Translocations involving the MLL
gene in leukemia are uniquely characterized by the expression of both the MLL-fusion and the reciprocal fusion product in majority of the cases. The lack of detectable reciprocal fusions in up to 20% of cases may be due to complex rearrangements involving multiple genes that have been recently identified(18
). Given that the der11 MLL-fusion protein is expressed in all 11q23 rearranged leukemias, all experimental models have been based on expressing this MLL-fusion gene and the data from these models demonstrate that the MLL-fusion gene indeed promotes transformation of the host cell. However, the role of the reciprocal gene has not been evaluated in detail. Our results indicate that in established t(4;11) leukemias the reciprocal product – AF4-MLL is not essential for leukemia cell growth or survival. We observed no significant effects of siRNA mediated knockdown of AF4-MLL in both RS4;11 and SEMK2-M1 cell lines. The lack of an antibody to detect AF4-MLL precludes our ability to evaluate the knockdown of the protein, but our real-time quantitative RT-PCR data show that we achieved consistent knockdown of the mRNA by >50%. Moreover, sequential electroporations with the AF4-MLL siRNA failed to show any effect on SEMK2-M1 cell growth while under identical conditions, MLL-AF4 knockdown resulted in significant growth inhibition. In a recent publication, Bursen et al report that murine Lin−
bone marrow cells were transformed by retrovirally expressed AF4-MLL, resulting in the development of acute leukemia in a third of the mice transplanted with AF4-MLL or AF4-MLL + MLL-AF4 expressing cells(11
). While at first our results appear to contradict these published results, several considerations highlight that we cannot compare the study by Bursen et al to ours. Firstly, the two studies address different questions. In their study, Bursen et al show that leukemia may be initiated by retrovirally expressed AF4-MLL, while our results demonstrate that established t(4;11) leukemias are no longer dependent on AF4-MLL for continued growth. It is possible that events downstream of the translocation that culminate in the development of leukemia render the reciprocal fusion redundant. Additionally, the cellular origin of MLL-rearranged leukemia remains unknown. The Lin−
bone marrow fraction used by Bursen et al is a heterogeneous population largely composed of proliferating progenitor cells. Recently published results suggest that t(4;11) leukemias might originate in relatively more primitive cells that are pre-hematopoietic in their lineage commitment (20
). Thus, the patient-derived leukemia cell lines used in our study might differ significantly in their developmental origins compared to the cells transformed by retroviral expression of AF4-MLL in the study by Bursen et al. However, it is worth noting that in contrast to the results of Bursen et al, other groups have successfully generated leukemia in murine models driven by the MLL-AF4 fusion alone (i.e. without the reciprocal fusion) either via a conditional knock-in approach or via retroviral expression(10
). Finally, MLL-rearranged leukemias are characterized by increased H3K79 methylation at specific loci that correlate with increased expression of the corresponding genes(10
). Recent data indicate that the carboxy terminus of AF4 plays an important role in recruiting the H3K79 methyltransferase DOT1L to these loci of increased transcription, implicating the der11 MLL-AF4 as the major oncogene in t(4;11) leukemias.
Overall, our results show that leukemias with MLL-translocations remain dependent on the der11 MLL-fusion protein for continued growth while the reciprocal fusion appears to be non-essential. Therapies targeting the MLL-fusion protein itself, its interacting proteins, or its downstream mediators may thus be beneficial for treating patients with these leukemias.