MYCN Regulates Cell Survival Via EIF4G1 in Inv(16) Acute Myeloid Leukemia

Transcription factors are frequently deregulated in leukemia and are pivotal to its pathogenesis. The core-binding factor (CBF) transcription factor is a heterodimer of CBFβ and RUNX proteins, is critical to hematopoiesis, and dysregulated in AML. The fusion oncogene CBFβ-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells by sequestering RUNX1 by protein-protein interaction to induce AML. The CBFβ-SMMHC inhibitor, AI-10-49, specifically disrupts its binding to RUNX1 resulting in genome-wide RUNX1 binding, apoptosis induction, and enhanced survival in inv(16) mouse model [Illendula* and Pulikkan* et al, Science 2015]. While previous studies have demonstrated that AI-10-49 induced cell death is partly mediated by repression of MYC expression in inv(16) AML cells [Pulikkan et al, Cell 2018], the function of other MYC family members, in inv(16) cell survival, and overall in AML, remains poorly understood.

By conducting Real Time RT-PCR in CD34+ bone marrow cells from human AML samples, we identified MYCN upregulation in inv(16) AML. Analysis of RNA-seq data from inv(16) AML cells treated with AI-10-49 revealed that MYCN expression is downregulated (7-fold). MYCN silencing via CRISPR/Cas9 induced apoptosis in inv(16) AML cells. Furthermore, MYCN genetic targeting by CRISPR/Cas9-RNP approach in human primary inv(16) AML cells transplanted in humanized NSGS xenograft mouse model displayed significant delay in leukemia development, underlining the role of MYCN in inv(16) AML maintenance.

ChIP-seq analysis conducted in inv(16) AML cells treated with AI-10-49 demonstrated increased RUNX1 occupancy at MYCN distal genomic element located at +26 Kb (named 'MYCN-e26') downstream from MYCN TSS without any RUNX1 binding at MYCN TSS. ChIP-seq analysis for histone marks such as H3K27ac and H3K4Me1, and chromatin accessibility by ATAC-seq suggested MYCN-e26 acts as a transcriptionally active enhancer element. Analysis of DNase I hypersensitive sites (DHSs) representing accessible chromatin regions in primary AML samples revealed that MYCN-e26 is a bona fide AML enhancer. Finally, deletion of a genomic element in MYCN-e26 surrounding the RUNX1 binding site (200 bp) by CRISPR/Cas9 genome editing downregulated MYCN transcript levels and inhibited inv(16) AML cell survival. Taken together, MYCN-e26 is instrumental in MYCN transcription and cell survival in inv(16) AML cells as a RUNX1 target.

To identify the mechanism(s) by which MYCN regulates inv(16) AML cell survival, we performed CUT&Tag sequencing for MYCN and H3K27ac histone mark in inv(16) AML cells. Gene ontology analysis for MYCN binding peaks in transcriptionally active regions revealed mRNA metabolism and stability as the major pathways orchestrated by MYCN in inv(16) AML cells. By comparing the mRNA metabolism/ stability signature genes with transcripts significantly deregulated in human primary inv(16) AML samples, we identified eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) as one of the major genes regulated by MYCN in inv(16) AML cell survival. Our CUT&Tag sequencing analysis demonstrated MYCN binds to EIF4G1 promoter and enhances its transcription. MYCN silencing by CRISPR/Cas9 approach in inv(16) AML cells showed significant downregulation of EIF4G1, suggesting MYCN is required for EIF4G1 expression in inv(16) AML cells. EIF4G1 genetic silencing via CRISPR/Cas9 approach induced apoptosis in inv(16) AML cells in vitro, and significantly delayed leukemia development in NSGS xenograft mouse model, suggesting EIF4G1 is required for the survival of inv(16) leukemia-initiating cells.

Our studies demonstrate that CBFβ-SMMHC modulates MYCN levels by inhibition of RUNX1 binding to MYCN distal enhancer in inv(16) AML cells. In addition, our studies bring novel function of MYCN in inv(16) leukemic cell survival by regulating EIF4G1. Future therapeutic approaches targeting MYCN/EIF4G1 axis in combination with AI-10-49 warrant better clinical outcome for the treatment of inv(16) AML.

No relevant conflicts of interest to declare.