AKAP95 condensates regulate transcription and can be targeted in MLL-fusion driven oncogenesis (ChIP-Seq)

Oncogenic transcription represents a crucial event in MLL rearranged (MLLr) leukemia, yet the precise role of transcription co-activators in MLL-fusion protein-associated transcription and their contribution to leukemogenesis remain incompletely understood. Here, we demonstrate that AKAP95 is notably up-regulated in both MLLr patients and in MLL-AF9-transformed mouse bone marrow. AKAP95 proves essential for initiating mouse MLL-AF9 leukemia, exhibiting widespread DNA binding across the genome, particularly at gene promoters. Interaction with the translocated MLL1 fragment primarily occurs through phase separation mechanisms. AKAP95's RNA-binding properties, coupled with its phase separation behavior, govern its binding to pre-mRNA and concurrent transcriptional regulation. Importantly, we engineered a peptide to disrupt its phase separation, effectively attenuating AKAP95-mediated transcriptional co-activation and inhibiting the growth of MLLr cell lines. These findings illustrate a vivid example of an RNA-binding protein orchestrating transcription and tumorigenesis via phase separation, underscoring the potential for targeting oncogenic condensation mechanisms therapeutically. Overall design: Flag-mEGFP was knocked into the N-terminus of the AKAP95 locus in HeLa cells to tag the endogenous AKAP95. Lentiviruses pLKO.1-shAKAP95, pCDH-CMV-HA-JD-PI95-puro, and pCDH-CMV-HA-H32Q-PI95-puro were packaged in 293T cells with the envelope plasmids psPAX2 and pMD2.G. One million HeLa cells were transduced with 200 µL of concentrated virus by spin infection at 1000 × g for one and a half hours. Overexpression of wild-type AKAP95 and phase separation mutants was performed in HeLa cells to establish the significance of AKAP95's phase separation ability in promoting tumorigenesis. The binding targets of AKAP95 in genomic loci were determined by ChIP-seq.