PI3K, mTOR and NOTCH pathways are frequently dysregulated in T-cell acute lymphoblastic leukaemia (T-ALL). Blockade of PI3K and mTOR with the dual inhibitor PI-103 decreased proliferation in all 15 T-ALL cell lines tested, inducing cell death in three. Combined PI3K/mTOR/NOTCH inhibition (with a γ-secretase inhibitor (GSI)) led to enhanced cell-cycle arrest and to subsequent cell death in 7/11 remaining NOTCH mutant cell lines. Commitment to cell death occurred within 48-72 h and was maximal when PI3K, mTOR and NOTCH activities were inhibited. PI-103 addition led to upregulation of c-MYC, which was blocked by coincubation with a GSI, indicating that PI3K/mTOR inhibition resulted in activation of the NOTCH-MYC pathway. Microarray studies showed a global increase in NOTCH target gene expression upon PI3K/mTOR inhibition. NOTCH-MYC-induced resistance to PI3K/mTOR inhibition was supported by synergistic cell death induction by PI-103 and a small molecule c-MYC inhibitor, and by reduction of the cytotoxic effect of PI-103+GSI by c-MYC overexpression. These results show that drugs targeting PI3K/mTOR can upregulate NOTCH-MYC activity, have implications for the use of PI3K inhibitors for the treatment of other malignancies with activated NOTCH, and provide a rational basis for the use of drug combinations that target both the pathways.

Novel inhibitors of PI3K, Akt and mTOR have been developed recently, some of which have entered clinical trials. Although such compounds inhibit cell proliferation, their effects on cell survival, an important determinant of clinical response, are less clear-cut. Using a broad panel of myeloma cell lines and primary patient samples we show that dual PI3K and mTOR inhibition can induce cell death. The effects are most marked in cells expressing the t(4;14) translocation, whereas t(11;14) cells are largely resistant. Using specific inhibitors of individual pathway components, we show that optimal induction of cell death requires inhibition of both PI3K and mTOR. This is due to a PI3K-independent component of mTOR activation downstream of the MAP Kinase pathway. Novel mTOR kinase inhibitors, which block both TORC1 and TORC2 complexes thereby also reducing Akt activity, are less effective than dual PI3K/mTOR inhibitors because of feedback activation of PI3K signalling. Dual PI3K/mTOR inhibitors sensitise t(4;14) and t(14;16), but not t(11;14), expressing cells to the cytotoxic effects of dexamethasone. We have identified a robust cytogenetic biomarker for response to PI3K/mTOR inhibition - these results will inform the design and prioritisation of clinical studies with novel inhibitors in genetic subgroups of myeloma.

1358 The PI3K/mTOR and NOTCH pathways are promising therapeutic targets for the treatment of T-ALL. Hyperactivation of the PI3K/mTOR pathway occurs frequently, predominantly due to loss of PTEN function through deletion, mutation, microRNA induced downregulation or post-translational modification. NOTCH signalling is aberrantly activated in the majority of patients, most commonly due to mutation of Notch-1. Activation of NOTCH signalling can also positively regulate mTOR activity and increase PI3K/Akt signalling via downregulation of PTEN expression. We examined the effects of PI3K/mTOR blockade, using the dual inhibitor PI-103, on the proliferation and survival of T-ALL cell lines with various combinations of NOTCH and PTEN abnormalities. There was marked reduction in the proliferation of all T-ALL cell lines tested, regardless of their PTEN status or level of activated Akt. However, using Annexin-V/PI staining, we observed significant induction of cell death (<50% survival) in only 3/15 cell lines. Blockade of NOTCH signalling, using a gamma secretase inhibitor (GSI), had no effect on cell survival and only a modest effect on cell proliferation: only 5/13 NOTCH deregulated cell lines, all of which expressed wild-type PTEN, showed a clear reduction in cell number. By contrast only 1 of the 8 GSI-resistant cell lines expressed wild-type PTEN. We tested the effect of combined blockade of PI3K/mTOR and NOTCH pathways using PI-103 + GSI to determine the extent of any non-overlapping effects. In NOTCH-mutant/PTEN-WT cells, combined blockade led to a reduction in cell size and number with a more rapid and marked increase in cell cycle arrest and reduced levels of Cdk4 and Cyclin D3, than achieved with either agent alone. Further, 8/13 cell lines with deregulated Notch showed a significant reduction in cell survival with PI-103+GSI compared with PI-103 alone. In 5/6 NOTCH-mutant/PTEN-WT cell lines survival fell to ≤50% below control, with rapid commitment to cell death (48–72 hours). This mutational context represents the majority of primary T-ALL samples at presentation - confirmation of the enhanced effect of dual PI3K/mTOR and NOTCH blockade was obtained in primary T-ALLs cultured in suspension or on stromal support. Utilizing selective inhibitors of the PI3K (PIK90) or mTOR (rapamycin) enzymes in combination with GSI we found that blockade of both enzymes was required to achieve maximal levels of cell death. c-MYC is an important oncogene in T-ALL - it is a direct transcriptional target of Notch signaling and protein stability is modulated via the PI3K/Akt/GSK3 module. Therefore, we examined the effects of PI3K/mTOR and NOTCH blockade on levels of nuclear c-MYC in cells sensitive to inhibition of both pathways. c-MYC was downregulated by GSI but, surprisingly, increased after 48h of PI-103 treatment. This followed increased levels of nuclear Notch intracellular domain and could be abolished by the addition of GSI, indicating a NOTCH-dependent mechanism. Gene expression microarray analysis confirmed global upregulation of NOTCH signalling in PI-103 treated cells and the suppression of this effect by addition of GSI. These findings were confirmed for Notch target genes (Deltex-1, c-MYC, Hes-1, CD21 and GIMAP5) by qPCR and flow cytometry (CD21). Upregulation of Notch-MYC proliferation and survival signals could explain why T-ALL cells survive after PI3K/mTOR blockade. We found that maintenance of c-MYC levels by retroviral expression counteracted the effects of PI3K/mTOR/NOTCH blockade and comparable levels of cell death, to those seen with PI-103 plus GSI, were seen in cells treated with PI-103 + the c-MYC inhibitor, 10058-F4. Our data show that targeting PI3K/mTOR can upregulate NOTCH-MYC activity in T-ALL cells with aberrant NOTCH signalling. These finding have implications for the use of PI3K inhibitors for the treatment of T-ALL, and other malignancies with activated NOTCH signaling, and provide a rational basis for the use of drug combinations that target both pathways. Disclosures: No relevant conflicts of interest to declare.

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