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NOTCH1 and the pathobiology of ALK-positive ALCL
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Figure 6. ALK inhibitor sensitive and resistant anaplastic large cell lymphoma cell lines are sensitive to γ-secretase inhibitors. (A) Western blot for cleaved intracel- lular NOTCH1 (ICN) and α-tubulin in ALK+ anaplastic large cell lymphoma (ALCL) cell lines when treated with 1 mM GSI-I for 48 h. Only the relevant sections of the whole blot are shown and the contrast of the whole image was modified in order to improve legibility. Data are representative of three biological repeats. (B) Proliferation of a panel of ALK+ and ALK– ALCL cell lines treated with 1 mM of GSI-I for 48 h, compared to vehicle control, as measured by the MTT assay (***P<0.0001; n=3). (C) Quantification of the percentage of cells positive for annexin V (AV) and/or propidium iodide (PI) when treated with either vehicle control or 1 mM GSI-I for 48 h (***P<0.001; n=3). (D) BLISS matrix showing the combination index on treating the indicated ALK+ ALCL cell lines with crizotinib and PF- 03084014 for 72 h (using a range of concentrations from 25 to 100 nM for crizotinib, and from 100 nM to 10 mM for PF-03084014). A combination index of <1 indicates synergy between drugs, 1 indicates additive effects, >1 indicates antagonistic effects (n=3). (E) Quantification of the percentage of cells positive for AV and/or PI when treated with either vehicle control, 50 nM crizotinib, 2 mM PF-03084014 or a combination of PF-03084014 and crizotinib for 48 h (NS: not significant; *P<0.05; ***P<0.00; n=3). (F) Proliferation over vehicle control of wild-type or crizotinib-resistant Karpas-299 cells when treated with increasing concentrations of GSI-I, as measured by the MTT assay (n=3). (G) Ten-year event-free survival of patients with ALK+ anaplastic large cell lymphoma showing either little or no (n=56), or strong (n=33) NOTCH1 expression. All bar plots display the mean of biological replicates and error bars represent standard deviations.
S5C). In keeping with these data, we show that silencing NOTCH1 by shRNA in the ALK+ ALCL cell lines DEL, SU-DHL1 and SUP-M2 (and the ALK– ALCL cell line FEPD) leads to significant decreases in both MYC and DTX1 transcript levels (Figure 5G and H, Online Supplementary Figure S5A) and protein levels (Online Supplementary Figure S5D), suggesting that NOTCH1 in ALCL signals through a number of pathways beyond HES1 and HEY1. Indeed, analysis of published microar- ray data suggests that the expression of MYC and NOTCH1, and DTX1 and NOTCH1 correlates in both ALK+ and ALK– ALCL, but not in reactive lymph nodes (Online Supplementary Figure S5E-J).
Given the reduced cell proliferation and increased cell death observed on shRNA-mediated knockdown of NOTCH1 expression, ALCL cell lines were incubated with two different GSI: GSI-I (Z-LLNle-CHO) and PF- 03084014.41 GSI-I inhibited NOTCH1 cleavage, as shown by a decrease in expression of the intracellular domain of
NOTCH1 (ICN) 48 h after drug treatment (Figure 6A), concomitant with a decrease in cell proliferation, as shown by the MTT assay (Figure 6B), and an increase in apoptosis, as determined by positive cell surface staining for annexin V and/or intracellular propidium iodide (Figure 6C, Online Supplementary Figure S6A). In contrast, PF-03084014 did not significantly affect cell proliferation or apoptosis on its own at any of the concentrations assessed, ranging from 10 nM to 10 mM for up to 72 h of incubation (data not shown). None of the ALCL cell lines used in the research described here carries either the NOTCH1 T311P or T349P mutation.
Gamma secretase inhibitors synergize with ALK inhibitors to induce cell death
Co-incubation of three of four ALCL cell lines with either PF-03084014 or GSI-1 with crizotinib led to additive to synergistic activity in reducing cell proliferation, as indi- cated by a Bliss Independence Index of less than one
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