Abstract Topic: 3. Acute myeloid leukemia - Biology & Translational Research
Background: Calcium (Ca2+) is a ubiquitous messenger that regulates a wide range of cellular functions including proliferation, cell migration and apoptosis. Abnormal expression of proteins involved in Ca2+ signaling have been associated with oncogenesis in many solid tumor models. However, its involvement in the pathophysiology of acute myeloid leukemia (AML) has been less investigated.
Aims: In this work, we aim (i) to identify actors of Ca2+ signaling involved in the pathophysiology of AML, and (ii) to study their function in normal and malignant hematopoiesis.
Methods: We first analyzed the TCGA and GTEx databases and identified STIM2 as negatively correlated with overall survival. We investigated using the nCounter® PlexSet™ technology expression of the main Ca2+ regulators in a cohort of 407 patients in the alpha 07-02 protocol with evaluable ELN17 risk. We observed that STIM2 expression was higher in patients with intermediate and unfavorable ELN17 risk (p=.0007) and that increasing STIM2 expression tended to be associated with a lower complete remission when adjusting for ELN17 risk and leukocytosis. Since high STIM2 was associated in databases with AML-M4 and M5, we investigated the role of STIM2 in two leukemic cell lines with monocytic potential, THP1 and OCI-AML3, as well as in in vitro monocytic differentiation of peripheral blood-derived CD34+ cells.
Results: In THP1 and OCI-AML3 cells, STIM2 expression increased at RNA and protein level upon monocyte differentiation induced by vitamin D. In CD34+ cells driven in vitro into monocyte differentiation, STIM2 expression increased simultaneously to the acquisition of CD64/CD14 markers. In order to decipher STIM2 function, we used a lentiviral-based shRNA knockdown (KD) strategy. Expression study on 770 genes using the nCounter® PanCancer Pathways Panel in THP1 cells after STIM2 KD identified a transcriptomic signature characterized by a decrease in the expression of genes associated with the MAPK, JAK/STAT, Wnt and Notch pathways, an increased expression of genes regulating the cell cycle and apoptosis. Phenotypically, STIM2 KD inhibited THP1 cell proliferation, induced a cell cycle blockage in G2/M and a massive apoptosis. Moreover, STIM2 KD significantly inhibited monocytic differentiation in the presence of Vitamin D. In primary cells, STIM2 KD induced a similar phenotype, including inducing apoptosis and blockage in G2/M. In THP1 and primary cells, STIM2 KD was associated with decreased level of p-Rb, Bcl2, Bcl-XL and MCL-1, increased level of the pro apoptotic Bad and Bax, and cleavage of caspase 9 without cleavage of caspase 8, revealing activation of mitochondrial apoptosis. Assessing cell cycle regulators, we observed a decreased expression of Cyclin B1, CDK1 and the phosphatase cdc25c, involved during the G2/M transition. We hypothesized that dysregulation in Ca2+ level mediated by STIM2 KD induced a genotoxic stress, leading to DNA double stranded breaks and p53 induction. Using Western Blot, we observed that STIM2KD increased p53, and induced pH2AX foci. Chemical inhibition of p53 using pifithrin-α partially reverted the apoptotic death, as well as the G2/M blockage induced by STIM2KD in both THP1 and in primary cells after STIM2 KD.
Summary/Conclusion: Our data evoke a negative prognostic role of STIM2 in AML. We highlight a role of STIM2 in cell proliferation and survival via protection against genotoxic stress and p53-dependent control of apoptosis and cell cycle in leukemic and myeloid cells. STIM2 acts as a Ca2+ censor, detecting reticular calcium depletion and opening of ORAI channels at cell membrane. Studies are underway to determine whether the role of STIM2 described here involve a modification of calcium fluxes via this capacitive entry of Ca2+ from the extracellular medium.
Keywords: Acute myeloid leukemia, Calcium, Leukemogenesis