Activation of AKT/mTOR pathway in Ph+ acute lymphoblastic leukemia (ALL) leads to non-mutational resistance

Document Type

Article

Department

Centre for Regenerative Medicine

Abstract

Introduction: The t(9;22) (q34;q11) translocation results in the constative active BCR/ABL tyrosine kinase. Der22 involves the Breakpoint Cluster Region (BCR) gene locus with two principal breaks: a. M-bcr, encoding for the p210-BCR/ABL and b. m-bcr, encoding for the p185-BCR/ABL fusion proteins, respectively. BCR/ABL is the oncogenic driver of Chronic Myeloid Leukemia (CML) and 30% of adult Acute Lymphatic Leukemia (ALL). Activated BCR/ABL kinase is responsible for aberrant activation of multiple signaling pathways, such as JAK/STAT, PI3K/AKT and RAS/MAPK which eventually result in leukemic transformation. Successful targeting of BCR/ABL by selective tyrosine kinase inhibitors (TKIs) such as Imatinib, Nilotinib, Dasatinib and Ponatinib are used for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Most patients with CML in the early stage (CML-CP) treated with TKIs have increased overall survival. However, TKIs have not been as effective in patients with CML blast crisis (CML-BC) or Ph+ ALL. Point mutations in the tyrosine kinase domain (TKD) of BCR/ABL have emerged as the predominant cause of acquired resistance. These mutations are observed in up to 80% of patients with CML-BC and Ph+ ALL and in ~ 50% of Imatinib-resistant patients. In the remaining 20-50% of patients the mechanism of resistance to TKIs remains elusive. The aim of this study was to investigate the mechanism of non-mutational resistance in Ph+ ALL.
Methods: As models for non-mutational resistance, we used patient derived long term cultures (PDLTCs) from Ph+ ALL patients with different levels of non-mutational drug resistance and the SupB15RT, a Ph+ ALL cell-line rendered resistant by exposure to increasing doses of Imatinib and cross-resistant against all approved ABL Kinase Inhibitors (AKIs). Cell proliferation was assessed by XTT/MTT and trypan blue dye exclusion. Signaling pathway proteins were assessed by Western Blot analysis. Chromosomal karyotyping was undertaken on single cell genomes using multi-color FISH (M-FISH) technology. Mutation analysis on the ABL kinase domain was done by sequencing the heminested PCR products obtained from SupB15-WT and SupB15RT cell-lines.
Results: A non-mutational resistance cell line SupB15RT, was developed by exposing SupB15 cells to an increasing concentration of Imatinib over a 3 month period. SupB15RT were able to grow in 10 µM Imatinib. SupB15RT cells were karyotypically and mutationaly identical to SupB15 WT. All approved AKIs and allosteric inhibitors like GNF-2, ABL001 and Crizotinib were unable to inhibit growth of these cells, except for Dasatinib (IC50 40nM), a multi-target kinase inhibitor. Experiments to determine the mode of resistance revealed high level (3 fold) of activation of AKT/mTOR enabling these cells to grow and proliferate. We targeted the AKT/mTOR pathway using BKM-120 (PI3 Kinase inhibitor), BEZ-235 (PI3 Kinase and mTOR pathway) and Trorin1/Torin2 (mTORC1 and mTORC2) and found that Torin-1 and Torin-2 significantly inhibited proliferation of SupB15RT, in a dose dependent manner, with an IC50 of 11-20 nM. As Dasatinib alone inhibited growth of SupB15RT cells at 40-50nm concentrations, we combined Dasatinib with Torin1 and found that the combination of these two compounds had an additive inhibitory effect on cell growth. Following this we examined clinical samples from patients. We used three different Ph+ PDLTCs: a. HP (BCR/ABL negative), b. PH (BCR/ABL positive and responsive to TKIs) and c. BV (BCR/ABL positive and non-mutational resistant to TKIs). Interestingly, we found that AKT/mTOR pathway was activated in BV cells and its proliferation was inhibited by Torin1 with IC-50 of 50nM.
Conclusion: Our experiments revealed an additional pathway involved in the evolution of non-mutational resistance in Ph+ ALL which could assist in developing novel targeted therapy for Ph+ ALL patient(s) with non-mutational resistance.

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Publication (Name of Journal)

Blood

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