Chromatin-Based Cellular Dependency of HOXA9/MEIS1-Driven Acute Myeloid Leukemia
Authors: Valigi Federica , Kücüktas Fulya Mina , Fliamer Ilya , Jevtic Zivojin , Chatel-Soulet Hughes-Etienne , Bovay Amandine , Sivalingam Rathick , Baosic Andreja , Grebien Florian , Slany Robert K. , Tzankov Alexandar , Juge Sabine , Seguin Jonathan , Wang Menghan , El Taher Athimed , Giorgetti Luca , Schwaller Juerg 
Published: December 2024

Acute myeloid leukemia (AML) driven by KMT2A or NUP98 fusions, as well as mutant NPM1, depends on aberrant levels of MEIS1 and HOXA9. To gain insight into the initiation and progression of these types of AML, we characterized the pre-symptomatic phase in a DOX-inducible iKMT2A-MLLT3 transgenic AML mouse model. Within just 10 days, during the initiation phase, we observed a significant yet temporary expansion of mature Mac1+Gr1+ myeloid cells in the bone marrow (BM). This was followed by a progression phase (&gt;50 days) where predominantly Mac1+Gr1- blast-like cells appeared, leading to symptomatic disease after 70 days (±6). Single cell RNA sequencing (scRNAseq) illustrated these changes by depicting the expansion of BM granulocytic cell populations at the initiation phase and by identifying a limited set of significantly dysregulated genes when comparing iKMT2A-MLLT3 to control mice on DOX. During the progression phase (day 60) an increased number of differently expressed genes (DEGs) was observed in BM granulocytic- and monocytic progenitor-restricted cells. Integrative mining of various public datasets (RNAseq, ChIPseq, AML patient expression signatures, DepMap) allowed us to identify 38 genes as potential “disease drivers”. Primary validation using CRISPR-mediated inactivation via viral expression of two sgRNAs in KMT2A-MLLT3+ murine RN2-Cas9 AML cells revealed that, similar to the well-known primary target fusion genes Hoxa9, Meis1 & Pbx3, inactivation of Pds5a, encoding for a cohesin-associated protein, significantly induced myeloid differentiation as shown by increased in Mac1 and decreased Kit levels. Notably, inactivation of Pds5a reduced clonogenic growth to a much higher extent in AML cells than in normal BM HSPC in vitro. qRT-PCR and immunoblotting showed that reduction of Hoxa9 did not affect Pds5a levels and vice versa. in vivo validation by transplanting sgRNA-transduced iKMT2A-MLLT2/Cas9-GFP cells showed that inactivation of Pds5a significantly impaired the induction and maintenance of the disease in mice. Further validation by shRNA-mediated Pds5a knockdown in Hoxa9-dependent AML mouse (NUP98-NSD1+) and human (MOLM13: KMT2A-MLLT3+, OCI-AML3: NPM1c+) cell lines showed reduced clonogenic growth and increased differentiation. Surprisingly, leukemic cells with perturbed Pds5b expression, displayed no significant changes in regulating cell growth or differentiation, adding further evidences for the specific role of Pds5a in HOXA9/MEIS1-expressing AML. Given its role in controlling chromatin architecture and consequently in promoter-enhancer interactions, we investigated changes in 3D-genome organization by genome-wide chromosome conformation capture (Micro-C), and correlated them to transcription chromatin states by RNA-seq, Cut&Run and ATAC-seq upon Pds5a inactivation in KMT2A-MLLT3+ RN2-Cas9 AML cells. Transcriptional profilingidentified 2302 higher- and 2398 lower differentially expressed genes (FDR&lt;0.05) when comparing to control cells. However, no significant changes were observed for primary KMT2A-MLLT3 target genes like Hoxa9 and Meis1. In contrast, GSEA revealed a significant reduction in the expression of Hoxa9 (p=4.45-40) and Meis1 (p=5.95-31) target genes. Among these, Myc (p=1.61-4), a key regulator of AML transformation, stood up. Conversely, the myeloid differentiation transcription factor Cebpe was higher expressed (p=4.12-5), mirroring the terminal differentiation phenotype in Pds5a-depleted AML cells. ATAC-seq data from Pds5a-depleted cells showed increased accessibility at motifs for binding for Pu.1, AP-1, Ets, and Cebp, while the accessibility at the Myc locus was reduced. Remarkably, the long-range looping around the Myc gene locus appeared weakened in Pds5a-inactivated murine leukemic cell compared to control cells. In particular, the contact probability of Myc with BENC, its hematopoietic enhancer region, was drastically reduced. Collectively, this study unveils for the first time a role of Pds5a during initiation and progression of MEIS1/HOXA9-dependent AML. We show that the cohesin associated factor Pds5a, unlike its paralog Pds5b, is critical for the leukemia transformation. The deeper study in looping activity mediated by Pds5a could reveal a fundamental mechanism that regulates a critical genome architecture in MEIS1/HOXA9-dependent AML.