Receptor Tyrosine Kinases, Notch Mutations and PTEN Loss Converge On mTOR in T-ALL and Cause Addiction to Cap Dependent mRNA Translation.

High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL) and has been linked to c-myc independence, as well as resistance of T-ALL to Glucocorticoids and Notch inhibition. The master regulator of PI3K-AKT signaling is the lipid phosphatase PTEN. Despite impaired or lost PTEN activity in T-ALL experimental evidence suggests that input from receptor tyrosine kinases (RTKs) is needed to sustain high mTOR activation. Here we investigated how RTK signaling contributes to the pathogenesis of T-ALL. To mimic RTK signaling we expressed ΔTrkA, a constitutively active nerve growth factor RTK in primary murine hematopoietic cells. Injection of ΔTrkA-transduced cells into C57BL6 mice induced transplantable T-ALL with a latency of 120 days.

Signaling studies showed that ΔTRKA activates mTORC1 over the MAPK pathway, but not mTORC2. In contrast, ΔTRKA⁺ T-ALL showed a profound shift in the use of downstream signaling cascades, displaying a very high activation of mTORC1 and mTORC2 and absent MAPK signaling. To understand the rewired signaling network we first ruled out contribution of insertional mutagenesis by sequencing vector integration sites. Array-CGH revealed hetero- or homozygous loss of PTEN in 4 of 6 T-ALL lines. In the remaining 2 T-ALL lines, which showed normal PTEN protein levels, we detected two PTEN mutations in the phosphatase domain, D92G and G165E, the latter of which has not been described before. Re-expression of the mutants in PTEN^−/− cells revealed complete loss of function for D92G and a partial loss for the G165E mutant. Restoration of wt PTEN expression in the T-ALLs resulted in reversion of the signaling pattern. Sequencing of Notch1 revealed both, PEST domain mutations and 5'-deletions in cis, in all but one investigated T-ALL. Northern and Western Blots confirmed the expression of truncated Notch1 transcripts and protein. Notch inhibition in PTEN^−/− cell lines resulted in robust inhibition of mTORC2, showing that Notch contributes to mTORC2 activation independently of the Hes1-PTEN axis. Despite the loss of PTEN most T-ALLs remained addicted to the input of the leukemia initiating RTK, ΔTRKA. Importantly, the cooperation of three oncogenic pathways converging on mTOR culminated in an oncogenic addiction to mTOR. We screened several inhibitors of the mTOR pathway and found that ΔTRKA⁺ PTEN⁻ Notch⁺ T-ALL are highly susceptible to an inhibition of cap dependent translation by 4EGI-1, whereas primary hematopoietic cells and freshly isolated thymocytes were far less susceptible. Polysome profiling indicated a profound decrease in ribosome occupancy upon treatment with 4EGI-1. Microarray analysis of polysome fractions revealed that mRNAs belonging to genes that were strongly upregulated between T-ALL and normal thymocytes were specifically shifted out of the actively translated transcriptome by 4EGI-1. These genes included members of the translational apparatus itself, mitochondrial matrix proteins, cell cycle regulators such as CyclinD1 and c-myc as well as Bcl-2. Western blots confirmed early loss of these proteins after cessation of cap dependent translation.

Biochemical and genetic dissection of T-ALLs induced by constitutive RTK signaling uncovered activating Notch mutations and PTEN loss as cooperating events and revealed a strong, druggable addiction to cap-dependent translation downstream of oncogenic mTOR. The striking overlap of genes defining the molecular difference between T-ALL blasts and normal thymocytes and genes that preferentially lost ribosomal occupancy upon 4EGI-1 treatment suggests that this drug targets oncogenic core pathways in T-ALL and deserves further investigation.

No relevant conflicts of interest to declare.