Acute myeloid leukemia (AML) is a typically lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report enhanced toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi’s ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.

There is a desperate need for new and effective therapeutic approaches to acute myeloid leukemia (AML) in both children and adults. Epigenetic aberrations are common in adult AML, and many novel epigenetic compounds that may improve patient outcomes are in clinical development. Mutations in epigenetic regulators occur less frequently in AML in children than in adults. Investigating the potential benefits of epigenetic therapy in pediatric AML is an important issue and is discussed in this review.

SummaryAcute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, most AML retain wild-type TP53, encoding the pro-apoptotic tumor suppressor p53. MDM2 inhibitors (MDM2i), which activate wild-type p53, and BET inhibitors (BETi), targeting the BET-family co-activator BRD4, both show encouraging pre-clinical activity, but limited clinical activity as single agents. Here, we report synergistic toxicity of combined MDM2i and BETi towards AML cell lines, primary human blasts and mouse models, resulting from BETi’s ability to evict an unexpected repressive form of BRD4 from p53 target genes, and hence potentiate MDM2i-induced p53 activation. These results indicate that wild-type TP53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML.

Background: There remains a critical requirement for novel therapies for Acute Myeloid Leukemia (AML). Bromodomain and extra-terminal domain (BET) inhibitors are emerging as exciting therapeutic agents for hematopoietic malignancies. 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 oncogenic loci. Although a heterogeneous disease, most AML retains wild type p53. However, p53 is often rendered functionally deficient by over-expression of MDM2. Potentiating the p53 response though MDM2 antagonism is therefore potentially beneficial to most AML subtypes. We hypothesized that dual inhibition of MDM2 and BET would be synthetic lethal to p53 wild type AML. Methods: For in vitro experiments CPI203 (BET inhibitor, Constellation Pharmaceuticals) and nutlin-3 (MDM2 antagonist, Sigma) were assessed on p53 wild type cell lines (OCI-AML3, MOLM-13 and MV411) and p53 wild type primary murine AML. To assess the combination's dependency on wild type p53; p53 mutated cell lines (KG1a, KASUMI-1 and THP1) were tested. Cell viability was assessed using resazurin (Alamar blue dye) across numerous dose ratios on the OCI-AML3 cell line and analysed using the Envision Fluorescent Reader. Drug combination indices (CI) were evaluated using Calcusyn (version 2.0). Apoptosis was assessed using flow cytometry staining for Annexin V and propidium iodide (PI) on all p53 wild type and mutated cell lines. For in vivo experiments CPI0610 (clinical grade BET inhibitor, Constellation Pharmaceuticals) and RG7112 (MDM2 inhibitor, Roche) were tested as single agents, in combination and with relevant vehicle controls. RNA seq was performed on the GAIIX sequencer and gene ontology analysis was performed using DAVID/INGENUITY pathway analysis (IPA). Results: In the OCI-AML3 cell line, resazurin analysis demonstrated that combining CPI203 with nutlin-3 was potently synergistic in decreasing viable cells for a 1:12.5 (mean CI=0.07) and 1:25 ratio (mean CI=0.299), and synergistic for a 1:50(mean CI=0.44) and 1:100(mean CI= 0.66) ratios. There was no benefit in using the combination treatment on the p53 mutated cell lines. Apoptosis was enhanced at least 1.5 fold (median 1.7, range 1.5-2.65) by the drug combination versus the single agents, in the panel of p53 wild type cell lines tested. Analysis of whole genome RNA seq on OCI-AML3 treated cells, showed that genes up-regulated by the combination of CPI203 and nutlin-3, had a thirty-fold enrichment for p53 signalling (FDR (<0.05). Down-regulated genes were enriched for FOXM1-dependent cell cycle progression genes. To evaluate the combination in vivo, we used a Trib-2 driven primary AML where leukemogenesis is induced through inhibition of C/EBPα. Myeloblasts were transduced with GFP on the same retroviral construct asTrib-2 for disease tracking. Treatment was commenced in all mice (n=40), post confirmation of disease engraftment. Three mice from each treatment group were sacrificed after 48hrs and cells sorted for GFP to perform RNA seq in this in vivo setting. After 21 days of treatment all mice were sacrificed (n=27, one vehicle control succumbed to disease 15 days post engraftment). End of treatment results (primary read out was the GFP% which equates to the blast%) demonstrated superior in vivo efficacy of dual inhibition of MDM2 and BET in comparison with controls in eradicating AML, p<0.0001, (see figure). Importantly, normal haematopoiesis was spared - as evidenced by normal full blood counts and comparable myeloid, B-cell and T-cell populations with our C57bl6 wild type controls. RNA seq of the murine blasts revealed that many more genes significantly (FDR<0.05) changed expression in the combination treated mice than single agent treated mice. The p53 pathway was the most common up-stream regulator of genes changing expression post combination treatment, p<0.0001. The combination affected many more genes in the p53 pathway than RG7112 alone (120 genes versus 20 genes respectively), in line with our in vitro results. Conclusion: This combination of BET and MDM2 inhibition is effective and superior to single agent therapy on all p53 wild type AMLs tested, in vitro and in vivo. In both contexts this is associated with potentiating the p53 response and could be relevant to many patients with p53 wild type AML. Figure 1. Figure 1. Disclosures Latif: Novartis: Honoraria. Copland:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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.