MLL Fusion Driven Activation of CDK6 Potentiates Proliferation in MLL-rearranged Infant ALL

Abstract 1426

MLL-rearranged acute lymphoblastic leukemia represents a highly aggressive and clinically unfavorable type of childhood leukemia, displaying unique gene expression signatures. Nevertheless, the overwhelming number of differentially expressed genes made it difficult to elucidate the actual “drivers” of the leukemia. However, recent advances demonstrated that MLL fusion proteins recruit the histone methyltransferase DOT1L, leading to H3K79 methylation. Hence, genomic regions displaying aberrant enrichment of H3K79 methylation are prone to mark genes transcriptionally activated by the MLL fusion protein itself. Based on this concept, two independent studies recently identified gene signatures consisting of genes likely to represent direct MLL fusion targets. Yet, functional validation of such genes so far remains unacknowledged. In the present study we confirmed that CDK6 (cyclin-dependent protein kinase 6) represents a direct target of MLL-AF4 in t(4;11)-positive ALL cells. In contrast to its functional homologue CDK4, ChIP-sequencing analysis showed the presence of both MLL and AF4, as well as H3K79 methylation at the CDK6. Moreover, CDK6 mRNA appeared significantly (p<0.001) higher expressed in primary MLL- rearranged infant ALL patient samples when compared with other childhood ALL subtypes without translocations of the MLL gene. Next, using RNA interference, we performed MLL-AF4 and MLL-ENL knockdown experiments in ALL cell lines bearing these corresponding fusion transcripts, resulting in CDK6 down-regulation, whereas CDK4 expression was unaffected. These results emphasize that CDK6 is indeed a genuine transcriptional target of the MLL fusion protein itself. Moreover, direct knockdown of CDK6 itself significantly inhibited proliferation in MLL-rearranged ALL cells, whereas knockdown of CDK4 virtually had no effect on the cell cycle in these cells.

Taken together we conclude that CDK6 up-regulation in MLL-rearranged ALL is directly mediated by the MLL fusion itself and provides these cells with a proliferation advantage.