ARTS, a Pro-Apoptotic Mitochondrial Septin-Like Protein That Binds to XIAP, Is Silenced in Acute Lymphoblastic and Primitive Acute Myeloblastic Leukemia Cells.
Authors: Shikami Masato , Konopleva Marina , Samudio Ismael , Hoshino Koyu , Garcia-Manero Guillermo , Kojima Kensuke , Tsao Twee , McQueen Teresa , Andreeff Michael 
Published: October 2019

Abstract
ARTS (pro-apoptosis-related protein in the TGF-b signaling pathway) is a septin-like protein that is localized in mitochondria and was reported to promote apoptosis by antagonizing the Inhibitor-of-Apoptosis protein XIAP (Nat Cell Biol 2:915, 2000, EMBO Journal 23:1627, 2004, Oncogene 1:2004). Here, we analyzed the expression and function of ARTS in leukemic and normal cells. We determined the expression levels in leukemic cell lines and in primary leukemia samples by Western blot analysis using an antibody against the C-terminal region of ARTS that identifies 27 unique amino-acids. ARTS was expressed in all AML cell lines studied (OCI-AML3, HL-60, Kasumi-1, NB4, U937, K562), but expression was undetectable in two ALL cell lines (ALL1 and Jurkat). We extended expression studies to primary samples from 34 AML and 14 ALL samples. ARTS was absent or expressed at very low levels in all ALL samples analyzed. In contrast, ARTS was found to be expressed in 50% of primary AML samples. Interestingly, FAB M0 and M1 AML infrequently expressed ARTS (n=1/11), while expression was higher in FAB M2-M5 samples (13/23). These data suggest that ARTS expression is repressed in ALL and in immature AML cells. There was no apparent correlation with cytogenetics. In normal bone marrow and blood cells, ARTS was expressed at low levels. The ARTS gene has a CpG island near its promoter and was reported to be regulated by methylation. We therefore examined ARTS methylation in leukemic cells by methylation specific PCR (MSP). In U937 cells, MSP analysis demonstrated only a methylated product, but ARTS was expressed, while in Jurkat cells the promoter was unmethylated, but ARTS was not expressed. NB4 and Raji cell lines had both, unmethylated and methylated ARTS promoters. In contrast, ARTS mRNA was detected in all cell lines, normal cells and leukemic samples. Our data demonstrate that the methylation status of the ARTS gene is not related to the expression of ARTS. It has been reported that Etoposide induces overexpression of ARTS and binding to XIAP. We therefore treated leukemic cell lines (OCI-AML3 and ALL1), and MCF7 breast cancer cells with Etoposide, Ara-C and Doxorubicin. ARTS expression was increased in MCF7, but not in leukemic cell lines. In MCF7, ARTS was localized in mitochondria before treatment and after 16 hours was partially translocated to the nucleus. On the other hand, Etoposide, Ara-C, and Doxorubicin did not induce ARTS expression in leukemic cells. Co-culture of leukemic cell lines and mesenchymal stem cells increased ARTS expression not only in OCI-AML3, but also in ALL1 cells in 3/3 experiments.
Conclusions: This study suggests that ARTS expression is absent in ALL and immature AML cells, but is variably expressed in more differentiated AML and in normal hematopoietic cells. Its expression is not methylation dependent and it is not upregulated by Etoposide, Ara-C and Doxorubicin. However, co-culture with MSC increased ARTS levels. Results, so far, are not consistent with the proposed pro-apoptotic function of ARTS.