CBFβ-SMMHC Inhibition Disrupts Enhancer Chromatin Dynamics and Represses MYC Transcriptional Program in Inv(16) Leukemia
Authors: Pulikkan John Anto , Hegde Mahesh , Belaghzal Houda , Illendula Anuradha , Yu Jun , Ahmed Hafiz , O'Hagen Kelsey , Ou Jianhong , Müller-Tidow Carsten , Wolfe Scot A. , Zhu Julie , Dekker Job , Bushweller John H. , Castilla Lucio H. 
Published: April 2021

Chromatin complexes regulate gene expression in normal and malignant hematopoiesis. The significance of transcription factor deregulation on the control of epigenetic dynamics in leukemia is poorly understood. The leukemia fusion CBFβ-SMMHC is expressed in inv(16) acute myeloid leukemia (AML), and deregulates the activity of the transcription factor RUNX1. This fusion protein blocks myeloid differentiation, expands pre-leukemic myeloid progenitor cells, and drives AML development. The CBFβ-SMMHC inhibitor, AI-10-49, specifically disrupts its binding to RUNX1 resulting in an acute RUNX1 release, inducing apoptosis in inv(16) AML cells and delaying leukemia latency in mice. However, the mechanism by which AI-10-49 induces apoptosis is unknown. In this study, we utilize pharmacologic, genomic and genetic approaches to demonstrate a RUNX-dependent mechanism by which CBFβ-SMMHC maintains leukemia survival, and that this mechanism can be pharmacologically targeted in the treatment of inv(16) AML.

Gene Set Enrichment Analysis of RNA-seq data from inv(16) AML cells treated with AI-10-49 identified the deregulation of a MYC signature, including cell cycle, ribosome biogenesis and metabolism, and the reduction of MYC transcript levels (10-fold). MYC shRNA knockdown induced apoptosis of inv(16) AML cells, and MYC expression rescued AI-10-49 induced apoptosis. Furthermore, mouse leukemia cells transduced with Myc shRNAs showed significant delay in leukemic latency upon transplantation, validating the requirement of MYC in inv(16) AML maintenance in vivo. Similarly, pharmacologic inhibition of MYC activity, using a combined treatment with AI-10-49 and the BET-family bromodomain inhibitor JQ1, revealed a strong synergy in inv(16) AML cells and significant delay in leukemia latency in mice. Analysis of chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) revealed that AI-10-49 treatment increased RUNX1 occupancy at three MYC distal enhancer elements (including two new enhancers: +0.18 Mb and +0.5 Mb, and the E3 enhancer of the BDME superenhancer at +1.7 Mb) downstream from MYC TSS but not at the MYC TSS or the T-cell leukemia associated N-ME enhancer. The open-chromatin and active nature of these regions was further validated by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and H3K27Ac ChIP-seq.

Since the MYC locus is occupied by a number of tumor-specific enhancers, we analyzed the enhancer interactions in a 4 Mb region (-1Mb to +3 Mb of MYC TSS) by chromosome conformation capture carbon copy (5C) in inv(16) AML cells. The 5C analysis revealed that the three enhancers are physically connected with each other and with the MYC promoter. Again, the T-cell ALL associated N-ME enhancer was not active in these cells. Furthermore, the six-hour AI-10-49 treatment had not altered these interactions, indicating that MYC repression was probably not due to changes in open chromatin but to alterations in regulatory factors. Therefore, we analyzed the occupancy of chromatin complex components at the three enhancers, N-ME and MYC TSS regions, utilizing ChIP-quantitative PCR. We demonstrate that AI-10-49 treatment results in the repression of the SWI/SNF complex component BRG1 and recruitment of polycomb-repressive complex 1 (PRC1) component RING1B at the three MYC enhancers. This switch was associated with a reduction of active enhancer mark H3K4me1 and an increase of the repressive mark H3K27me3 at these sites. Finally, deletion of a small region in each of these enhancers surrounding the RUNX1 binding site (41 bp, 67 bp and 295 bp) by genome editing (CRISPR/Cas9) reduced MYC transcript levels by 50% and the viability of inv(16) AML cells in vitro, indicating that each one of these enhancers plays a critical role in regulating MYC levels and sustaining the survival of inv(16) AML cells.

Our results demonstrate that CBFβ-SMMHC regulates MYC levels by repression of RUNX1 activity at three distal MYC enhancers in inv(16) AML cells. AI-10-49 treatment induces an acute release of RUNX1, increases RUNX1 occupancy at the distal enhancers, and disrupts enhancer chromatin dynamics which in turn induces apoptosis by repressing MYC. Furthermore, this study suggests that combined treatment of inv(16) AML with AI-10-49 and BET-family inhibitors may represent a promising targeted therapy.

Disclosures

No relevant conflicts of interest to declare.