Abstract
Mutations that deregulate Notch1 and Ras/phosphoinositide 3 kinase (PI3K)/Akt signalling are prevalent in T-cell acute lymphoblastic leukaemia (T-ALL), and often coexist. Here we show that the PI3K inhibitor GDC-0941 is active against primary T-ALLs from wild-type and KrasG12D mice, and addition of the MEK inhibitor PD0325901 increases its efficacy. Mice invariably relapsed after treatment with drug-resistant clones, most of which unexpectedly had reduced levels of activated Notch1 protein, downregulated many Notch1 target genes, and exhibited cross-resistance to γ-secretase inhibitors. Multiple resistant primary T-ALLs that emerged in vivo did not contain somatic Notch1 mutations present in the parental leukaemia. Importantly, resistant clones upregulated PI3K signalling. Consistent with these data, inhibiting Notch1 activated the PI3K pathway, providing a likely mechanism for selection against oncogenic Notch1 signalling. These studies validate PI3K as a therapeutic target in T-ALL and raise the unexpected possibility that dual inhibition of PI3K and Notch1 signalling could promote drug resistance in T-ALL.
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Acknowledgements
This work was supported by grants from the William Lawrence and Blanche Hughes Foundation to J.C.A. and K.S.; by Specialized Center of Research (SCOR) awards 7019 and 7703 from the Leukaemia and Lymphoma Society of America; by National Institutes of Health grants R37 CA72614 and R01 CA180037 (to K.S.), K99 CA157950 (to M.D.), K08 CA134649 (to Q.L.) and P01 CA119070 (to J.C.A. and W.S.P.), and by the ALSAC of St. Jude Children’s Research Hospital (J.R.D.). K.A. is supported by the Ohio Supercomputer Center (#PAS0425) and is an Ohio Cancer Research Associate (#GRT00024299); and J.X received a Research Fellowship from the American Cancer Society (ACS). K.S. is an ACS Research Professor. We are grateful to T. Jacks and D. Tuveson for KrasG12D mice; to L. Wolff for the MOL4070 virus; and to D. Largaespada and G. Narla for sharing their advice and expertise.
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M.D., J.R.D., D.S. and K.S. designed experiments and analysed the data. M.D., J.W., J.L., D.O’C., J.N. and L.B.L. performed experiments. S.-C.C. and K.A. provided bioinformatics analysis. J.X., J.C.A., W.S.P., Q.L. and D.S. provided reagents. J.C.A., W.S.P., J.R.D. and D.S. provided conceptual advice. M.D. and K.S. wrote the manuscript.
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D.S. and L.B.L. are fulltime employees of Genentech Inc.
Extended data figures and tables
Extended Data Figure 1 Generation of primary T-ALLs and preclinical trial design.
a, Neonatal KrasWT and Mx1-cre, LSL-KrasG12D mice that were injected with the MOL4070LTR retrovirus at birth were observed until death as described previously5. Half of KrasWT T-ALLs harbour Notch1 mutations. KrasG12D expression was induced at age 21 days in Mx1-cre, LSL-KrasG12D mice, and they died from an aggressive myeloproliferative neoplasm around 90 days of age. Bone marrow harvested at the time of death induced T-ALL when transferred into sublethally irradiated recipient mice. All of these KrasG12D T-ALLs harbour Notch1 mutations that are acquired post-transplant. See also ref. 11. b, Primary leukaemias are first expanded in vivo (factory mouse), and are then transplanted into multiple recipient mice of identical strain background. These mice are then randomly assigned to treatment with vehicle (control arm) or experimental drugs (GDC-0941 or GDC-0941 plus PD901). c, Overview of GDC-0941 plus PD901 treatment regimen. Mice were transplanted at day zero and started treatment on day 4. GDC-0941 was administered daily at a dose of 100 mg kg−1 (blue bar), and PD901 (5 mg kg−1 day−1) was cycled 4 days on and 3 days off each week (red boxes). Treatment was continued for 8 weeks or until death.
Extended Data Figure 2 Proliferation of parental and resistant E2 T-ALL cells and primary leukaemias.
a, E2 cells were plated in DMSO or 1 μM GDC-0941 for 16 h, labelled with 10 μM BrdU for 90 min, then analysed for BrdU incorporation and DNA content (7-AAD) by flow cytometry. The parental E2 line demonstrates a ∼90% reduction (from 53.4 to 5.8%) in BrdU-positive cells at this concentration of GDC-0941, while resistant lines (E2-R3 and E2-R5) continue to proliferate robustly. b, Recipient mice engrafted with T-ALL JW81 parental and resistant clones were treated with GDC-0941 and injected with BrdU. in vivo treatment of these primary T-ALLs reduced the percentage of proliferating cells in the parental leukaemia by ∼50% (from 20% to 11.1%), but had minimal effects on the two drug-resistant clones.
