CARMA1 Is Required for Notch1-Induced NF-κb Activation in T-Cell Acute Lymphoblastic Leukemia

Background

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm originated from early T cell progenitors. Constitutive activation of the Notch1-signaling pathway plays an important role in T-ALL. After three shears, Notch1 generates intracellular fragment of Notch1 (Notch1-IC), which translocates to the necleus and exerts its biological functions. NF-κB pathway is an important regulator of cell survival and a major downstream target of Nothc1 in T-ALL. However, the molecular mechanism of Notch1 activating NF-κB pathway is still unknown. CARMA1 forms a complex with MALT1 and BCL10 (CBM complex), which plays a role in activation of NF-κB pathway in leukomonocyte. Therefore, we asked whether CARMA1 mediates Notch1-induced NF-κB pathway.

Methods

Microarray gene expression data were obtained from GEO database (accession number GSE26713) to assess expression of CARMA1, BCL10, MALT1 in T-ALL patients. In addition, we apply microarray dataset (GEO accession number GSE42328) to examine the relationship between gene expression and patients outcome. Western blot was used to analyze CRAMA1 expression in different cell lines. CCK-8 assay, EdU assay, cell cycle and colony formation experiments were performed in CARMA1-shRNA and control-shRNA T-ALL cell lines to investigate the effect of CARMA1 in T-ALL. We intravenously injected the Control-shRNA and CARMA1-shRNA MOLT-4 cells into NOD-SCID/IL2Rg^null (NSG) mice to evaluate the influence of CARMA1 knockdown on overall survival. Dual luciferase reporter assay was applied to detect transcriptional activity of NF-κB.

Results

We found that CARMA1 was highly expressed in T-ALL cell lines, but not observed in B-ALL and AML cell lines. The T-ALL patients with high CARMA1 expression had better outcome compared with the low expression group. However, we failed to observe any significant differences in the expression of BCL10 and MALT1 between primary normal and T-ALL cells or relationship of survival with MALT1 and BCL10 in T-ALL. A previous finding showed that Notch1 mutations are correlated with an improved long-term prognosis (J Clin Oncol 2013;31:4333). The above results suggested both high expression of CARMA1 and activating Notch1 mutaions relate to favorable prognosis, indicating the possible relevance between CARMA1 expression and Notch1 activation.

To elucidate the effect of CARMA1 on cell proliferation, we performed CARMA1 knockdown experiment with shRNAs in T-ALL cells. CARMA1 knockdown in MOLT-4 cells significantly decrease the quantity and size of cell colonies. MOLT-4 and Jurkat cell lines showed significant reductions in cell viability through CARMA1 silencing compared with control-shRNA cells. The EdU proliferation assay demonstrated that knockdown of CARMA1 in MOLT-4 and Jurkat cell lines markedly reduced the number of EdU-positive cells. Interestingly, CARMA1 had no significant effect on cell proliferation in CCRF-CEM cells. This finding is consistent with a recent report, which demonstrated that CARMA1 knockdown showed no difference in proliferation of CCRF-CEM cells, but only one cell line was used (Leukemia. 2017;31:255). CCRF-CEM is a SIL-TAL1 fusion gene positive cell line and its survival depends on the activation of TAL1 transcriptional complex. Therefore, the proliferation of SIL-TAL1 fusion gene positive cell lines may be independent of CARMA1 expression.

We next explored the significance of CARMA1 in cell cycle and in vivo tumorigenic ability. Compared to the control-shRNA cells, CARMA1 knockdown arrested T-ALL cell lines in G1 phase. CARMA1 knockdown remarkably led to the downregulation of the percentage of CD45 positive cells and prolonged overall survival of NSG mice. To further determine the role of CARMA1 in Notch1-induced NF-Κb activation, we transfected Notch1-IC into control-shRNA and CARMA1-shRNA cells, respectively. We found that Notch1-IC significantly upregulated the activity of NF-κB in control-shRNA cells. However, the Notch1-induced NF-κB was abolished in CARMA1 knockdown T-ALL cells. The results demonstrate that CARMA1 is involved in Notch1-mediated NF-κB activation.

Conclusion

Taken together, CARMA1 markedly contributes to cell survival in SIL-TAL1 negative T-ALL cells and may play a crucial role in Notch1-induced activation of NF-κB pathway. Furthermore, CRAMA1 might be a prognostic marker and a prospective therapeutic target for T-ALL.