Role of Signal Transducing Adaptor Protein-1 (STAP-1) in Chronic Myelogenous Leukemia Stem Cells

Abstract

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder caused by hematopoietic stem cells expressing the BCR-ABL fusion oncoprotein, which constitutively activates multiple signal transduction pathways such as mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and Janus kinase/signal transducer and activator of transcription (JAK/STAT). Although tyrosine kinase inhibitor (TKI) therapy results in dramatic clinical success, studies have shown that TKIs are unable to eradicate leukemic stem cells (LSCs). Several key signaling molecules and pathways have been proposed to regulate the survival of CML LSCs in the presence of TKI; however, the details remain unclear. It is necessary to elucidate the mechanisms that maintain LSCs to better understand the pathogenesis of CML and develop new treatment approaches.The family of signal-transducing adaptor proteins (STAPs), which includes STAP-1 and STAP-2, has been implicated in various intracellular signaling pathways. In 2003, we cloned STAP-2 as a c-fms interacting protein and reported that STAP-2 binds to BCR-ABL and enhances activity, leading to the activation of downstream molecules such as ERK, STAT5, BCL-xL, and BCL2. STAP-1 was cloned as a c-kit interacting protein from a hematopoietic stem cell library, but it is unknown whether STAP-1 plays a role in CML. Given the structural homology between STAP-1 and STAP-2 and the hematopoietic expression of STAP-1, we hypothesized that STAP-1 might contribute to the leukemogenesis of CML.A STAP-1-deficient (KO) CML mouse model was developed. To generate this model, lineage (Lin)− Sca-1+ c-Kithigh (LSK) fraction isolated from bone marrow (BM) cells was infected with a retrovirus carrying BCR-ABL1 and GFP and subsequently transplanted into congeneric recipients. STAP-1 KO CML mice showed significantly longer survival than WT CML mice and displayed less severe splenomegaly and lung hemorrhages compared with WT mice. In recipient BM, absolute numbers of STAP-1 KO LSCs (GFP+ LSK cells) were significantly lower than WT LSCs. In the colony-forming assay, STAP-1 KO LSCs generated fewer colonies compared to WT LSCs. Using flow cytometric analysis, we found that STAP-1 KO LSCs had a higher apoptotic rate than WT LSCs. These findings suggest that the suppression of apoptosis induced by STAP-1 mediates longer survival of LSCs. To further understand the effects of STAP-1, we performed a gene expression analysis using RNA-sequence (RNA-seq) and compared WT and STAP-1 KO CML LSCs. When canonical pathways were analyzed with Ingenuity Pathway Analysis, various pathways associated with inflammatory cytokines were observed to be regulated in STAP-1 KO CML LSCs. Changes in mRNA expression, including that of SOS1, SOS2, FOXO3, FASLG, NFKB2, and BCL-xL, indicated that the PTEN signaling pathway, known to play a tumor suppressive role in CML, was significantly activated by STAP-1 KO (p=1.096E-3, activation Z-score=2.611). The pathway related to JAK/STAT signaling was also affected (p=2.04E-5, activation Z-score=-3.286). Downstream genes in the JAK/STAT signaling pathway, such as STAT5B and BCL-xL, were downregulated more than 2-fold in STAP-1 KO LSCs, suggesting that the deletion of STAP-1 inhibits the expression of STAT5-targeted anti-apoptotic protein and induced apoptosis of CML LSCs. To confirm the results of the RNA-seq experiment, an intracellular flow cytometric assay with CML Lin− cells was conducted. The frequency of cells positive for phosphorylated STAT5 was reduced for STAP-1 KO compared with that for WT. Quantitative PCR with CML LSCs confirmed the downregulation of BCL2 and BCL-xL, which are STAT5-targeted anti-apoptotic genes, in STAP-1 KO CML LSCs.In conclusion, we show that STAP-1 plays a crucial role in the maintenance of CML LSCs using a murine model of CML. STAP-1 deficiency results in the reduction of phosphorylated STAT5, downregulation of anti-apoptotic genes BCL-2 and BCL-xL, and induced apoptosis of CML LSCs. These findings suggest that STAP-1 and related signaling pathways could be potential therapeutic targets for CML LSCs. DisclosuresIchii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Fujimoto Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.