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J. Hu et al.
synergistic effect with MK1775. Immunofluorescence analysis and immunoblots of cleaved Caspase-3 in HPB- ALL and CUTLL1 cells manifested robust apoptosis upon combined treatments as compared to single treatment (Figure 4B and Online Supplementary Figure S7). Annexin V staining confirmed strong synergism by dual treatments in six T-ALL cell lines, while sparing normal BM cells (Figure 4C). Consistently, another GLS1 inhibitor, bis-2-(5-pheny- lacetamido-1,3,4-thiadazol-2-yl) ethyl sulfide (BPTES), elicited synergistic cell death in combination with MK1775 (Figure 4D). Therefore, these findings indicate the strong anti-T-ALL efficacy by co-inhibition of WEE1 and GLS1.
Targeting of WEE1 and glutaminase suppresses HPB-ALL xenografts
To evaluate the in vivo efficacy of the combination strat- egy, we established a human xenograft using HPB-ALL cells. In order to visualize leukemia cell expansion in vivo,
lentiviruses expressing firefly luciferase were infected into HPB-ALL cells and five million luciferase-expressing cells were intravenously injected into each NPG mouse. The intensities of luciferase luminescence signals were equiva- lent in all mice at day 6 post engraftment, and these ani- mals were then randomly divided into four groups sub- jected to control, MK1775, BPTES or combination treat- ment (Figure 5A and B). Whereas single administration showed a moderate inhibition of leukemia progression, MK1775/BPTES combination elicited a much stronger anti-leukemia effect, as evidenced by bioimaging signals at day 13 and day 20 post xenograft (Figure 5B). When the control cohort became moribund, all the mice were sacri- ficed to assess the leukemia burden in vivo. Administration of either MK1775 or BPTES alone moderately inhibited engraftment, and MK1775/BPTES combination induced a synergistic and remarkable suppression of leukemogenesis (Figure 5C). As a result, dual treatment ameliorated spleen enlargement and resulted in more reddish bones (Figure
A
B
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D
Figure 6. Synergistic anti-leukemia effects of MK1775 and CB-839 on a patient-derived T-cell acute lymphoblastic leukemia (T-ALL) xenograft. Human primary T-ALL cells were injected into irradiated NPG mice. (A) Human CD45+ cells from bone marrow (BM) and spleen were analyzed by flow cytometry when the control- treated mice became moribund around day 25 post xenograft. (Right) Quantifications of CD45+ percentages. Combo: combination. (B) Representative immunohisto- logical images of PCNA and cleaved Caspase-3 (c-Caspase 3) in the spleen sections from mice receiving different treatments. Scale bar, 50 mm. (A and B) Quantifications shown represent the means (± standard deviation) of biological triplicates. (C) Representative spleen and bone images of mice subjected to different treatments (left) with spleen weights (right). (D) Kaplan-Meier survival curves of T-ALL PDX treated with MK1775 and/or CB-839 (n=5 in each group). Dotted lines define the treatment time frame. **P<0.01.
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haematologica | 2021; 106(7)