The T(4;11) Fusion Protein MLL/AF4 Regulates TERT Expression

Abstract

About 50% of all infants suffering from acute lymphoblastic leukaemia (ALL) show the translocation t(4;11)(q21;q23) which creates the fusion genes MLL/AF4 and AF4/MLL. This reciprocal translocation identifies a therapy resistant form of leukaemia with a poor prognosis. In order to gain a better insight into the molecular mechanisms of t(4;11) leukaemias we used the two ALL cell lines SEM and RS4;11, both harbouring the t(4;11) translocation, however with different fusion sites. Specific small interfering RNAs (siRNAs) against the two fusion site variants of the MLL/AF4 fusion transcript were designed and transfected into the respective cells via electroporation, using very mild conditions. Serial electroporations at two-day intervals resulted in sustained depletion of the MLL/AF4 transcript up to 70–80%, and depending on the experimental setup, cells were analysed after two or three electroporations. Knock-down of MLL/AF4 resulted in strong inhibition of proliferation and clonogenicity in the two t(4;11)-positive cell lines SEM and RS4;11, along with induction of apoptosis. MLL/AF4 depletion resulted in a 65% decrease in telomerase activity in both SEM and RS4;11 cells, with telomerase reverse transcriptase (TERT) expression being reduced twofold on both transcript and protein levels. Notably, TERT reduction was even stronger (90% depletion) when apoptosis caused by MLL/AF4 knock-down was suppressed with the caspase inhibitor zVAD. In contrast, levels of the RNA component of the telomerase, TERC, were not affected by MLL/AF4 knock-down. Additionally, MLL/AF4 knock-down was associated with reduced expression of several members of the HOXA gene cluster, HOXA6 (65% reduction), HOXA7 (85% reduction), HOXA9 (60% reduction) and HOXA10 (75% reduction). Interestingly, siRNA-mediated knock-down of the MLL/AF4 target gene HOXA7 also induced apoptosis and resulted in a 70% decrease of TERT levels in two t(4;11) positive cell lines without affecting MLL/AF4. Chromatin immunoprecipitation assays revealed HOXA7 binding to the promoter of TERT. Therefore, MLL/AF4 regulates TERT expression, at least in part, via HOXA7. These data suggest that t(4;11) positive cells with substantially lower TERT expression undergo apoptosis, and that TERT may play an antiapoptotic role in t(4;11) positive ALL. Furthermore, these studies identify TERT as a putative new therapeutic target in this therapy-resistant infant leukaemia.