The Transcriptional Activators p300/Crebbp Are Context-Dependent Functional Repressors of Interferon Genes, Offering Therapeutic Promise in AML

The lysine acetyltransferase (KAT) enzymes p300 and CREBBP, primarily recognized as histone acetyltransferases, are pivotal co-activators in acute myeloid leukemia (AML) maintenance. However, the traditional view of histone acetylation as a basic mechanism for transcriptional activation is increasingly being challenged, while the role of p300/CREBBP in acetylating non-histone proteins remains largely unexplored. This gap confounds the rationale for advancing novel p300/CREBBP KAT inhibitors to clinical trials, as a comprehensive understanding of p300/CREBBP functions is crucial for their effective clinical evaluation. Here, we set out to dissect the roles of p300/CREBBP in AML beyond histone acetylation, assess the implications of inhibition of their KAT domain and explore potential effective therapeutic combinations to facilitate their clinical application in treating AML.

Initially, we methodically analyzed the impact of p300/CREBBP inhibition across various AML subtypes using RNASeq and qPCR as preliminary screens, followed by confirmatory single-cell RNASeq and mass spectrometry-based total proteomics. We assessed the effects of KAT domain inhibition (hereafter KATi - via A-485 or CPI-1612) in comparison to a p300/CREBBP bromodomain inhibitor and a PROTAC degrader. All these compounds suppressed key AML maintenance genes, but uniquely, KATi also induced a massive expression of interferon-stimulated genes (ISG). This was particularly pronounced in monocytic AML and contributed to cell cycle arrest, differentiation and apoptosis. In SLAMSeq and qPCR, we observed that ISG induction began shortly after KATi and intensified progressively within the first 24 hrs, illustrating a dynamic ‘feed-forward’ transcriptional activation.

We hypothesized that this unexpected induction of ISG upon KATi might reveal a previously unrecognized suppressive role exerted by the p300/CREBBP KAT activity. To investigate this, we employed a multidimensional approach, comparing cells treated with KATi to DMSO controls. We began with acetylproteomics and RIME-based chromatin pull-down of p300 to pinpoint the interactors of p300/CREBBP in monocytic AML and complemented these with ChIPSeq of identified acetylation-interactors, PROSeq to track real-time transcription at promoters and enhancers, and a PerturbSeq screen paired with scRNASeq to assess the functional importance of the interactors. p300 and CREBBP emerged as their own most significant interactors and targets for (auto)acetylation. They were followed by the cBAF remodeling complex members ARID1A and SMARCA4. Other significant partners included transcription(al) (co)factors like RUNX1, IRF8, PU.1, MEF2D or IRF2BP2. PROSeq revealed a pronounced and almost exclusive increase in transcription at ISG promoters. At these sites, ChIPSeq revealed KATi-induced ‘trapping’ of p300 and CREBBP, increased remodeling activity by ARID1A/SMARCA4 and exclusion of IRF8 from the chromatin. In PerturbSeq, the ISG induction triggered by KATi was particularly reduced when disrupting ARID1A/SMARCA4, highlighting the cBAF complex's critical role in mediating ISG responses to KATi. This was further validated by employing PROTAC-mediated degradation of the cBAF subunit SMARCA4, which mitigated the ISG release and alleviated AML cell death associated with KATi. Finally, RIME-based chromatin pull-down and mass spectrometry for the cBAF subunit ARID1A revealed new interactions with STAT1 and other positive ISG regulators upon KATi, while ChIPSeq showed exclusive new binding of STAT1 to critical ISG promoters, confirming a shift to a transcriptionally active state.

Testing the therapeutic potential of this ISG release beyond single compound treatment, we combined KATi with Interferon alpha (IFNa). This demonstrated an exponential increase of the ISG response and a significant, highly synergistic induction of apoptosis in human and murine monocytic AML cell lines and primary samples, suggesting a promising clinical combination. In vivo treatment combinations are currently underway.

In conclusion, active p300/CREBBP maintain a repressive environment for ISGs in conjunction with the cBAF complex in monocytic AML. p300/CREBBP KAT domain inhibition shifts this to a permissive state, releasing ISGs, which can be further amplified by IFNa. In our view, this combination holds significant translational potential for AML treatment.

Vassiliou:STRM.BIO: Consultancy; AstraZeneca: Research Funding. Kühn:Pfizer: Consultancy; Kura-Oncology: Research Funding; Jazz: Consultancy, Other: Speaker activity; Janssen: Consultancy; Giliead: Other: Speaker Activity; BMS/Celgene: Consultancy, Other: Speaker Activity; Abbvie: Consultancy, Other: Speaker activity; Servier: Consultancy, Other: Speaker activity; Syndax: Research Funding. Huntly:Amphista: Consultancy; Janpix: Membership on an entity's Board of Directors or advisory committees; Istesso: Consultancy; Menerini: Consultancy; AstraZeneca: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Oellerich:Roche: Consultancy, Honoraria; Merck KGaA: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Genmab: Consultancy, Honoraria; Beigene: Consultancy, Honoraria; Kronos Bio: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; Gilead: Research Funding. Sasca:Astellas: Honoraria; AstraZeneca: Honoraria; Gilead: Honoraria; BMS: Honoraria; Abbvie: Honoraria.