Role of Signal Transducing Adaptor Protein (STAP) Family in Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder that is caused by hematopoietic stem cells expressing the BCR-ABL fusion oncoprotein, which constitutively activates multiple signal transduction pathways, regulates cell proliferation, and is involved in apoptosis. Since the approval of tyrosine kinase inhibitors for the management of CML, treatments have been dramatically improved. However, some proportion of patients still cannot achieve an optimal response, and the discovery of novel targets for CML treatment is required. In this study, we showed that the signal-transducing adaptor protein (STAP) family is a potent therapeutic target for CML. STAP-1, one of the two related proteins, is a c-kit interacting protein that was cloned from a hematopoietic stem cell cDNA library in 2000. Three years later, we cloned STAP-2 as a c-fms interacting protein from a fetal liver library. These proteins share 33% of overall amino acid identity, and have a pleckstrin homology (PH) and Src-homology 2 (SH2) -like domain, which are typical structures of an adaptor protein. Recently, we have reported that STAP-2 binds to BCR-ABL via its SH2-like domain and enhances BCR-ABL activity (Sekine et al. Oncogene 2012), while the function of STAP-1 is largely unknown. In this study, we aimed to elucidate the roles of STAP family members in CML.

First, a STAP-2 deficient (KO) CML mouse model was characterized. We isolated the lineage (Lin)⁻ Sca-1⁺ c-Kit^high (LSK) fraction of bone marrow (BM) cells, infected them with a retrovirus carrying MSCV-BCR-ABL-ires-GFP, and then transplanted them into congeneric recipients. In our CML model mice, the recipients developed and succumbed to CML within three weeks after the transplantation. When we compared the CML model mice derived from wild type (WT) and the STAP-2 KO mice, there were no significant differences in survival. Interestingly, we noticed that the absolute numbers of STAP-2 KO leukemic stem cells (LSCs; GFP⁺ LSK cells) in recipient BM were significantly lower than those of control cells (WT vs. STAP-2 KO; 2080.1 ± 379.6 cells vs. 715.5 ± 153.6 cells). In an in vitro colony-forming assay, STAP-2 KO LSCs generated fewer colonies in the first and second plating when compared to WT LSCs. These findings imply a possibility that STAP-2 may play a role in the regulation of LSCs. The structural homology between STAP-1 and STAP-2 as well as the hematopoietic-specific expression of STAP-1 suggested that STAP-1 may affect the function of LSCs more strongly than STAP-2. Thus, we next investigated the role of STAP-1 in CML in the same manner as that for STAP-2. We observed that STAP-1 KO CML mice showed significantly longer survival than WT CML mice. These mice displayed less severe splenomegaly and lung hemorrhages when compared to WT mice, suggesting that loss of STAP-1 attenuates extramedullary progression of CML. Regarding the effects on LSCs, the absolute numbers of STAP-1 KO LSCs were significantly lower than those of the controls (WT vs. STAP-1 KO; 2090.3 ± 694.07 cells vs. 412.57 ± 114.07 cells). In the colony-forming assay, STAP-1 KO LSCs generated less colonies compared to WT. We also investigated the cell cycle status and apoptosis of LSCs to understand how maintenance of CML stem cells is impaired by the STAP family. Neither STAP-1 nor STAP-2 affected the cell cycle status, and we found that STAP-1 and STAP-2 KO LSCs had a higher apoptotic rate than WT LSCs, by flow cytometric analysis of cells stained for annexin V and 7AAD. These findings suggest that suppression of apoptosis induced by STAP family members mediate longer survival of LSCs. We also measured the expression of STAP-1 and STAP-2 in BM cells derived from patients in the chronic phase of CML. As a result, STAP-1 and STAP-2 mRNA were abundant in the LSC (CD34⁺ CD38⁻ Lin⁻) compartment.

Here, we show that the STAP family plays a crucial role in the maintenance of CML LSCs using CML model mice. The expression of STAP-1 and STAP-2 in human CML stem cells suggest that STAP family members could be used as therapeutic targets in CML treatment. Our previous work revealed that STAP-2 modulates various signals related to inflammatory responses as well as transcription factors, including STAT5 and c-CBL, which have been reported to be BCR-ABL related signaling molecules. In this study, the signal transduction pathway that the STAP family uses to regulate the maintenance of LSCs remains unclear, and will be clarified in future studies.