Acute myeloid leukemia (AML) with complex karyotype is characterized by high genomic complexity, including frequent TP53 mutations and chromothripsis. Genomic rearrangements can reposition active enhancers near proto-oncogenes, leading to their aberrant expression; however, a comprehensive understanding of these events in AML is still incomplete. To facilitate the discovery of such "enhancer hijacking" events, we developed Pyjacker, a computational tool, and applied it to 39 AML samples with complex karyotype. Pyjacker identified several enhancer hijacking events in AML patient samples, including aberrant expression of MNX1, which can result from del(7)(q22q36) and is associated with hijacking of a CDK6 enhancer. MNX1 activation occurred in 1.4% of patients with AML and showed significant co-occurrence with BCOR mutations. Through a xenograft mouse model, we demonstrated that MNX1 is required for leukemia cell fitness. Pyjacker is an easy-to-use, accurate, and broadly applicable tool for identifying consequences of genomic events driving tumorigenesis, especially when germline genomic data are missing. SIGNIFICANCE:This study examines the consequences of structural alterations in AML and demonstrates that proto-oncogene activation by enhancer hijacking is an understudied pathomechanism. MNX1 overexpression demonstrates that deletions on chromosome 7q can not only lead to haploinsufficiency but also to activation of oncogenes by enhancer hijacking.

Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners (MNX1 and ETV6) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6. The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking may be a more widespread consequence of translocations where no oncofusion product was identified, including e.g. t(1;3) or t(4;12) AML.

Stabilin‐1 (Stab1) and Stabilin‐2 (Stab2) are scavenger receptors expressed by liver sinusoidal endothelial cells (LSECs). The Stabilin‐mediated scavenging function is responsible for regulating the molecular composition of circulating blood in mammals. Stab1 and Stab2 have been shown to influence fibrosis in liver and kidneys and to modulate inflammation in atherosclerosis. In this context, circulating and localized TGFBi and POSTN are differentially controlled by the Stabilins as their receptors. To assess Stab1 and Stab2 functions in inflammatory and fibrotic skin disease, topical Imiquimod (IMQ) was used to induce psoriasis‐like skin lesions in mice and Bleomycin (BLM) was applied subcutaneously to induce scleroderma‐like effects in the skin. The topical treatment with IMQ, as expected, led to psoriasis‐like changes in the skin of mice, including increased epidermal thickness and significant weight loss. Clinical severity was reduced in Stab2‐deficient compared to Stab1‐deficient mice. We did not observe differential effects in the skin of Stabilin‐deficient mice after bleomycin injection. Interestingly, treatment with IMQ led to a significant increase of Stabilin ligand TGFBi plasma levels in Stab2−/− mice, treatment with BLM resulted in a significant decrease in TGFBi levels in Stab1−/− mice. Overall, Stab1 and Stab2 deficiency resulted in minor alterations of the disease phenotypes accompanied by alterations of circulating ligands in the blood in response to the disease models. Stabilin‐mediated clearance of TGFBi was altered in these disease processes. Taken together our results suggest that Stabilin deficiency‐associated plasma alterations may interfere with preclinical disease severity and treatment responses in patients.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by a block in differentiation and accelerated proliferation of myeloid progenitor cells. Genes encoding for epigenetic regulators are among the most frequent targets for mutations and structural variations in AML, giving rise to profound epigenetic heterogeneity between and within tumors. Deletions of chromosome 5q [del(5q)] are among the most common copy number alterations in AML and are associated with extremely poor clinical outcome and therapy resistance, however the mechanisms linking del(5q) to leukemic progression are not understood. Analyzing DNA methylation profiles from 477 elderly AML patients using DNA methylome deconvolution, we discovered that del(5q) AML is an epigenetically distinct subgroup characterized by a signature of DNA hypermethylation, which we propose may be linked to dysregulation of H3K9me1/2 and overexpression of the leukemic stem cell marker, DNMT3B. Interrogation of the minimally deleted 5q region highlighted the H3K9me1/2 demethylase KDM3B as a likely target for haploinsufficiency in this subgroup. Our data suggest that del(5q) AML should be reconsidered as an epigenetically dysregulated subgroup, driven by heterozygous loss of KDM3B, and that the resulting imbalance of H3K9me1/2 may contribute to the progression of these aggressive leukemias. ### Competing Interest Statement The authors have declared no competing interest.

