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.

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Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation and minimize toxicity via MDM2i, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. By integrating the results of these two screens, we found inhibition of BRD4 activates p53 and its target genes. BRD4 (Bromodomain-containing protein 4) is a member of the bromodomain and extraterminal (BET) family proteins, which has typically been reported to activate genes, such as c-MYC, BCL2 and CDK4/6. However, we unexpectedly reveal that BRD4 acts as a transcriptional repressor of p53 target genes. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type p53 in vitro, against two mouse models of AML in vivo, and against several primary human patient blasts in vitro. Synergistic cell killing was associated with synergistic activation of p53 target genes, and cell killing and p53 target gene expression. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, targetable wild-type p53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML. Citation Format: Sha Li, Anne Latif, Ashley Newcombe, Kathryn Gilroy, Neil Robertson, Xue Lei, Darren Finlay, Helen Stewart, Karina Barbosa, Brian Higgins, Tim Chevassut, Xu Huang, Mhairi Copland, Karen Keeshan, Ani Deshpande, Peter Adams. A synthetic lethality approach to eradicate AML via synergistic activation of pro-apoptotic p53 by MDM2 and BET inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3426.