Background:There is growing evidence that chronic myeloid leukemia stem cells (CML-SCs) use survival signals distinct from the BCR-ABL1 kinase to maintain their viability in the presence of ABL-tyrosine kinase inhibitors (TKIs). Understanding the mechanisms through which CML-SCs are resistant to ABL TKIs would help in designing future therapies leading to complete eradication. Induced pluripotent stem cells (iPSCs) are promising for studying stem cell signaling pathways. When reprogramming is performed with CML cells, CML-derived iPSCs offer a unique system for modeling CML pathogenesis and developing high throughput drug screening. ABL001(asciminib) is a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development including CML. In the present study, we investigated the molecular mechanisms by which ABL001 and ponatinib regulate the CML-SCs developing from CML-derived iPSCs.Methods:We generated iPSCs from bone marrow mononuclear cells of three CML patients including CML-CP and CML-AP. Karyotyping analysis revealed that CML-derived iPSCs have Philadelphia chromosome (Ph). In vitro re-differentiation of CML-iPSCs was performed with differentiation media (30 ng/ml VEGF, 30 ng/ml BMP-4, 40 ng/ml SCF, 50 ng/ml Activin) for 4 days. At day 14, a single cell suspension expressing CD34+CD38- was achieved with hematopoietic cytokine (300 ng/ml Flt-3 ligand, 10 ng/ml IL-3, 10 ng/ml IL-6, 50 ng/ml G-CSF, 25 ng/ml BMP-4).Results:We generated the phenotypically immature cell population (CD34+CD38-CD90+CD45+) and mature cell population (CD34-CD45+) from CML-derived iPSCs. The single treatment of ABL001 or ponatinib significantly reduced the mature cell population (CD34-CD45+) from CML-derived iPSCs. Although the single treatment of ABL001 or ponatinib alone have minimum effect on immature cell population (CD34+CD38-CD90+CD45+), co-treatment with ABL001 and ponatinib significantly reduce the cell population (CD34+CD38- CD90+CD45+) (p<0.001). Also co-treatment with ABL001 and ponatinib significantly reduced the colony formations of mature erythroid, granulocyte-macrophade, and mixed of these hematopoietic cells derived from CML-iPSCs for CFC assay. Further, NOD/SCID mice were injected with CML-iPSCs, and then treated with ABL001 and ponatinib on day 21 for 7 days. Co-treatments with ABL001 and ponatinib significantly reduced the population of CD34+CD38-CD90+ cells compared with ABL001 or ponatinib as single agents. To identify the mechanisms that limit the CML immature cell population by ABL001 and ponatinib, CD34+CD38-CD90+CD45+ cell fractions from CML-iPSCs were cultured with ABL001 and or ponatinib for 72 hrs. Co-treatment with ABL001 and ponatinib reduced the expression of BMI1, c-Myc, Oct4 and Nanog and induced the expression of p21Cip1. Finally, we investigate the engraftment potential of CML-derived iPSCs treated with ABL001 and ponatinib. One million of CD34+ cells treated with ABL001 and ponatinib were intravenously transplanted into NOD/SCID mouse. Transient engraft was observed from control mice, however, co-treatment of ABL001 and ponatinib never shows the engraftment.Conclusion:Our preclinical results indicate that combination with ABL001 and ponatinib have potential as an important option for controlling the immature CML cell population. It is expected that ABL001 with catalytic-site inhibitors may become extremely useful therapeutic interventions to achieve treatment-free remission in patients with CML. DisclosuresTauchi:Otsuka pharmaceutical KK: Speakers Bureau; Phizer KK: Research Funding, Speakers Bureau; Celegen KK: Speakers Bureau; Bristol-Myers Squibb KK: Speakers Bureau; Novartis pharma KK: Research Funding, Speakers Bureau. Ohyashiki:Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Jansen pharmaceutical KK: Honoraria, Research Funding; Fujirebio KK: Honoraria; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; Fujirebio KK: Honoraria; Kyowa Hakko Kirin KK: Honoraria; Taiho pharmaceutical KK: Honoraria; Alexion: Honoraria; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Otsuka pharmaceutical KK: Honoraria; Jansen pharmaceutical KK: Honoraria, Research Funding; Chugai pharmaceutical KK: Research Funding; Otsuka pharmaceutical KK: Honoraria; Alexion: Honoraria; Taiho pharmaceutical KK: Honoraria; Bristol-Myers Squibb KK: Honoraria, Research Funding, Speakers Bureau; Nippon Shinyaku: Honoraria, Research Funding, Speakers Bureau; Dainippon Sumitomo KK: Honoraria, Research Funding, Speakers Bureau; MSD KK: Honoraria; Kyowa Hakko Kirin KK: Honoraria; Chugai pharmaceutical KK: Research Funding; MSD KK: Honoraria; Phizer KK: Honoraria, Research Funding, Speakers Bureau; Novartis pharma KK: Honoraria, Research Funding, Speakers Bureau.