Acute myeloid leukemia with complex karyotype (ckAML) is characterized by high genomic complexity, including frequent TP53 mutations and chromothripsis. We hypothesized that the numerous genomic rearrangements could reposition active enhancers near proto-oncogenes, leading to their aberrant expression. We developed pyjacker, a computational tool for the detection of enhancer hijacking events, and applied it to a cohort of 39 ckAML samples. Pyjacker identified motor neuron and pancreas homeobox 1 ( MNX1 ), a gene aberrantly expressed in 1.4% of AML patients, often as a result of del([7][1])(q22q36) associated with hijacking of a CDK6 enhancer. MNX1 -activated cases show significant co-occurrence with BCOR mutations and a gene signature shared with t(7;12)(q36;p13) pediatric AML. We demonstrated that MNX1 is a dependency gene, as its knockdown in a xenograft model reduces leukemia cell fitness. In conclusion, enhancer hijacking is a frequent mechanism for oncogene activation in AML. Statement of significance This study examines the consequences of structural alterations and demonstrates that proto-oncogene activation by enhancer hijacking is an overlooked pathomechanism in AML. MNX1 overexpression demonstrates that deletions on chromosome 7q can not only lead to haploinsufficiency, but also to activation of oncogenes by enhancer hijacking, providing a novel leukemogenic mechanism. ### Competing Interest Statement UHT is currently employed at Oxford Nanopore Technologies. EJ is currently employed at AstraZeneca. LB has received honoraria from AbbVie, Amgen, Astellas, BristolMyers Squibb, Celgene, Daiichi Sankyo, Gilead, Hexal, Janssen, Jazz Pharmaceuticals, Menarini, Novartis, Pfizer, Roche, and Sanofi, as well as research support from Bayer and Jazz Pharmaceuticals. DBL received honoraria from Infectopharm GmbH. All other authors declared no conflict of interest. [1]: #ref-7

Background: The genomic landscape of acute myeloid leukemia (AML) has been previously mostly studied in younger patients who received intensive chemotherapy. Data in older patients receiving less intensive therapies are scarce. In addition, no genetic risk classification for older AML patients has so far been established. Aims: To characterize the genomic landscape and leukemogenic pathways of AML in older patients, and to study the clinical implications of these biological features. Methods: Targeted sequencing of 263 genes was performed in 604 patients enrolled in the randomized, multi-center phase 3 ‘ASTRAL-1’ trial (NCT02348489) evaluating the second-generation hypomethylating agent guadecitabine (SGI-110) in treatment-naïve AML pts not eligible for intensive chemotherapy in comparison to a treatment choice of decitabine, azacitidine, or low-dose cytarabine. Results: Recurrent mutations were found in ASXL1 (28%), TET2 (27%), SRSF2 (22%), DNMT3A (21%), RUNX1 (20%), TP53 (19%), NPM1 (15%) and FLT3 (13%; [internal tandem duplication (ITD) 8%, tyrosine kinase domain 6%]). DDX41mut were found in 5.5% of the patients, 61% of whom with suspected germline variant. Adverse cytogenetic features such as complex karyotype (29%), -7/del(7q) (22%), 5q/del(5q) (20%), -17/del(17p)/abn(17p) (12%) were common. Based on the 2022 ELN classification, patients were stratified into the adverse (73%), intermediate (14%) or favorable (13%) risk group. Based on the International Consensus Classification (ICC), most patients were classified as AML with myelodysplasia-related gene mutations (45%), AML with mutated TP53 (17%), AML with mutated NPM1 (16%), AML not otherwise specified (10%) and AML with myelodysplasia-related cytogenetic abnormalities (5%). A modelling algorithm yielded a stable oncogenetic tree (Figure 1A) identifying distinct leukemogenic trajectories with ASXL1, DDX41, DNMT3A, TET2 and TP53 mutations as leukemia-initiating events. ASXL1, DNMT3A, and TET2 gave rise to further clones, whereas DDX41 and TP53 terminated at the node, suggesting independence from further events that drive leukemogenesis. Supporting the ICC classification, the ASXL1 subtree contained 8 of 9 genes that define the new category of AML with myelodysplasia-related gene mutations. When assessing clinical impact, both the 2017 and 2022 ELN risk classifications failed to identify clinically meaningful prognostic groups. In Cox regression models, older age (HR 1.02 [1.01-1.04], p=.009), male sex (HR 1.32 [1.09-1.6], p=.004), ECOG score ≥2 (HR 1.55 [1.28-1.88], p<.001), higher white blood cell counts (HR 1.63 [1.34-1.97], p<.001), FLT3-ITD (HR 1.7 [1.2-2.4], p=.003), SRSF2 (HR 1.36 [1.06-1.76], p=.017), and TP53 mutations (HR 1.59 [1.24-2.05], p<.001) had an adverse impact on overall survival, whereas DDX41 mutations (HR 0.41 [0.24-0.69], p<.001) were exceptionally beneficial. To simplify the model, a subsequent backward elimination based on the Akaike information criterion led to delineation of 3 genetically defined risk groups (favorable: DDX41mut, adverse: TP53mut or FLT3-ITDpos, intermediate: all other) with predicted survival curves (Figure 1B). Summary/Conclusion: Using different modelling algorithms, our comprehensive analysis of the so far largest study in older, treatment naïve AML patients identified distinct trajectories of leukemia development, provided support for AML with mutated DDX41 as a new clinico-pathologic entity and a basis for the development of a risk stratification that may be applicable for the numerous older patients receiving less intensive therapies.Keywords: Tumorigenesis, Age, AML, Prognostic groups

