Loss of Ahi-1 Impairs the Self-Renewal Activity of Normal and BCR-ABL+ Stem Cells in Vitro and in Vivo

Abstract

The resistance of chronic myeloid leukemia (CML) leukemic stem cells (LSC) to ABL tyrosine kinase inhibitor (TKI) monotherapy remains a challenge in curing CML. We have identified AHI-1 (Abelson helper integration site-1) oncogene as a potential therapeutic target in CML. It encodes a scaffold protein with a SH3 domain, multiple proline rich motifs and several WD40 repeats. Its expression is highly increased in BCR-ABL+ LSCs. Overexpression of Ahi-1 in primitive hematopoietic cells enhances cell proliferation in vitro and induces leukemia in vivo, and these effects can be enhanced by BCR-ABL. We have also demonstrated that AHI-1 interacts with multiple kinases, including BCR-ABL, β-catenin and JAK2 to mediate LSC activities and TKI resistance in vitro and in vivo . Interestingly, mouse Ahi-1 is highly expressed in hematopoietic stem cells (HSCs, CD45+EPCR+CD48-CD150+ (E-SLAM)) and progenitors (lin-Sca-1+c-kit+ (LSK)), but expressed at low levels in differentiated cells. In addition, Ahi-1 transcripts are found to be expressed in human embryonic stem cells (ES) and at all stages of mouse embryo development, with increasing expression just prior to birth, suggesting that Ahi-1 expression is developmentally regulated. To investigate the precise role of Ahi-1 in regulating normal and LSC self-renewal and differentiation, we have developed a new conditional Ahi-1 knockout mouse model. Ahi-1 deficient mice (AKO) were viable and fertile, but displayed around 30% reduction of bone marrow (BM) and thymus cellularity and altered lineage differentiation as compared to control wild-type mice (AWT). Interestingly, cell cycle analysis showed that loss of Ahi-1 reduced quiescent G0 cells by about 17% in HSC (lin-Sca-1+c-kit+CD48-) as compared to AWT cells. Ahi-1 deficient LSK cells did not exhibit differences in output of colony forming cells (CFCs) but did have dramatically reduced CFC secondary replating efficiency as compared to control cells. In vivo limited dilution assays (LDA), measured by bone marrow transplantation, demonstrated that Ahi-1 loss did not result in significant changes in the frequency of HSC numbers in primary transplanted mice (AWT vs AKO: 1/85000 vs 1/74000), but greatly reduced the reconstitution ability of these cells in serial transplantation assays, since no detectable donor cells were found in tertiary transplanted mice with AKO cells, while engrafted donor cells were still detected in tertiary transplanted mice with AWT cells. These results suggest that Ahi-1 plays a key role in regulating long-term repopulation activity of normal HSCs.The effects of Ahi-1 on LSC growth and maintenance were more profound when BCR-ABL was expressed in Ahi-1-deficient LSCs. Primitive Ahi-1-deficient BM cells transduced with BCR-ABL retroviruses (AKOBA) displayed reduced cell proliferation in the presence of growth factors, especially 14 days after in vtiro culture, compared to BCR-ABL-expressing BM cells from AWT mice (AWTBA). The FACS analysis for these cells after 7 days in culture showed reduced LSK numbers (30%) in AKOBA cells compared to AWTBA control cells. CFC assays revealed a 40% reduction in colonies in AKOBA as compared to AWTBA cells, but the effect was strikingly enhanced in the second and third replating assays (~70% reduction) in AKOBA cells. Most interestingly, we demonstrated that loss of Ahi-1 significantly enhanced survival of leukemic mice generated by crossing of Ahi-1 conditional KO mice with BCR-ABL-transgenic mice as compared to Ahi-1 wild-type/BCR-ABL control mice (65 days vs. 14 days, P=0.008). These results demonstrate that Ahi-1 plays a critical role in regulating HSC functionality and that its deletion perturbs self-renewal and leukemogenesis of BCR-ABL+ LSCs. DisclosuresNo relevant conflicts of interest to declare.