Figure 1.
LAM-003 display antileukemic activity in AML cells harboring FLT3-ITD and synergistic activity with venetoclax. (A) Dot plot of average EC50 values in AML cell lines harboring WT FLT3 (WT) (n = 12) or FLT3-ITD (n = 3) or primary blasts (FLT3 WT, n = 7; FLT3-ITD and/or D835 mutation, n = 18) treated with LAM-003 for 72 hours. Cell lines were tested in duplicate a minimum of 2 independent times, and primary samples were tested once, in duplicate. The geometric mean ± 95% confidence interval is shown. Open circles are primary samples harboring D835 mutations, half circles are primary samples harboring FLT3-ITD and D835 mutation. EC50 values were calculated as described in the supplemental Methods and materials and shown in supplemental Table 1. *P < .05 using unpaired Student t test with Welch’s correction. (B) Expression of FLT3 and phospho-S6, phospho-AKT, phospho-STAT5, and phospho-SYK/ZAP-70 was evaluated by using flow cytometry in MV-4-11 (green bars) and MOLM-13 (pink bars) cells after treatment with LAM-003 for 24 hours. Average data ± standard deviation (SD) from 2 independent experiments are shown. (C) Normalized isobologram at the EC75 of three FLT3-ITD–harboring cell lines treated with a combination of LAM-003 and venetoclax for 72 hours before viability was assayed by using CellTiter-Glo. Each data point is the average of 2 independent experiments, each performed in duplicate. (D) Western blot analysis of MOLM-14 cells treated with LAM-003 (1 µM), venetoclax (20 nM), or the combination for 24 hours. Lysates were probed with antibodies to PARP or vinculin, which was used as a loading control. Upper and lower arrows denote full-length PARP and cleaved PARP, respectively. Representative data shown from 2 independent experiments. (E) EC50 values of LAM-003 or venetoclax in parental MOLM-13 or MV-4-11 cells and venetoclax-resistant cell lines (Ven-R) treated for 72 hours before viability determined by using CellTiter-Glo luminescent cell viability reagent. Experiments were performed a minimum of 2 independent times, each in duplicate, and averaged data ± SD are shown. (F) Western blot analysis of MOLM-14 cells treated with LAM-003 (1 µM), venetoclax (20 nM), or the combination for 24 hours. Lysates were probed with the indicated antibodies. Vinculin was used as a loading control. Representative blot shown from 2 independent experiments.

LAM-003 display antileukemic activity in AML cells harboring FLT3-ITD and synergistic activity with venetoclax. (A) Dot plot of average EC50 values in AML cell lines harboring WT FLT3 (WT) (n = 12) or FLT3-ITD (n = 3) or primary blasts (FLT3 WT, n = 7; FLT3-ITD and/or D835 mutation, n = 18) treated with LAM-003 for 72 hours. Cell lines were tested in duplicate a minimum of 2 independent times, and primary samples were tested once, in duplicate. The geometric mean ± 95% confidence interval is shown. Open circles are primary samples harboring D835 mutations, half circles are primary samples harboring FLT3-ITD and D835 mutation. EC50 values were calculated as described in the supplemental Methods and materials and shown in supplemental Table 1. *P < .05 using unpaired Student t test with Welch’s correction. (B) Expression of FLT3 and phospho-S6, phospho-AKT, phospho-STAT5, and phospho-SYK/ZAP-70 was evaluated by using flow cytometry in MV-4-11 (green bars) and MOLM-13 (pink bars) cells after treatment with LAM-003 for 24 hours. Average data ± standard deviation (SD) from 2 independent experiments are shown. (C) Normalized isobologram at the EC75 of three FLT3-ITD–harboring cell lines treated with a combination of LAM-003 and venetoclax for 72 hours before viability was assayed by using CellTiter-Glo. Each data point is the average of 2 independent experiments, each performed in duplicate. (D) Western blot analysis of MOLM-14 cells treated with LAM-003 (1 µM), venetoclax (20 nM), or the combination for 24 hours. Lysates were probed with antibodies to PARP or vinculin, which was used as a loading control. Upper and lower arrows denote full-length PARP and cleaved PARP, respectively. Representative data shown from 2 independent experiments. (E) EC50 values of LAM-003 or venetoclax in parental MOLM-13 or MV-4-11 cells and venetoclax-resistant cell lines (Ven-R) treated for 72 hours before viability determined by using CellTiter-Glo luminescent cell viability reagent. Experiments were performed a minimum of 2 independent times, each in duplicate, and averaged data ± SD are shown. (F) Western blot analysis of MOLM-14 cells treated with LAM-003 (1 µM), venetoclax (20 nM), or the combination for 24 hours. Lysates were probed with the indicated antibodies. Vinculin was used as a loading control. Representative blot shown from 2 independent experiments.

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