T-cell acute lymphocytic leukemia protein 1 (TAL1) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL). Its deregulation can occur through diverse cis-alterations, including SIL-TAL1 microdeletions, translocations with T-cell Receptor loci, and more recently described upstream intergenic non-coding mutations. These mutations consist of recurrent focal microinsertions that create an oncogenic neo-enhancer accompanied by activating epigenetic marks. This observation laid the groundwork for an innovative paradigm concerning the activation of proto-oncogenes via genomic alterations of non-coding intergenic regions. However, for the majority of T-ALL expressing TAL1 (TAL1+), the deregulation mechanism remains 'unresolved'. We took advantage of H3K27ac and H3K4me3 chromatin immunoprecipitation sequencing data of eight cases of T-ALL, including five TAL1+ cases. We identified a putative novel oncogenic neo-enhancer downstream of TAL1 in an unresolved monoallelic TAL1+ case. A rare but recurrent somatic heterozygous microinsertion within this region creates a de novo binding site for MYB transcription factor. Here we demonstrate that this mutation leads to increased enhancer activity, gain of active epigenetic marks, and TAL1 activation via recruitment of MYB. These results highlight the diversity of non-coding mutations that can drive oncogene activation.

To characterize the genomic landscape and leukemogenic pathways of older, newly diagnosed, non-intensively treated patients with AML and to study the clinical implications, comprehensive genetics analyses were performed including targeted DNA sequencing of 263 genes in 604 patients treated in a prospective Phase III clinical trial. Leukemic trajectories were delineated using oncogenetic tree modeling and hierarchical clustering, and prognostic groups were derived from multivariable Cox regression models. Clonal hematopoiesis-related genes (ASXL1, TET2, SRSF2, DNMT3A) were most frequently mutated. The oncogenetic modeling algorithm produced a tree with five branches with ASXL1, DDX41, DNMT3A, TET2, and TP53 emanating from the root suggesting leukemia-initiating events which gave rise to further subbranches with distinct subclones. Unsupervised clustering mirrored the genetic groups identified by the tree model. Multivariable analysis identified FLT3 internal tandem duplications (ITD), SRSF2, and TP53 mutations as poor prognostic factors, while DDX41 mutations exerted an exceptionally favorable effect. Subsequent backwards elimination based on the Akaike information criterion delineated three genetic risk groups: DDX41 mutations (favorable-risk), DDX41wildtype/FLT3-ITDneg/TP53wildtype (intermediate-risk), and FLT3-ITD or TP53 mutations (high-risk). Our data identified distinct trajectories of leukemia development in older AML patients and provide a basis for a clinically meaningful genetic outcome stratification for patients receiving less intensive therapies.