Extended Data Figure 3 Pharmacokinetics and pharmacodynamics of GDC-0941.
a, Plasma concentration of GDC-0941 after a single dose in sv129/Blk6 F1 (FVB) mice and nu/nu mice (control). Each time point represents three mice and error bars show the s.e.m. b, Levels of Akt phosphorylation (pAkt) on serine 473 normalized to total Akt in murine bone marrow cells exposed to GDC-0941 ex vivo for 15 min before stimulation with 10 ng ml−1 GM-CSF. The inhibitory concentration at which is reduced by 90% (IC90) for GDC-0941 is ∼100 nM. Error bars show s.d. of three technical replicates.
Extended Data Figure 4 Resistant T-ALLs retain the same immunophenotypes as the corresponding parental leukaemias.
Flow cytometric analysis of leukaemia cells demonstrates identical patterns of CD4 and CD8 in parental and resistant leukaemias (n = 4). Duplicates represent different transplant recipients.
Extended Data Figure 5 GDC-0941-resistant leukaemias that emerge in vivo are cross-resistant to compound E.
a, Western blot analysis of six resistant clones (R1–R6) isolated at death from recipients that were transplanted with T-ALL 2M and treated. Note that the resistant T-ALLs R1–R3 and R5 have lost or markedly downregulated NICD and Myc expression. Each lane contains bone marrow lysate isolated from an independent recipient of the indicated leukaemia (n = 2 for parental 2M, n = 2 for 2M-R2, n = 3 for 2M-R3). b, c, Parental T-ALL 2M and resistant leukaemias were exposed to compound E (b) or GDC-0941 ± compound E (c) ex vivo for 48 h and proliferation was measured using an MTS assay. Each graph depicts percentage maximum MTS units and error bars show s.e.m. of three technical replicates. b, Resistant leukaemias R1–R4 (dashed lines) are less sensitive than the parental leukaemia (solid line) to compound E treatment. c, The additive effect of combining GDC-0941 with 0.1 μM compound E (dashed line) in the parental leukaemia (P) is absent in resistant leukaemias 2M-R1 and 2M-R3. d, Resistant leukaemias with insertions in Aph1a (2M-R1) and Notch1 (JW81-R5) have markedly lower NICD and Myc protein levels than the parental leukaemias (P).
Extended Data Figure 6 Increasing the concentration of GDC-0941 inhibits Akt phosphorylation in GDC-0941-resistant T-ALL.
Primary drug-resistant leukaemia cells were harvested from recipient mice and exposed to a range of GDC-0941 doses for 30 min (triangles; dose range 0–5.0 μM). Western blotting reveals higher basal levels of pAkt S473 in resistant leukaemias compared to the corresponding parental T-ALL. Higher doses of GDC-0941 were required to suppress pAkt levels in the resistant leukaemias.
Extended Data Figure 7 Notch1 modulates sensitivity to GDC-0941 in T-ALL cell lines.
a, b, Exposing PTEN-positive cell lines T-ALL 7, T-ALL 12 and PF382 to 0.1 and 1 μM of compound E (CE; a), and lines E2 and TIMI to 1 μM (b) consistently reduced (01. µM concentration) or eliminated (1 µM concentration) NICD expression and had variable effects on PTEN expression and pAkt S473 levels as assessed by western blotting. c, E2 and Jurkat T-ALL cell lines were treated with GDC-0941 (0941; triangles, 0.01, 0.1 and 1 μM) for 24 h. Western blotting indicates that GDC-0941 does not reduce NICD levels. The 1 µM dose of GDC-0941 induces extensive cell death in E2 cells as indicated by reduced actin. d, S1A, S49 and BW cells were transduced with a construct co-expressing NICD and GFP. Transduced cells were plated in varying doses of GDC-0941. After 3 days, numbers of viable GFP+ (green line) and GFP− cells (blue line) were counted by flow cytometry and equalized to DMSO control (% maximum). e, Exposing Jurkat cells to 0.1 or 1.0 μM compound E for 24 h resulted in a dose dependent reduction in NICD protein levels. f, Jurkat cells were exposed to a range of GDC-0941 concentrations with 0.1 µM compound E or control vehicle (DMSO) for 72 h and proliferation was measured by MTS assay. The addition of compound E significantly reduced the efficacy of GDC-0941 at multiple concentrations. g, Stable Jurkat clones expressing either control vector (MIG) or NICD were treated with GDC-0941 and compound E according to the same protocol in f. Note that enforced expression of NICD rescues the compound E-mediated growth inhibition. f, g, Error bars show s.e.m. of three technical replicates. Asterisks indicate significant differences (two-sided t-test, P values < 0.05).
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Dail, M., Wong, J., Lawrence, J. et al. Loss of oncogenic Notch1 with resistance to a PI3K inhibitor in T-cell leukaemia. Nature 513, 512–516 (2014). https://doi.org/10.1038/nature13495
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DOI: https://doi.org/10.1038/nature13495
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