Significance of Fusion Genes for Maintenance of Leukaemia

MLL/AF4 and AML1/MTG8 are fusion genes most frequently found in infant acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML), respectively. We have previously shown that transient siRNA mediated knock-down of MLL/AF4 and AML/MTG8 impairs proliferation and clonogenicity in vitro and causes a significant increase in median survival in a xeno-transplantation model.

We investigated the role of MLL/AF4 and AML1/MTG8 in leukaemic maintenance and progression of established disease in vivo. We used an inducible lentiviral shRNA expression system to determine the effects of knockdown of MLL/AF4 in the t(4;11)-positive SEM cell line and of AML1/MTG8 in the t(8;21)-positive human leukaemic cell line Kasumi-1. In addition, to allow in vivo imaging SEM and Kasumi-1 cells were labelled with luciferase.

shRNA cassettes specifically targeting the MLL/AF4 and the AML1/MTG8 fusions were cloned into the pTRIPZ vector where shRNA expression and RFP expression are both induced by doxycycline. Luciferase labelled SEM and Kasumi-1 cells were transduced with lentiviral particles, selected with puromycin and transduction efficiency was determined by quantification of RFP positive cells using flow cytometry. Experiments were initiated when more than 65% of the cell populations expressed RFP. Knockdown and expression of known targets of the fusion genes were verified at both the RNA and protein level by qPCR and western blotting, respectively. Cell growth was monitored by cell counts. Immunodeficient NSG mice were given doxycycline in the diet (625ppm) from two days prior to intrafemoral transplantations with 10⁶ luciferase labelled SEM and Kasumi cells transduced with pTRIPZshAML1/MTG8 or pTRIPZshMLL/AF4. The food was changed every other day and disease progression was monitored using in vivo bioluminescence imaging.

Upon induction of shRNA expression in vitro Kasumi-1 cells transduced with pTRIPZshAML1/MTG8 showed decreased expression of AML1/MTG8 at protein and RNA levels, which correlated with impaired proliferation. Furthermore, the AML1/MTG8 knockdown resulted in decreased CD34 expression and increased levels of IGFBP7 and PRG2. Induction of shMLL/AF4 expression in SEM cells resulted in decreased expression levels of MLL/AF4 with concomitant decreased expression of HOXA7. However, the number of RFP positive SEM pTRIPZshMLL/AF4 cells decreased over time. When we investigated the in vivo consequences of fusion-gene knockdown in transplanted NSG mice we found no significant differences in overall survival. Notably, mice transplanted with pTRIPZshAML1/MTG8-transduced Kasumi-1 cells showed a lower grade of disseminated disease compared to mice transplanted with pTRIPZshMLL/AF4-transduced Kasumi-1 cells. Furthermore, tumours from these mice had significantly lower RNA and protein levels of AML1/MTG8 (p<0.05). We could not verify knockdown of MLL/AF4 in tumour cells harvested from mice transplanted with SEM pTRIPZshMLL/AF4. Interestingly, these cells showed a complete loss of RFP expression compared to SEM transduced with pTRIPZshAML1/MTG8 (p<0.05).

Knockdown of MLL/AF4 and AML1/MTG8 in vitro using an inducible shRNA system led to decreased proliferation and affected genes associated with differentiation. However, the effects were delayed compared to transient siRNA knockdown. In vivo optical imaging is a useful tool to monitor leukaemic progression in vivo. The technique gives information about location and degree of disease dissemination in addition to the overall survival. The loss of RFP expression particularly in SEM pTRIPZshMLL/AF4 cells both in vitro and in vivo highlights the significance of MLL/AF4 in leukaemic maintenance. Further in vitro and in vivo experiments are currently ongoing with knockdown of AML1/MTG8 and MLL/AF4 in alternative cell lines and primary patient-derived material.

No relevant conflicts of interest to declare.