Stabilin-1 (Stab1) and Stabilin-2 (Stab2) are two major scavenger receptors of liver sinusoidal endothelial cells that mediate removal of diverse molecules from the plasma. Double-knockout mice (Stab-DKO) develop impaired kidney function and a decreased lifespan, while single Stabilin deficiency or therapeutic inhibition ameliorates atherosclerosis and Stab1-inhibition is subject of clinical trials in immuno-oncology. Although POSTN and TFGBI have recently been described as novel Stabilin ligands, the dynamics and functional implications of these ligands have not been comprehensively studied. Immunofluorescence, Western Blotting and Simple Western™ as well as in situ hybridization (RNAScope™) and qRT-PCR were used to analyze transcription levels and tissue distribution of POSTN and TGFBI in Stab-KO mice. Stab-POSTN-Triple deficient mice were generated to assess kidney and liver fibrosis and function in young and aged mice. TGFBI and POSTN protein accumulated in liver tissue in Stab-DKO mice and age-dependent in glomeruli of Stabilin-deficient mice despite unchanged transcriptional levels. Stab-POSTN-Triple KO mice showed glomerulofibrosis and a reduced lifespan comparable to Stab-DKO mice. However, alterations of the glomerular diameter and vascular density were partially normalized in Stab-POSTN-Triple KO. TGFBI and POSTN are Stabilin-ligands that are deposited in an age-dependent manner in the kidneys and liver due to insufficient scavenging in the liver. Functionally, POSTN might partially contribute to the observed renal phenotype in Stab-DKO mice. This study provides details on downstream effects how Stabilin dysfunction affects organ function on a molecular and functional level.

Background: Epigenetic enzymes, including histone modifiers and regulators of DNA methylation, are among the most frequent targets for mutations and structural variations in acute myeloid leukemia (AML). However, studies of complex karyotype AML, an aggressive subgroup defined by ≥3 unrelated chromosome abnormalities in the absence of other class-defining genetic abnormalities, have focused largely on its genetic rather than epigenetic characteristics. Within this subgroup, the most common abnormality is a deletion in the long arm of chromosome 5; an early event in leukemogenesis, which is invariably associated with poor clinical outcome and therapy resistance. While several candidate 5q tumor suppressors have been previously investigated, the pathogenic mechanisms underlying this deletion have not yet been elucidated. Aims: Here we aimed to assess the utility of DNA methylation profiles to improve the molecular classification of AML, with a focus on patients with complex karyotype. Methods: A reference-free methylome deconvolution method (MeDeCom) was applied to a cohort of 480 older AML patients which had been profiled by Infinium MethylationEPIC array. This approach allows decomposition of bulk methylomes to reveal a set of latent methylation components (LMCs) that might otherwise be obscured within epigenetically heterogeneous tumors. To decipher their likely origin, each methylation component was then interpreted by comparison to known cell-type-specific methylomes, and association with mutational and cytogenetic features. Results: Deconvolution of the AML methylome identified a distinct hypermethylation signature enriched in del5q AML. This encouraged us to further investigate candidate 5q tumor suppressor genes from an epigenetic perspective. Based on the overlap of deleted 5q segments in own and public datasets, we defined a minimally deleted region within 5q31.2, which peaks at a locus encoding the H3K9me1/2 demethylase, KDM3B. Differential gene expression analysis comparing del5q to 5q-retaining AML identified KDM3B as the most significantly downregulated of all genes within the minimally deleted interval. We thus hypothesized that KDM3B depletion could result in an increase in H3K9me1/2 and consequently trigger de novo DNA methylation at its targets, giving rise to the observed hypermethylation signature. Supporting this model, the del5q methylation signature correlated not only with reduced KDM3B gene expression but also with increased expression of its opposing H3K9me1/2 methyltransferases, EHMT2 (G9a) and PRDM16, and their downstream effector of de novo DNA methylation, DNMT3B. Furthermore, we could support our hypothesis by comparison to AML subgroups whose epigenetic alterations have already been described: Firstly, we note that the del5q methylome shows similarity to IDH mutant AML; a subgroup in which KDM3B activity is inhibited due to its metabolic dependency on alpha-ketoglutarate. Secondly, we found a similar methylation signature in patients overexpressing MECOM (EVI-1), an oncogene which is known to drive DNA hypermethylation through cooperation with DNMT3B. Summary/Conclusion: Our findings suggest that haploinsufficiency of KDM3B might represent an underlying epigenetic event in deletion 5q AML. We hypothesize that the resulting imbalance in H3K9me1/2 could confer epigenetic plasticity that might favor leukemic progression.Keywords: Complex aberrant karyotype, DNA methylation, AML

