Project description:E1A binding protein (p300) and CREB binding protein (CBP) are two homologous histone acetyl transferases that co-activate key oncogenes such as MYC and IRF4, which are relevant transcription factors in a number of haematological malignancies. In this work, we studied the effects of CCS1477 (also known as Inobrodib), a first in class orally available inhibitor of the p300/CBP bromodomains. We found that this compound causes cell cycle arrest in a panel of different hematologic cell lines and primary samples, together with a concomitant induction of myeloid differentiation in most of the AML samples. Mechanistically, we discovered that 6 hours exposure to CCS1477 triggers a rapid and selective loss of EP300 from MYB-bound enhancers in THP1 AML cells. Interestingly, after 48 hours treatment EP300 redistributes to alternate sites to induce up regulation of differentiation genes. Displacement of EP300 from MYB-bound enhancers is significantly associated with the rapid collapse in the expression of nearby genes, most of which correspond to key myeloid identity transcription factors such as MYC, MYB, CEBPA, GFI1, SPI1 and IRF8. In most of these down-regulated genes after CCS1477 treatment, the RNA-Pol II is stalled along the gene body thus failing in completing the transcriptional elongation. In OPM2 myeloma cells, CCS1477 releases EP300 from FGFR3, the target of the common (4;14) translocation, with loss of EP300 from enhancer sites bound by IRF4. We also showed that in leukaemia patients this drug causes a substantial increase in blood neutrophil count and up-regulation of MYB-regulated differentiation genes in the population of blast cells, while in myeloma patients it conferred a sustained reduction in urinary free light chains. Overall, our study describes the molecular activity of a novel epigenetic drug in different haematological malignancy settings, with promising effects in vivo both as single agent and in combination with standard of care agents such as azacitidine and venetoclax.

Project description:EP300 and CBP are paralogous, multidomain histone acetyltransferases (HATs) that regulate gene expression by binding to and acetylating diverse proteins. With widespread effects on gene regulation, these enzymes are attractive targets for therapeutic development. Discovery efforts for chemical inhibitors typically target protein domains that are amenable to probe binding. In some cases, probes have been identified for more than one domain within a single protein, including high potency inhibitors of both the catalytic HAT domain and bromodomain (BRD) that mediate the enzymatic and protein binding (“reader”) activities of EP300/CBP. In cancer cell systems, combination inhibition of both domains is more toxic than single inhibitor alone, indicating that inhibition of individual domain functions may elicit differential effects. Thus, we hypothesized that determining the relative effects of domain-specific inhibition of EP300/CBP on tumor cell growth may elucidate exceptional-responding tumor types to one inhibitor or the other. This would permit a broader therapeutic index by enabling reduced dosing of domain-targeted inhibitors, while maintaining enhanced inhibition of cancer cell growth. Here, using high-throughput cell line-based screening, we demonstrate that targeting the HAT or BRD domains of EP300/CBP using the chemical probes A485 and CCS1477 has differential effects in select tumor types. Group 3 medulloblastoma (G3MB) cells were especially sensitive to EP300/CBP BRD inhibition, as compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, the effects of bromodomain and HAT-domain specific inhibition are distinct, with bromodomain inhibition causing rapid early disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB cells.

Dataset Information

Activity of EP300/CBP bromodomain inhibitor CCS1477 across diverse hematologic malignancies (ChIP-Seq)

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