Differential effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway in acute lymphoblastic leukemia

Purpose: Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established. Experimental design: We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative. Results: Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2. Conclusions: Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes

Impact of ABL-kinase inhibition on PI3K/AKT/mTOR signaling in BCR-ABL and TEL-ABL positive ALL LTCs.

<p>BCR-ABL+ (BV, PH, KW, CM, BV und DW), TEL-ABL+ (VG) and BCR-ABL- (HP, KR, RL, CR und SK) LTCs were treated with 1µM Imatinib for 20h. Lysates of these cells were used for the detection of phosphorylated and total AKT, S6 and 4E-BP1 by Western blotting. Because of the constitutively activated PI3K/AKT/mTOR pathway in Jurkat cells, lysates of untreated Jurkat cells were used as positive controls and that of cells treated for 2h with 1µM Wortmannin (WM), a PI3K inhibitor, were used as negative controls. β-Actin was used as loading control.</p

Effect of ABL-directed tyrosine kinase inhibitors on BCR-ABL+ ALL LTCs.

<div><p>Ph+ ALL cells with the T315I mutation showed no growth inhibition (A) or induction of cell death (B) in response to any of the TKI. The BCR-ABL+ ALL-LTC PH was used as a positive control (A and B). </p> <p>Response to ABL-directed TKI of 6 non-mutated BCR-ABL+ LTCs (BV, CM, DW, KW, PH and VB) and the LTC KÖ with the T315I mutation (C). Cell death was examined on day 4 of exposure to increasing concentrations of imatinib, dasatinib, and nilotinib. </p> <p>(A, B, C) Cell proliferation was assessed by XTT assay and induction of cell death was measured by Annexin-V/propidium iodide staining. The data shown represent the means <u>+</u> SD of 3 experimental replicates from one representative experiment out of 3 performed. </p></div

The impact of mTORC1 inhibition in B-ALL is independent of the presence of an ABL translocation.

<div><p>BCR-ABL+ (BV, PH, KW, CM, BV und DW), TEL-ABL+ (VG) and BCR-ABL- (HP, KR, RL, CR und SK) LTCs were exposed to increasing concentrations of the mTORC1 inhibitor RAD001. (A) Proliferation and (B) cell death were measured after 4 days of drug treatment. The (A) proliferation rate and (B) rate of cell death of the ABL-translocated cells (BCR-ABL+/TEL-ABL+) and the BCR-ABL- cells did not differ in their response to treatment with RAD001 at 25nM or 5µM, respectively (corresponding approximately to the IC₅₀) values determined for growth inhibition and induction of apoptosis, respectively). </p> <p>(A, B) Cell proliferation was assessed by XTT assay, induction of apoptosis was measured by Annexin-V/propidium iodide staining. The data shown represent the means + SD of 3 experimental replicates from one representative experiment out of 3 performed. </p> <p>(C) BCR-ABL+ (PH, BV and KÖ), TEL-ABL+ (VG), BCR-ABL- (HP) and Jurkat cells were treated with increasing concentrations of RAD001 for 2h. Lysates of these cells were used for the detection of phosphorylated and total AKT, S6 and 4E-BP1 by Western blotting. Lysates of untreated Jurkat cells were used as positive controls and those of cells treated with the PI3K inhibitor Wortmannin (WM) for 2h at 1µM were used as negative controls. β-Actin was used as loading control.</p></div

The impact of combined PI3K, mTORC1 and mTORC2 inhibition in B-ALL is independent of the presence of an ABL translocation.

<div><p>BCR-ABL+ (BV, PH, KW, CM, BV und DW), TEL-ABL+ (VG) and BCR-ABL- (HP, KR, RL, CR und SK) LTCs were exposed to increasing concentrations of the PI3K/mTORC1/C2 inhibitors NVP-BGT226 and NVP-BEZ235. (A) Proliferation was measured after 4 days of drug treatment. Inhibition of proliferation by 10nM NVP-BGT226 (corresponding approximately to the IC₅₀) was more pronounced in the ABL-translocated cells (BCR-ABL+/TEL-ABL+) than in BCR-ABL- cells (p=0.0283 (*)). In contrast, treatment with 50nM NVP-BEZ235 (corresponding approximately to the IC₅₀) showed no difference between BCR-ABL+/TEL-ABL+ and BCR-ABL- cells. (B) Apoptosis was measured after 4 days of drug exposure. The rate of cell death induced by NVP-BEZ235 or NVP-BGT226 was not significantly different in ABL-translocated cells (BCR-ABL+/TEL-ABL+) and BCR-ABL- cells (A, B) Cell proliferation was assessed by XTT assay, induction of cell death was measured by Annexin-V/propidium iodide staining. The data shown represent the means + SD of 3 experimental replicates from one representative experiment out of 3 performed. </p> <p>(C, D) BCR-ABL+ (PH, BV and KÖ), TEL-ABL+ (VG), BCR-ABL- (HP) and Jurkat cells were treated with increasing concentrations of (C) NVP-BGT226 or (D) NVP-BEZ235 for 2h. Lysates of these cells were used for the detection of phosphorylated and total AKT, S6 and 4E-BP1 by Western blotting. Lysates of untreated Jurkat cells were used as positive controls and those of cells treated for 2h with 1µM Wortmannin (WM) served as negative controls. β-Actin was used as loading control. d = DMSO control. </p></div

The impact of additional inhibition of mTORC2 on combined PI3K and mTORC1 inhibition in B-ALL is independent of the presence of an ABL translocation.

<div><p>BCR-ABL+ (PH and BV) and BCR-ABL- (HP) cells were treated with 0.5µM or 2µM NVP-BKM120 (PI3K inhibitor), 0.5µM or 2µM RAD001 (mTORC1 inhibitor) alone or in combination. For combined PI3K/mTORC1/C2 inhibition, cells were treated with 0.5µM or 2µM of NVP-BGT226 or NVP-BEZ235. (A) Proliferation and (B) cell death were measured after 4 days of drug treatment. </p> <p>(A, B) Cell proliferation was assessed by XTT assay, induction of cell death was measured by Annexin-V/propidium iodide staining. The data shown represent the means + SD of 3 experimental replicates from one representative experiment out of 3 performed. </p> <p>(C) Lysates were prepared after 2h of drug treatment for the detection of phosphorylated and total AKT, S6 and 4E-BP1 by Western blotting. Lysates of untreated Jurkat cells were used as positive controls, those of cells treated for 2h with 1µM Wortmannin (WM) were used as negative controls. β-Actin was used as loading control. </p></div