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Deletions in the long arm of chromosome 7 (del7q) are recurrent events in acute myeloid leukemia (AML) which are associated with an unfavorable outcome. Since the search for recessive tumor suppressor genes located within the deleted region was unsuccessful, the prevalent idea currently is that haploinsufficiency of one or more tumor suppressor genes drive this leukemia. Other studies showed that structural rearrangements such as translocations and inversions can lead to the activation of oncogenes through relocation of cis-regulatory elements. A similar mechanism would also be plausible for chromosomal deletions, specifically del(7q) in AML. Aims: We investigated the hypothesis that del(7q) could activate a proto-oncogene located outside the deleted region due to restructuring of topologically associating domains and relocation of cis-regulatory elements. Methods: Whole genome sequencing (WGS), RNA sequencing (RNAseq) and Infinium MethylationEPIC array were used to study 13 patients with isolated del(7q) or monosomy 7. Epigenomic profiling using antibody-guided chromatin tagmentation with sequencing (ACT-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq) and circular chromosome conformation capture (4C) was done on primary AML samples with MNX1 activation. CRISPR/Cas9 genome editing was used to generate model systems for the putative enhancer hijacking event. Results: We identified a subgroup of del(7q)-AML that aberrantly expresses Motor Neuron and Pancreas Homeobox (MNX1), which is a key developmental homeobox gene located on chromosome 7q36.3 coding for the transcription factor MNX1. MNX1 expression is usually tightly regulated and restricted to the brain, gastrointestinal tract and pancreas. The majority of MNX1-expressing cases had breakpoints within CDK6 and upstream of MNX1, thus, the deletions juxtapose MNX1 with a region containing a strong hematopoietic enhancer. To verify the presence of an enhancer near the breakpoint, we used epigenomic techniques to map accessible chromatin and histone modifications in primary AML. We identified a putative enhancer in CDK6 and demonstrated interaction between the CDK6 region and the MNX1 promoter in these samples. To strengthen the evidence that the enhancer drives MNX1 activation, we inserted the putative enhancer upstream of MNX1 in a human induced pluripotent cell line (hiPSC). Upon differentiation into CD34+ hematopoietic stem and progenitor cells (HSPC), MNX1 was activated in the hiPSC line containing the inserted enhancer. This activation was not observed in the control, i.e. the parental hiPSC line without the inserted enhancer. Summary/Conclusion: We showed that chromosomal deletions in AML can lead to enhancer hijacking events through reshuffling of cis-regulatory elements. Specifically, we showed that MNX1 is activated in a subgroup of del(7q)-AML by CDK6 enhancer. Further validation and insights into the effects of MNX1 activation will be gained from single cells RNA sequencing (scRNA-seq) and single cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) in the hiPSC line with the inserted enhancer fragment. Keywords: Gene regulation, Cytogenetic abnormalities, Acute myeloid leukemia, Epigenetic

Liver sinusoidal endothelial cells (LSECs) control clearance of Transforming growth factor, beta-induced, 68kDa (TGFBi) and Periostin (POSTN) through scavenger receptors Stabilin-1 (Stab1) and Stabilin-2 (Stab2). Stabilin inhibition can ameliorate atherosclerosis in mouse models, while Stabilin-double-knockout leads to glomerulofibrosis. Fibrotic organ damage may pose a limiting factor in future anti-Stabilin therapies. While Stab1-deficient (Stab1-/-) mice were shown to exhibit higher liver fibrosis levels upon challenges, fibrosis susceptibility has not been studied in Stab2-deficient (Stab2-/-) mice. Wildtype (WT), Stab1-/- and Stab2-/- mice were fed experimental diets, and local ligand abundance, hepatic fibrosis, and ligand plasma levels were measured. Hepatic fibrosis was increased in both Stab1-/- and Stab2-/- at baseline. A pro-fibrotic short Methionine-Choline-deficient (MCD) diet induced slightly increased liver fibrosis in Stab1-/- and Stab2-/- mice. A Choline-deficient L-amino acid-defined (CDAA) diet induced liver fibrosis of similar distribution and extent in all genotypes (WT, Stab1-/- and Stab2-/-). A hepatic abundance of Stabilin ligand TGFBi correlated very highly with liver fibrosis levels. In contrast, plasma levels of TGFBi were increased only in Stab2-/- mice after the CDAA diet but not the MCD diet, indicating the differential effects of these diets. Here we show that a single Stabilin deficiency of either Stab1 or Stab2 induces mildly increased collagen depositions under homeostatic conditions. Upon experimental dietary challenge, the local abundance of Stabilin ligand TGFBi was differentially altered in Stabilin-deficient mice, indicating differentially affected LSEC scavenger functions. Since anti-Stabilin-directed therapies are in clinical evaluation for the treatment of diseases, these findings bear relevance to treatment with novel anti-Stabilin agents.

