domain of Stim1. Induction of hematopoietic-specific expression of mutant Stim1 via Vav-Cre resulted in recapitulation of hematological features of SS, including thrombocytopenia and a mild bleeding tendency, with confirmation of increased SOCE activity and calcium flux. Notably, we confirmed the development of bone marrow fibrosis, as well as findings of severe osteosclerosis, in these mice. TEM imaging confirmed that platelets from Stim1R304W/+Vav-Cre mice exhibit abnormal granularity, similar to what we observed from our SS patients. Noting that we identified increased STIM1 expression in samples from MPN patients harboring either JAK2 or CALR mutations, we further characterized the role of STIM1 in MPN disease development. NSGS mice were engrafted with CALR-mutant CD34+ cells subjected to CRISPR ablation of STIM1. Recipient mice exhibited decreased human CD45+ cell engraftment in conjunction with prolonged survival. In contrast, targeting of STIM1 in JAK2-mutant CD34+ cells led to exacerbated disease phenotypes, as manifested by enhanced human CD45+ cell engraftment, worsened splenomegaly, and early lethality. Taken together, these findings suggest a striking, differential relationship between mutant JAK2 and CALR and STIM1 activity and their relationship to MF development. Altogether, this study represents the first demonstration of MF development in patients with Stormorken Syndrome, and reveals a previously unrecognized hallmark of altered calcium signaling via aberrant STIM1 activation underlying SS and MPNs. We further demonstrate recapitulation of these hematologic features in a novel knock-in Stim1R304W/+Vav-Cre mouse model. These findings implicate an important role for STIM1and SOCE activity in MF development, and further uncover important distinctions between JAK2 vs CALR mutations and their interaction with altered STIM1 activity. These studies may contribute to the development of novel therapeutic approaches for these disorders.