Reduced expression of miR-185 predicts therapy response in CD34⁺ CML cells and miR-185 reexpression restores TKI sensitivity in these cells. (A) TaqMan qRT-PCR was performed to validate differentially expressed miR-185 and miR-340 in CD34⁺ cells from IM responders (n = 11) and IM nonresponders (n = 11). (B). Random forest classifier analysis of miR-185 and miR-340, assessed by the leave-one-out cross-validation (LOO-CV) method, to predict IM responders and IM nonresponders. (C) Validation of miR-185 transcript levels in CD34⁺ cells from 47 responders and 11 nonresponders from a second, separate CML cohort before therapy with the TKI nilotinib (baseline) and 3 months posttreatment. (D) Results of CFC assays (± TKIs) of CD34⁺ CML cells from TKI nonresponders (n = 3) transduced with either a miR-185 expressing vector or a pRRL control vector, with or without 5 µM IM, 150 nM DA, or 5 µM NL treatment. The y-axis shows the frequency of colonies derived from erythroid-burst forming units (BFU-E) and granulocyte/macrophage-colony forming units (CFU-GM; left panel). Results of replating all the cells harvested from the primary CFC assays (left panel) into secondary CFC assays are shown in the right panel. (E) Results of LTC-IC assays performed on the same transduced cells as in panel D, with or without TKIs, as indicated. Data shown are mean ± standard error of the mean (SEM). CFC outputs were measured for cells from 4 individual patients with CML. P values were calculated using a 2-tailed unpaired Student t test.