Background: The total number of structural rearrangements in acute myeloid leukemkia (AML) is generally much lower than in other cancer types. However, AML with a complex karyotype (ckAML) is a rare AML subtype (10-15% of all cases) characterized by the presence of at least three cytogenetic alterations and has a dismal prognosis. It is still poorly understood. Many commonly deleted regions have been identified, for example in 5q, 7q, 12p and 17p, but it is still unclear how they may drive leukemia. In parallel, it has been discovered in the last decade that structural rearrangements can lead to aberrant expression of genes, for example of EVI1 in inv(3) or t(3;3), but no systematic search for such events has been undertaken. Since ckAML cases have many structural rearrangements, it is likely that some of them could lead to enhancer hijacking. Aims: We systematically searched for genes, which can be activated by enhancer hijacking in ckAML. Methods: We performed whole genome sequencing (WGS) and RNAseq of 42 ckAML cases. For each gene, we identified samples which had breakpoints located in the same topologically-associating domain, using the WGS. Then, we tested if the samples with breakpoints in the vicinity of the gene had an outlier high expression for this gene. Enhancer hijacking should lead to the expression of only the rearranged allele, so we filtered for genes with monoallelic expression, based on SNPs detected in WGS and RNAseq. Results: We detected many putative enhancer hijacking events in our cohort of 42 ckAML cases. Among the top 20 candidates were three genes, which have already been reported in the literature as being activated by enhancer hijacking in AML: EVI1, BCL11B and MNX1. This supports the reliability of our method. In addition, we detected several novel interesting candidates and we could for some of them find recurrence of these events in other cohorts. Several enhancers were hijacked, including strong hematopoietic enhancers like MYC, GATA2 or CDK6. Summary/Conclusion: We performed for the first time an exhaustive search for enhancer hijacking events in ckAML, and found both known and novel genes. This provides a better understanding of this deadly and poorly understood disease. Since these events are rare, sequencing even larger cohorts could lead to the identification of even more genes and could help estimate the frequency of these events.Keywords: AML, EVI1, TP53, Complex aberrant karyotype

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy resulting from a block in the differentiation of myeloid progenitors and activation of growth promoting genes. In clinics, hypomethylating agents (HMA) such as 5-azacitidine (AZA) or 2-deoxy-5-azacytidine (decitabine) are routinely used to treat patients with AML. Chromosomal rearrangements in AML can lead to aberrant activation of an oncogene by juxtaposition with an activating enhancer, a phenomenon known as enhancer hijacking. However, there is currently no therapeutic agent known to specifically target oncogenic enhancers in AML. Aims: To find an epigenetic inhibitor which abrogates enhancer hijacking leading to oncogenic overexpression of MNX1 (motor neuron and pancreas homeobox 1) in a subset of AML cases. Methods: 174 different epigenetic inhibitors were screened in the AML cell line GDM-1, which carries a translocation t(6;7) and overexpresses MNX1 due to hijacking of an enhancer from the MYB locus on chromosome 6. Candidate inhibitors were identified by cell viability assays. Western blot, real-time quantitative polymerase chain reaction (RT-qPCR) and microRNA (miRNA) sequencing were then applied to analyze changes of MNX1 and miRNA expression, respectively. Results: Top hits included compounds targeting DNMT, BET, KDM6A/6B, P300/CBP proteins and domains found in the SWI/SNF complex. Treatment with the HMA, decitabine (DAC), resulted in significant reduction of MNX1 expression at both RNA and protein level. DAC treatment induced activation of cancer testis antigens (CTAs), long terminal repeats (LTRs). Hypomethylation after DAC treatment will be confirmed through MiSeq of LINE-1. To investigate the mechanism of DAC-dependent MNX1 downregulation, we performed high throughput sequencing of microRNA (miRNA). However, we could not detect upregulated miRNAs upon DAC treatment precluding the contribution of miRNAs on MNX1 downregulation. Summary/Conclusion: We screened 174 epigenetic inhibitors in the AML cell line GDM-1 and showed that DAC treatment significantly reduces MNX1 expression, which is activated by enhancer hijacking. The contribution of activated miRNAs targeting MNX1 and DAC treatment mediated disruption of the topologically associated domain, which allows the enhancer-promoter interaction, are investigated as possible mechanisms for MNX1 expression. Further candidate compound validation will make use of a GDM-1 derivative control cell line without t(6;7) translocation but with ectopic MNX1 expression. Epigenetic inhibitor screening will be repeated in this isogenic cell line. The epigenetic inhibitor, which will not lead to reduction in cell viability of the isogenic line but result in a significant reduction in cell viability of wild type GDM1, will be identified as the inhibitor that disrupts the enhancer-promoter interactions driving oncogenic MNX1 overexpression. This inhibitor may offer clinical benefit in other MNX1-dependent AMLs with enhancer hijacking events.Keywords: Acute myeloid leukemia, decitabine, Epigenetic, Screening

