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1169 Altered Calcium Signaling Via STIM1 Drives Aberrant Megakaryopoiesis and Myelofibrosis

Program: Oral and Poster Abstracts
Session: 301. Platelets and Megakaryocytes: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Bleeding and Clotting, Translational Research, Platelet disorders, Diseases
Saturday, December 7, 2024, 5:30 PM-7:30 PM

Molly Brakhane, MSc1, Vittorio Abbonante, PhD2*, Angelo Laranjeira3*, Christian Andrea Di Buduo, PhD2*, Brooke Sadler, PhD4, Katrina Ashworth5*, Tim Kong, MSc3, Fan He, PhD1, Mary Fulbright, MSc3*, Dong Chen, MD, PhD6, Regina Clemens, MD7*, Elena Pegoraro8*, Michele P. Lambert, MD9, Kandace Gollomp, MD10, Jorge Di Paola, MD5, Alessandra Balduini, MD11 and Stephen Oh, MD, PhD12

1Division of Hematology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO
2University of Pavia, Pavia, ITA
3Division of Hematology, Department of Medicine, Washington University School of Medicine, St Louis, MO
4Pediatrics, Washington University School of Medicine, Saint Louis, MO
5Department of Pediatrics, Washington University School of Medicine, St Louis, MO
6Division of Hematopathology, Mayo Clinic, Rochester, MN
7Departmnet of Pediatrics, Washington University at St Louis, St Louis, MO
8University of Padova, Padova, ITA
9Children's Hospital of Philadelphia, Philadelphia, PA
10Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
11University of Pavia, Medford, MA
12Washington University School of Medicine, Saint Louis, MO

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.

Disclosures: Lambert: Octapharma, Dova, Principia, Rigel, Argenx, PDSA, 22qSociety and CdLS Foundation: Membership on an entity's Board of Directors or advisory committees; Novartis Dova, Principia, Argenx, Rigel, Sobi, Sanofi and Janssen: Consultancy; FWGBD, PDSA, NIH, Sysmex, Novartis, Principia, Argenx, Dova, Octapharma and Sanofi: Research Funding. Oh: Sierra Oncology: Consultancy; Novartis: Consultancy; AbbVie: Consultancy; Blueprint Medicines: Consultancy; Incyte: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Disc Medicine: Consultancy; Constellation: Consultancy; PharmaEssentia: Consultancy; Geron: Consultancy; Kartos Therapeutics: Consultancy; CTI BioPharma: Consultancy.

*signifies non-member of ASH