BET Inhibitors Potentiate Activation of p53 and Killing of AML By MDM2 Inhibitors — a Candidate Combination Therapy

Abstract

In 2016, there will be approximately 19,950 new cases of Acute Myeloid Leukaemia (AML) in the United States. After diagnosis, five-year survival is currently ~26%, with available therapeutic approaches. Therefore, there remains a critical requirement for novel therapies for AML. Bromodomain and extra-terminal domain inhibitors (BETi) are emerging as exciting therapeutic agents for haematopoietic malignancies, including AML. The BET protein family, BRD2, BRD3, BRD4 and BRDT, are best known as transcriptional co-activators whose targets include oncogenic loci, such as c-MYC, BCL2 and CDK4/6. Pharmacological inhibition of BET bromodomains targets malignant cells by preventing reading of acetylated lysine residues, thus disrupting chromatin-mediated signal transduction, which reduces transcription at these oncogenic loci. BETi alone have shown promising pre-clinical activity against diverse AML subtypes, and are now in clinical trial in AML and other hematological malignancies. Significantly, however, there is also some evidence that BET family proteins can also act as transcriptional repressors. Whether BET-mediated transcriptional repression is also a therapeutic target in cancer is not known. Although a heterogeneous disease, most human de novo AML (not due to relapse or secondary to myelodysplastic syndrome (MDS) or cancer therapy) harbor wild type TP53 (encoding the p53 tumor suppressor). TCGA reports that only 8% of de novo AML contain TP53 mutations (typically in AML of a complex karyotype). However, p53 is often rendered functionally deficient by over-expression of its negative regulator, MDM2. Accordingly, we hypothesized that dual inhibition of MDM2 and BET would be synergistically lethal to wild type TP53 AML. Confirming this hypothesis, we showed that a combination of MDM2 inhibitors (MDM2i) and BETi is synergistically lethal to human AML cell lines harbouring wild type TP53in vitro, against two mouse models of AML in vivo and against some primary human patient blasts in vitro. Importantly, in the mouse model, the drug combination did not affect normal haematopoiesis, suggesting a manageable level of toxicity. Synergistic cell killing was associated with synergistic activation of p53 target genes, and cell killing and p53 target gene expression both depended on expression of wild type TP53. Dual inactivation by CRISPR/Cas9 of two p53-activated pro-apoptotic genes, PUMA and NOXA, suppressed cell killing by the MDM2i/BETi drug combination. These data indicate that BETi potentiate activation of p53 by MDM2i to kill AML blasts. Regarding the mechanism of synergy, BETi did not enhance binding of p53 to its pro-apoptotic target genes and did not stabilize mRNAs of p53 target genes. Interestingly, however, we observed that in growing human AML cells, a target of BETi, BRD4, was constitutively bound to p53 target genes in control cells and MDM2i-treated cells, but displaced in BETi-treated cells. Accordingly, we hypothesized that BRD4 represses p53 target genes, preventing their activation by MDM2i alone but leading to synergistic activation and cell killing by combined MDM2i and BETi. Consistent with this hypothesis, knock down of BRD4 activated p53 target genes in the presence of MDM2i and ectopic expression of BRD4 repressed p53 target genes. Taken together, these data show that BETi potentiate activation of p53 by MDM2i by relieving BRD4-mediated repression of p53 target genes. This results in potent and specific toxicity towards AML cells harbouring wild type TP53. Disclosures No relevant conflicts of interest to declare.

Uncovering PROTAC Sensitivity and Efficacy by Multidimensional Proteome Profiling: A Case for STAT3

“…We also found that tumor suppressor p21, the downstream effector and cooperator of p53, was upregulated with 100 nM of ZBC260 and 24 h treatments (Figure S1H,I). These results were consistent with those of the previous studies, ,,− verifying the reliability of DIA-MPP for studying PROTAC efficacy.…”