Melanocytic neoplasms have been genetically characterized in detail during the last decade. Recurrent CTNNB1 exon 3 mutations have been recognized in the distinct group of melanocytic tumors showing deep penetrating nevus-like morphology. In addition, they have been identified in 1–2% of advanced melanoma. Performing a detailed genetic analysis of difficult-to-classify nevi and melanomas with CTNNB1 mutations, we found that benign tumors (nevi) show characteristic morphological, genetic and epigenetic traits, which distinguish them from other nevi and melanoma. Malignant CTNNB1-mutant tumors (melanomas) demonstrated a different genetic profile, instead grouping clearly with other non-CTNNB1 melanomas in methylation assays. To further evaluate the role of CTNNB1 mutations in melanoma, we assessed a large cohort of clinically sequenced melanomas, identifying 38 tumors with CTNNB1 exon 3 mutations, including recurrent S45 (n = 13, 34%), G34 (n = 5, 13%), and S27 (n = 5, 13%) mutations. Locations and histological subtype of CTNNB1-mutated melanoma varied; none were reported as showing deep penetrating nevus-like morphology. The most frequent concurrent activating mutations were BRAF V600 (n = 21, 55%) and NRAS Q61 (n = 13, 34%). In our cohort, four of seven (58%) and one of nine (11%) patients treated with targeted therapy (BRAF and MEK Inhibitors) or immune-checkpoint therapy, respectively, showed disease control (partial response or stable disease). In summary, CTNNB1 mutations are associated with a unique melanocytic tumor type in benign tumors (nevi), which can be applied in a diagnostic setting. In advanced disease, no clear characteristics distinguishing CTNNB1-mutant from other melanomas were observed; however, studies of larger, optimally prospective, cohorts are warranted.

Accurate classification of melanocytic tumors is important for prognostic evaluation, treatment and follow-up protocols of patients. The majority of melanocytic proliferations can be classified solely based on clinical and pathological criteria, however in select cases a definitive diagnostic assessment remains challenging and additional diagnostic biomarkers would be advantageous. We analyzed melanomas, nevi, Spitz nevi and atypical spitzoid tumors using parallel sequencing (exons of 611 genes and 507 gene translocation analysis) and methylation arrays (850k Illumina EPIC). By combining detailed genetic and epigenetic analysis with reference-based and reference-free DNA methylome deconvolution we compared Spitz nevi to nevi and melanoma and assessed the potential for these methods in classifying challenging spitzoid tumors. Results were correlated with clinical and histologic features. Spitz nevi were found to cluster independently of nevi and melanoma and demonstrated a different mutation profile. Multiple copy number alterations and TERT promoter mutations were identified only in melanomas. Genome-wide methylation in Spitz nevi was comparable to benign nevi while the Leukocytes UnMethylation for Purity (LUMP) algorithm in Spitz nevi was comparable to melanoma. Histologically difficult to classify Spitz tumor cases were assessed which, based on methylation arrays, clustered between Spitz nevi and melanoma and in terms of genetic profile or copy number variations demonstrated worrisome features suggesting a malignant neoplasm. Comprehensive sequencing and methylation analysis verify Spitz nevi as an independent melanocytic entity distinct from both nevi and melanoma. Combined genetic and methylation assays can offer additional insights in diagnosing difficult to classify Spitzoid tumors.