T-cell acute lymphoblastic leukemia (T-ALL) remains a therapeutic challenge. In particular, relapsed disease is refractory to further therapy and has a dismal outcome. High activation of the PI3K-AKT-mTOR pathway is a hallmark of T-ALL that has been linked to the resistance of T-ALL to glucocorticoids and chemotherapy. The master regulator of PI3K-AKT signaling is PTEN. PTEN loss (1) is accompanied by an adverse outcome, (2) occurs as secondary event in T-ALL relapses and (3) is selected for after xenotransplantation of human T-ALLs into NSG mice. Interestingly, even in the absence of PTEN, the PI3-Kinases γ and δ, which link receptor tyrosine kinases (RTK) and PIP3 signaling, are critical for T-ALL formation. In lung cancer, both PTEN loss and activation of RTK signaling are required for full activation of AKT and aggressive disease. However, whereas the role of PTEN in T-ALL is well appreciated, little is known about the contribution of RTK signaling. We previously demonstrated the aberrant expression of members of the Neurotrophin receptor tyrosine kinase family (tropomyosin-related kinases - TRKA/B/C) in primary human leukemias of myeloid and lymphoid origin. We detected high expression of TRKB in several T-ALL samples. Some samples also co-expressed intracellular BDNF (brain derived neurotrophic factor), the ligand for TRKB, suggesting the existence of autocrine loops between the receptor and its ligands.

Expression of the TRKB/BDNF autocrine loop or of a constitutively active form of the human TRKA receptor (ΔTrkA) in murine hematopoietic stem cells elicited T-ALL with a mean latency of 100 days in our murine transplantation model. Here we dissect the downstream signaling cascades in BDNF/TRKB or ΔTrkA induced T-ALL clones and show acquisition of activating Notch1 mutations and loss of PTEN during clonal evolution of T-ALLs induced by deregulated TRK-signaling. All three events contribute independently to activation of mTORC1 and mTORC2, demonstrating a strong selective pressure for enhanced mTOR signaling in T-ALL. To investigate the role of both mTOR complexes and downstream effectors, we used an improved Tet-regulated miR30-shRNA system in conjunction with a FACS-based reporter assay, allowing the rapid identification of shRNAs that give powerful knockdown at the single copy level. We identified several potent RNAi triggers against Rictor (mTORC2), Raptor (mTORC1) and members of the cap-initiation complex. Knockdown of Rictor or Raptor alone caused a transient decrease of proliferation and viability. In contrast, complete inhibition of mTOR with Torin1 or knockdown of eIF4E, the rate limiting subunit of the cap initiation complex, resulted in strong induction of apoptosis. This demonstrates that increased cap-dependent translation is a key effector of oncogenic mTOR in TRK+Notch+PTEN- T-ALL. Next, we investigated the efficacy of 4EGI-1E, a novel isoform of the previously described inhibitor of cap-dependent translation 4EGI-1, in this T-ALL model. 4EGI-1E induced apoptosis of T-ALL blasts in vitro with an EC50 of 3.5 mM, whereas the EC50 for global inhibition of cap-dependent translation was 50 mM, suggesting that 4EGI-1E targets a subset of mRNAs that are regulated by cap-dependent translation and are crucial for T-ALL survival. Microarray analysis of polysome fractions from DMSO and 4EGI-1E-treated cells in vitro revealed that 4EGI-1E treatment decreased the active translation of mRNAs for genes observed to be upregulated in T-ALL. These genes included members of the translational apparatus, mitochondrial matrix proteins, cyclins, c-Myc and Bcl-2. Polysome profiling in vivo recapitulated the in vitro results, showing that 4EGI-1E caused a global decrease in the ribosomal occupancy of cellular mRNAs. Network analysis suggested c-Myc as a central node within the depleted mRNAs. Abrogation of c-myc expression and induction of apoptosis were observed as soon as 4 hours after injection of 4EGI-1E (1.5 mg) into leukemia bearing animals. Depletion of T-ALL blasts from the bone marrow was achieved after 5 days of daily 4EGI-1E treatment, T-ALL (DMSO: 39% (SD 18%) vs. 4EGI-1E 0.54% (SD 0.62%) p=0.003). Finally, we investigated the effects of 4EGI-1E on healthy hematopoiesis, demonstrating the existence of a therapeutic window for inhibition of cap-dependent translation in vivo. Hence, inhibiting eIF4E is a promising approach to target c-myc in a genetically complex T-ALL model.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.