PMC

RFS as analyzed by the Cox proportional hazards regression model was significantly reduced by the presence of MRD (all patients, A) and higher percentage of CD34⁺CD123⁺ cells as a percentage of blasts in remission in the poor-risk cytogenetics patients (B). TTR, time to relapse (months).

(A) Percentages of CD123⁺ and CD131⁺ cells are shown for myeloid leukemia cell lines. (B) Annexin-V⁻/DAPI⁻ viable cells were measured by flow cytometry after treatment with SL-101 at indicated doses for 48 hours. The cell counts were normalized to those of untreated samples. (C) Correlation analysis was performed between expression of IL-3 receptor subunits and IC₅₀ values of SL-101 calculated by using Calcusyn software. (D) Expression of CD123 was measured in parental and CD123-overexpressing (K562^CG-CD123) K562 cells. Cells were left untreated or exposed to indicated concentrations of SL-101 for 48 hours, and viable cell counts were determined as described in (B).

(A) MOLM13 and MV4-11 cells were incubated (1 million/mL) with SL-101–DyLight 680 overnight, and internalized SL-101 was detected by flow cytometry on DyLight 680 signal after removing the membrane-bound SL-101. MFI, mean fluorescence intensity. (B) MOLM13 and MV4-11 cells were left untreated (untreat, or UNT) or treated with SL-101 at indicated concentrations for 24, 48, or 72 hours. Annexin-V⁺ apoptotic cells are shown. (C) MV4-11 and MOLM13 cells were treated with SL-101 (1 μg/mL) for 24 hours. Cycloheximide (100 μM) was used as a positive control. AHA (L-azidohomoalanine) reaction cocktails were applied to detect active protein synthesis. The signal was measured by a fluorescence microplate reader (485/520 nm) and the inhibition percentages were calculated in comparison with control group. (D) Mo7e cells (1 million/mL) were starved overnight and then stimulated with IL-3 (100 ng/mL) for 10 min. Cells were stained with antibodies after fixation and permeabilization.

(A) Percentages of CD123⁺ and CD131⁺ fractions within CD45^dim blast gate on primary AML samples are shown. (B) Samples were treated with SL-101 at indicated doses for 48 hours. Normalized viable cell counts (left) were determined in a cohort of primary AML samples. (C) Gating scheme for the LSC population (CD45^dimCD34⁺CD38⁻CD123⁺). Specific apoptosis was calculated based on percentage of Annexin-V⁺ cells using the following formula: percentage of specific apoptosis = 100 × (% apoptosis of treated cells − % apoptosis of control cells)/(100 − % apoptosis of control cells). Percentage of growth inhibition was calculated based on Annexin-V⁻/DAPI⁻ viable cells using the following formula: 100 – 100 × % viable cells of treated cells/% of viable cells of control cells in both CD45^dim blasts and LSC-enriched blasts. (D) Colony-forming cell assay was performed in primary AML samples and normal bone marrow (NBM) cells. Data were normalized to those from untreated control groups and represent the mean values of triplicate assays. The absolute colony numbers were shown in .

The primary AML patient sample 11 (4030094) was left untreated or pre-treated with SL-101 (1.0 μg/mL) overnight prior to transplantation via intravenous injection into NSG mice (1×10⁶ viable cells for each mouse). (A) Engraftment of human AML cells was measured by flow cytometry in blood by using anti-human CD45 antibody at week 5 after injection (left). CD123⁺ cells were gated on the human CD45⁺ fraction (right). (B) At week 6, all mice were sacrificed and engraftment of human AML was measured in both bone marrow (BM) and spleen. (C) Representative spleens from untreated (U) and SL-101-treated (S) mice are shown. (D) The gating scheme is shown for the CD45^dimCD34⁺CD38⁻CD123⁺ LSC-enriched population in untreated and SL-101–treated mice. (E) Percentages of LSCe in human CD45⁺ cells were determined in both bone marrow and spleen by flow cytometry. AML11 cells (0.6×10⁶) were injected into NSGS) mice that were treated intravenously with PBS or SL-101 at 0.1 mg/kg every other day for 6 doses, after confirming human CD45 engraftment. (F) Leukemia burden was measured after treatment (G) Body weight was monitored over treatment.