Efficacy and Mechanisms Of The mTOR Inhibitor AZD2014 Combined With L-Asparaginase Or JAK2 Inhibitor TG101348 In ALL

Despite impressive outcomes achieved with combination chemotherapy regimens in acute lymphoblastic leukemia (ALL), resistance and relapses occur frequently necessitating a quest for novel therapeutic targets. PI3K/AKT/mTOR signalling is a central regulator of cell metabolism, growth, proliferation and survival and has been shown to be constitutively activated in the aggressive T-ALL with Notch gain-of-function mutations (Palomero, Leukemia, 2006;20:1279). In a high-risk subgroup of B-ALL harbouring mutations in the CRLF2 gene, the frequent activation of mTOR and JAK/STAT pathways was recently reported (Roberts, Cancer Cell, 2012;22:153).

In this study, we tested the pre-clinical efficacy and mechanisms of action of the novel ATP-competitive mTOR inhibitor AZD2014 which effectively inhibits both TORC1 and TORC2 complexes. Pharmacological interactions with chemotherapeutic agent L-asparaginase (L-Asp) or JAK2 inhibitor TG101348 were further evaluated in high-risk immature T-ALL (PF-382, CCRF-CEM, RPMI-8402 cells, Notch1-mutated CUTLL1) and CRLF2-mutated B-ALL cells (MUTZ-5). Inhibition of mTOR kinase activity by AZD2014 used at sub-micromolar concentrations resulted in downregulation of mTORC1 substrates phospho-(p-)S6K (Ser240-244), p-4EBP1 (Thr37/46), mTORC2 target p-Akt (Ser473), and of the oncogenic transcription factor c-Myc. This translated into reduction of cell proliferation with G₀/G₁ cell cycle arrest in all tested ALL cells, with IC50_s ranging from 70 nM to 1,000 nM. In Notch-activated CUTLL1 T-ALL cells, L-Asp enhanced the cell growth inhibition by AZD2014 and further decreased p-S6K levels. In CRLF2-rearranged MUTZ-5 B-ALL cells, the AZD2014/TG-101348 combination enhanced the anti-proliferative effects of AZD2014 with further decrease of p-4EBP1 and down-regulation of Pim-1, a downstream target of Akt and JAK/STAT signaling, more prominently than in other tested pre-B-ALL cells, Nalm6 and REH. AZD2014 induced autophagy as shown by the conversion of the autophagosomal marker LC3-I to LC-II in CUTLL1 and MUTZ-5 cells.

To investigate the molecular modifications of the key cellular metabolic processes in response to mTOR blockade, we employed the proteomic technology of isobaric tags for relative and absolute quantitation (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry. Using iTRAQ platform, more than 1,770 unique proteins were identified in each of the four cell lines tested (T-ALL, PF-382 and CUTLL1; B-ALL, Nalm6 and MUTZ-5). AZD2014 frequently and significantly downregulated anti-apoptotic chaperone heat shock proteins HSP70 and HSP90 (p < 0.05) along with upregulation of the voltage-dependent anion channels (VDAC) (p < 0.01) and histone H1 (p < 0.05), both functioning as dynamic initiators of mitochondria-mediated apoptosis. In T-ALL cells, L-Asp alone or in combination with AZD2014 further upregulated VDAC, histone H1, and apoptosis related mitochondrial chaperone HSP10 (p < 0.05), and repressed translation initiation factors and proteasome-related proteins (p < 0.05). Interestingly, in Notch-mutant CUTLL1 cells L-Asp downregulated DNA methyltransferase Dnmt1 (p < 0.05), and the combination with AZD2014 further decreased histone-binding protein RBBP4 (p < 0.05) which interacts with histone deacetylase, suggesting previously unrecognized epigenetic anti-tumor modifications by AZD2014/L-Asp treatment. In B-ALL cells, the AZD2014/TG101348 combination induced significant repression of ribosomal and spliceosomal pathways (p < 0.001) along with marked repression of HSP70 and ubiquitin related protein E3 ubiquitin-protein ligase (p < 0.05), and increased expression of the apoptosis regulators VDAC (p < 0.01), histone H1 (p < 0.05) and chaperone HSP60 (p < 0.05).

In summary, these results suggest that mTOR blockade by AZD2014 inhibits ALL cell growth through multiple mechanisms, which include repression of translation initiation through potent inhibition of S6K and 4EBP1, induction of autophagy, modulation of HSP chaperone proteins and epigenetic modifications. These cellular alterations are further impaired by the concomitant inhibition of asparagine metabolism with L-Asp in T-ALL or through blockade of JAK2 signaling in B-ALL, providing avenues for novel rationally designed combinatorial regimens in ALL.