Program: Oral and Poster Abstracts
Session: 631. Chronic Myeloid Leukemia: Biology and Pathophysiology, excluding Therapy: Poster I
Cells from heavily-treated TKI-resistant CML-BC patients were stained with multiple cell surface markers including those defining typical LSC phenotype and intracellular survival signaling molecules and subjected to CyTOF analysis. β-catenin expression was found to be increased in various CML-BC stem/progenitor cell compartments and was highest in the CD34+CD38+CD123+Tim3+ (Tim-3+GMP) stem/progenitor subset. viSNE visualization demonstrated that overexpression of β-catenin was associated with high levels of p-CRKL, c-Myc, p-AKT, p-Tyr, p-STAT3, and p-STAT5 in CML-BC progenitor/stem cells. Cells from TKI-resistant primary CML-BC samples were then treated with C82, nilotinib, or both for 48 hours in vitro and analyzed by CyTOF. Nilotinib, as expected, had no effect on CML stem/progenitor cells in these samples. viSNE 3D visualization and quantified SPADE tree analysis revealed that C82 and more significantly the combined regimen decreased various stem/progenitor cells, especially Tim-3+GMP cells, a subset representing the candidate for CML-BC LSC. Next, NSG mice engrafted with human CML-BCT315I/E255V cells were treated daily with PRI-724 (ALZET pump, 30 mg/kg), nilotinib (oral gavage, 50 mg/kg), or combination. At the end of a 4-week treatment, bone marrow cells were analyzed by CyTOF. viSNE 4D visualization revealed that PRI-724 and the combination strikingly reduced various CML-BC stem/progenitor subsets. SPADE tree quantification analysis demonstrated that the combination, more profoundly than PRI-724 alone, reduced the leukemia burden, CD34+CD38-, CD34+CD38+,CD34+CD38+CD123+, and especially Tim-3+GMP stem/progenitor cells in NSG mice, which was further confirmed by conventional flow cytometry. Furthermore, PRI-724 alone and the combination with nilotinib induced CD11b expression in various CML-BC stem/progenitor subsets, suggesting CML-BC stem/progenitor cell differentiation. Notably, these treatments significantly inhibited CD44, a β-catenin downstream target gene and an important component in the leukemia/bone marrow niche interaction in CD34+CD38+CD123high and CD34+CD38+CD123highTim-3high subsets; and Tim-3, a novel marker for myeloid LSC in the CD34+CD38+CD123highTim-3high subset; respectively. Importantly, PRI-724 (P<0.05) and the PRI-724/nilotinib combination (P<0.01) significantly prolonged the overall survival of CML-BC-carrying NSG mice, as compared to control and the nilotinib only treated groups.
Collectively, results show that inhibition of β-catenin effectively targets CML-BC stem/progenitor cells and improves survival of NSG mice engrafted with human CML-BC cells. The combination of β-catenin and tyrosine kinase inhibitors markedly enhanced the effect even in cells resistant to TKIs. Furthermore, the combination induces differentiation, and may also interrupt the interaction of CML-BC LSC with the bone marrow niche. This study suggests that combined inhibition of β-catenin and Bcr-Abl represents a novel potential strategy for targeting CML-BC stem/progenitor cells and improving patient outcomes in CML-BC that merits further investigation in preclinical and clinical settings.
Disclosures: Carter: PrismBiolab: Research Funding .
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