Type 1 calreticulin mutations in myeloproliferative neoplasms promote glycolysis via intracellular calcium mediated upregulation of GLUT1

Abstract Myeloproliferative neoplasms (MPNs) comprise a group of cancers of the bone marrow that can transform into a post-MPN acute myeloid leukemia (AML), at which point the overall survival rate is less than 6 months. JAK2 inhibitors in MPN failed to alter disease progression, emphasizing the need for new treatments. Calreticulin (CALR) is one of the MPN driver mutations and encodes an endoplasmic reticulum (ER) calcium (Ca2+) binding protein. CALR mutations are classified as either type 1 or type 2. Only type 1 proteins exhibit loss of the C-terminal Ca2+ binding sites. Additionally, type 1 and 2 CALR mutations engender significant prognostic differences. Previously published work from our lab showed that type 1 CALR exhibits a loss of Ca2+ binding sites, rendering them unable to bind ER Ca2+ and leading to depletion of ER Ca2+ stores. We next wanted to evaluate where this Ca2+ is localized. Through imaging studies, we found that CALR type 1 expressing cells have significantly increased cytosolic Ca2+ and significantly lower mitochondrial Ca2+ compared to both CALR wildtype and CALR type 2 expressing cells. It has been well established in various cancer types that increased glycolytic activity is prompted in part by elevated cytosolic Ca2+. To test if this was the case in our model, we performed a vast array of metabolic assays in UT-7-MPL cells, a human megakaryocytic cell line, including but not limited to lactate secretion, Seahorse XF Glycolysis Stress Test assay, and metabolite tracing with 13-C labelled glucose. These all confirmed a highly glycolytic profile in UT-7-MPL cells expressing the CALR type 1 mutation. In addition, through evaluation of mitochondrial mass, membrane potential and several other assays, mitochondrial metabolism in CALR type 1 mutant cells appears severely dysfunctional. All the glycolytic and mitochondrial phenotypes are almost fully rescued upon the reintroduction of the Ca2+ binding sites of the CALRwt protein (P and C domains; P+C), indicating that perturbed Ca2+ homeostasis is the driver of these changes. Pharmacological treatment with cytosolic Ca2+ chelator BAPTA-AM or with kaempferol, an activator of the mitochondrial Ca2+ uniporter mimic the P+C rescue effect for both the glycolytic and the mitochondrial phenotypes. In our in vitro system CALR type 1 mutant cells express along with other glycolytic factors significantly higher cell surface levels of the glucose transporter GLUT1, and demonstrate enhanced sensitivity to pharmacological GLUT1 inhibition with BAY-876. Preliminary data in a transgenic mouse model as well as in primary patient samples show that GLUT1 expression is higher in CALR type 1 mutant mice and patient cells, and that the mitochondrial mass in this subset of patients is lower compared to CALR type 2 mutant patients. Finally, we validated that all these phenotypes are JAK/STAT pathway independent and that they are worsened by ruxolitinib treatment, providing a strong rationale for a combination treatment. Together, these data both expand the current knowledge of the role that Ca2+ dynamics play in metabolic reprogramming and can lead to improved targeted therapies for MPN patients. Citation Format: Michele Ciboddo, Jonathan Dowgielewicz, George Yan, Deborah Rodriguez, Hunter Blaylock, Nicole Arellano, Chad Coen, Chufan Cai, Nia Hammond, Alex Rosencrance, Elisa Rumi, Daniela Pietra, Daniele Vanni, Silvia Catricalà, Ilaria Carola Casetti, Oscar Borsani, Brandon Faubert, Shannon Elf. Type 1 calreticulin mutations in myeloproliferative neoplasms promote glycolysis via intracellular calcium mediated upregulation of GLUT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB256.