Background: The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member. Methods: Here we use functional genomics (RNA-seq, ChIP-seq and HiChIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss. Results: The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains that, in turn, led to altered genome compartmentalisation. These changes in genome structure resulted in altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway, resulting in increased sensitivity to MEK inhibition. Conclusions: The altered genomic architecture driven by the chronic loss of STAG2 results in altered gene expression that may contribute to leukaemogenesis and may be therapeutically targeted.

Background The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member. Methods Here we use functional genomics (RNA-seq, ChIP-seq and HiChIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss. Results The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains that, in turn, led to altered genome compartmentalisation. These changes in genome structure resulted in altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway, resulting in increased sensitivity to MEK inhibition. Conclusions: The altered genomic architecture driven by the chronic loss of STAG2 results in altered gene expression that may contribute to leukaemogenesis and may be therapeutically targeted.

Background The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member.Methods Here we use functional genomics (RNA-seq, ChIP-seq and HiChiIP) to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss.Results The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains that in turn led to altered genome compartmentalisation. These changes in genome structure lead to altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway resulting in increased sensitivity to MEK inhibition.Conclusions The altered genomic architecture driven by the chronic loss of STAG2 results in altered gene expression that may contribute to leukaemogenesis which may be therapeutically targeted.

Acute myeloid leukemia (AML) is a clonal heterogeneous disease of the myeloid white blood cells. It is characterised by an accumulation of immature blast cells and a number of chromosomal and genetic mutations have been identified. In both de novo and therapy-related AML, defective DNA repair mechanisms are responsible for some of these genetic abnormalities. Targeting the DNA repair mechanism has been shown to be successful against certain forms of solid tumors and may represent a novel therapeutic approach for AML.

14 The authors declare no potential conflicts of interest 2 15 ABSTRACT 16 The cohesin complex plays a major role in folding the human genome into 3D 17 structural domains. Mutations in members of the cohesin complex are known early 18 drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), 19 with STAG2 the most frequently mutated complex member. Here we use functional 20 genomics to investigate the impact of chronic STAG2 loss on three-dimensional 21 genome structure and transcriptional programming in a clinically relevant model of 22 chronic STAG2 loss. The chronic loss of STAG2 led to loss of smaller loop domains 23 and the maintenance/formation of large domains which in turn led to altered genome 24 compartmentalisation. These changes in genome structure were linked with altered 25 gene expression, including deregulation of the HOXA locus and the MAPK signalling 26 pathway, which may contribute to disease development and response to therapy. AUTHOR SUMMARY28 Acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS) are clonal 29 malignant diseases that affect the myeloid blood cell lineage. Around 40 different 30 mutations have been identified as being associated with MDS and AML; several 31 mutations effect genes in the cohesin complex particularly STAG2. STAG2 is an X-32 linked gene that is pivotal to the cohesin complex and the mutations result in a 33 truncated gene and loss of function.We have introduced a clinically relevant 34 truncating mutation into an isogeneic AML model. This has shown that changes in 35 the sizes of loop and domains formed in the genome resulting in appropriate gene 36 compartmentalisations. The associated changes in gene transcription has resulted 37 in deregulation of HOX genes, essential for development and differentiation, and in 38 the MAPK signalling pathway that could a therapeutic target. 3 39 INTRODUCTION 40 Acute Myeloid Leukaemia (AML) is a highly clonal disease characterised by the rapid 41 expansion of differentiation-blocked myeloid precursor cells, resulting in defective 42 haematopoiesis, and eventually, bone marrow failure [1]. In recent years, large-43 scale sequencing studies have identified a plethora of mutations within patient 44 derived AML cells, expanding our understanding of the genomic landscape, and 45 highlighting the complexity of the varying subtypes of this disease [2]. One of the 46 emerging genomic disease subgroups involves mutations within 47 chromatin/spliceosome related genes; including members of the cohesin complex. 48 Approximately 11% of patients diagnosed with a myeloid malignancy, including AML, 49 Myelodysplastic syndrome (MDS) or Myeloproliferative neoplasm (MPN), have been 50 shown to harbour a mutation within a member of the cohesin complex, with many 51 more showing significantly reduced expression of the complex members [3-5]. 52 Mutations within cohesin complex genes have also been identified in clinically 53 normal and aging individuals following sequencing analysis of large populations, 54 indicati...