Mixed lineage leukemia (MLL) gene encodes for a nuclear protein that regulates genes involved in development and hematopoiesis. De-regulation of MLL by chromosome translocations causes acute myeloid leukemia (AML). In addition, about 5–11% of AML patients have MLL-partial tandem duplication (PTD) mutations. MLL-PTD is associated with poor prognosis and shorter remission duration in AML. Preliminary studies from our group indicate that when MLL-PTD is knockdown in AML blasts we observe a mRNA expression profile that is very similar to that of bromodomain 4 (BRD4) inhibition in AML.
We hypothesized that MLL-PTD interacts with BRD4 forming a complex that regulate key genes in MLL-PTD positive AML. Therefore treatment with BRD4 inhibitors, such as JQ1, could be a valid therapeutic strategy for MLL-PTD AML patients.
To identify proteins forming the transcriptional complex regulated by BRD4 and MLL, we conducted a Rapid Immunoprecipitation Mass spectrometry of Endogenous protein (RIME) study using BRD4 and MLL antibodies. Successively, we treated MLL-PTD positive primary patient cells and CD34+ cord blood from healthy donors as controls with JQ1, and conducted RNA-seq and ChIP-seq studies. Finally, we conducted in vitro and in vivo studies on MLL-PTD harboring samples to assess JQ1 treatment efficiency in inducing cell death in MLL-PTD AML.
RIME analysis using BRD4 and MLL antibodies showed that the 75% of the proteins were in common between the two groups. PANTHER gene ontology analysis identified common pathways between them, suggesting that MLL and BRD4 could be part of similar protein complexes. Next, we treated MLL-PTD+ primary patient cells, and CD34+ cord blood cells as controls with JQ1 or vehicle, and conducted RNA-seq and ChIP-seq studies. RNA expression and peak intensity on promoter region results were combined to identify genes aberrantly expressed in MLL-PTD+ samples. We identified 98 up-regulated and 32 down-regulated genes that were differentially expressed d by JQ1 treatment only in AML samples. The top deregulated genes were validated by RT-qPCR and ChIP in both human and murine MLL-PTD+ samples. Next, we treated the MLL-PTD+ AML cell line (EOL1) and primary murine AML cells from Mll-PTD/Flt3-ITD model with JQ1 and showed that JQ1 treatment induced more apoptosis and reduction of living cells in MLL-PTD+ models respect the relative controls (fold change living cells at 48 hours WT: 0.81, Mll-PTD+: 0.42, p-value: 0.0247). Last, treatment of the Mll-PTD/Flt3-ITD xenotranplanted BoyJ mice with JQ1 significantly increased their survival compared with controls (median survival CTRL: 75 days, JQ1: 109 days, Mantel-Cox p-value: 0.0195). In addition, we observed a reduced engraftment in serial re-transplantation assays.
Our data support developing a therapeutic strategy to target BRD4 in MLL-PTD AML.