Telomerase is activated to maintain the long-term replicative potential in many human cancers and novel inhibitors of telomerase have recently entered clinical trials for a variety of malignancies. We investigated the therapeutic potential of targeting telomerase in AML and have uncovered a specific role for telomerase in leukaemia stem cell (LSC) maintenance.
Telomerase-deficient AML was generated by retroviral transduction of G3 Terc-/- LKS+ (Lin-Kit+Sca1+, enriched for hematopoietic stem cells) with pMIG-MLL-AF9. Transformed Terc-/- LKS+ colonies were reduced at early passage and were progressively eliminated with serial replating. In vivo, AML developed with delayed latency, but was fully penetrant in recipients of G3 Terc-/- and WT cells. The LSC frequency was similar between Terc-/- and WT AML.
Importantly, Terc-/- LSCs were unable to generate secondary AML and this was confirmed by limiting dilution analysis. Initial engraftment was similar between Terc-/- and WT LSCs, however in vivo leukemogenesis was prevented by cell cycle arrest, DNA damage and induction of apoptosis. Together, these findings demonstrate that telomerase is essential for the maintenance of LSC in vivo.
To determine the consequences of telomerase loss on AML LSCs, we performed gene expression profiling of purified LSCs. MLL-AF9-Terc-/- LSCs revealed enrichment of pathways controlling DNA damage/repair, cell cycle and apoptosis. Upstream analysis predicted activation of p53 and functionally, shRNA-mediated knockdown of p53 in Terc-/- LSCs partially rescued colony formation, cell cycle arrest and apoptosis.
To validate these findings in human AML we performed lentiviral shRNA knockdown of hTERT in the MLL-AF9-containing AML cell line Monomac6, followed by transplantation into NSGS (NOD/SCID/IL2Rgamma-/- transgenic for hSCF/hIL3/hGMCSF) xenograft recipients. Two independent shTERT constructs revealed significantly increased survival compared to non-transduced and non-targeting controls. hTERT knockdown correlated with reduced hCD45 engraftment, induction of DNA damage, cell cycle arrest and apoptosis.
These findings provide new mechanistic understanding into the effects of telomerase inhibition on MLL-rearranged AML and identify the telomerase complex as a novel therapeutic target for AML.