ALBERT

Beschreibung: The chromosomal translocation t(4;11) marks infant acute lymphoblastic leukemia associated with a particularly dismal prognosis. The leukemogenic role of the corresponding fusion gene MLL-AF4 is not well understood. We show that transient inhibition of MLL-AF4 expression with small interfering RNAs impairs the proliferation and clonogenicity of the t(4; 11)–positive human leukemic cell lines SEM and RS4;11. Reduction of mixed-lineage leukemia (MLL)–ALL-1 fused gene from chromosome 4 (AF4) levels induces apoptosis associated with caspase-3 activation and diminished BCL-XL expression. Suppression of MLL-AF4 is paralleled by a decreased expression of the homeotic genes HOXA7, HOXA9, and MEIS1. MLL-AF4 depletion inhibits expression of the stem-cell marker CD133, indicating hematopoietic differentiation. Transfection of leukemic cells with MLL-AF4 siRNAs reduces leukemia-associated morbidity and mortality in SCID mice that received a xenotransplant, suggesting that MLL-AF4 depletion negatively affects leukemia-initiating cells. Our findings demonstrate that MLL-AF4 is important for leukemic clonogenicity and engraftment of this highly aggressive leukemia. Targeted inhibition of MLL-AF4 fusion gene expression may lead to an effective and highly specific treatment of this therapy-resistant leukemia.

Beschreibung: 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.

Beschreibung: The chromosomal translocation t(4;11) marks a therapy-resistant infant leukemia with very poor prognosis. It results in the expression of two fusion-proteins, MLL-AF4 and AF4-MLL. We addressed the role of MLL-AF4 in t(4;11) positive SEM cells by siRNA-mediated suppression. Depletion of MLL-AF4 results in induction of apoptosis, inhibition of proliferation, decrease in colony formation and diminished leukemic engraftment in vivo. Currently, we are analyzing global changes in protein expression. For that, we compare the proteome of MLL-AF4 depleted SEM cells with those of control cells. The analysis is performed by 2D-gelelectrophoresis followed by mass spectrometry identification and immunoblot validation of differentially expressed spots. One of these spots was identified as Aldolase A. Comparison of MLL-AF4 depleted SEM cells with control cells showed neither change in mRNA levels nor in absolute protein levels of Aldolase A. Two-dimensional western blotting, however, revealed differences in the protein pattern, suggesting changes in Aldolase A modifications upon MLL-AF4 depletion. These analyses will provide us with a better insight into the effects of siRNA-mediated MLL-AF4 knockdown on the proteome, and may enable us to identify new targets for molecular therapeutic approaches.

Beschreibung: The chromosomal translocation t(4;11) marks infant acute lymphoblastic leukemia associated with a particularly dismal prognosis. We have recently shown that transfection of leukemic cells with MLL-AF4 siRNAs reduces leukemia-associated morbidity and mortality in xenotransplanted SCID mice suggesting that MLL-AF4 depletion negatively affects leukemia-initiating cells. In cell culture, transient suppression of MLL-AF4 expression impairs the proliferation of both t(4;11) cell lines SEM and RS4;11 more than tenfold. Clonogenicity of SEM cells was reduced fivefold and of RS4;11 cells more than twofold. One major reason for the antiproliferative consequences is the induction of apoptosis associated with caspase-3 activation and diminished BCL-XL expression. Caspase-3 was induced 3-fold and BCL-xL showed a 60% reduction after siRNA treatment compared to Mock-transfected controls in SEM cells. Analysis of intrinsic signaling pathways of apoptosis showed that MLL-AF4 depletion induces the expression of the proapoptotic genes APAF-1 and SEPT4 (ARTS). In both cell lines APAF-1 was induced more than twofold and SEPT4 more than fourfold. Our findings demonstrate that MLL-AF4 negatively interferes with an intrinsic apoptotic response and is, thus, important for the survival of these highly aggressive and chemoresistant leukemic cells. SiRNA-mediated MLL-AF4 inhibition may sensitize these cells for apoptosis-inducing agents. Therefore, complementing existing treatment strategies with a targeted inhibition of MLL-AF4 expression may lead to an improved and more specific treatment of this therapy-resistant leukemia.