DBD-α4 helix of EWSR1::FLI1 is required for GGAA microsatellite binding that underlies genome regulation in Ewing sarcoma.

Ewing sarcoma is the second most common bone cancer in children and young adults. In 85% of patients, a translocation between chromosomes 11 and 22 results in a potent fusion oncoprotein, EWSR1::FLI1. EWSR1::FLI1 is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWSR1 portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWSR1::FLI1. The FLI portion of the fusion contains a DNA binding domain shown to bind core GGAA motifs and GGAA repeats. A small alpha-helix in the DNA binding domain of FLI1, DBD-𝛼4 helix, is critical for the transcription function of EWSR1::FLI1. In this study, we aimed to understand the mechanism by which the DBD-𝛼4 helix promotes transcription, and therefore oncogenic transformation. We utilized a multi-omics approach to assess chromatin organization, active chromatinmarks, genome binding, and gene expression in cells expressing EWSR1::FLI1 constructs with and without the DBD-𝛼4 helix. Our studies revealed DBD-𝛼4 helix is crucial for cooperative binding of EWSR1::FLI1 at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWSR1::FLI1 such as formation of TADs, chromatin loops, enhancers and productive transcription hubs.

Seclidemstat blocks the transcriptional function of multiple FET-fusion oncoproteins.

Genes encoding the RNA-binding proteins FUS, EWSR1, and TAF15 (FET proteins) are involved in chromosomal translocations in rare sarcomas. FET-rearranged sarcomas are often aggressive malignancies affecting patients of all ages. New therapies are needed. These translocations fuse the 5' portion of the FET gene with a 3' partner gene encoding a transcription factor (TF). The resulting fusion proteins are oncogenic TFs with a FET protein low complexity domain (LCD) and a DNA binding domain. FET fusion proteins have proven stubbornly difficult to target directly and promising strategies target critical co-regulators. One candidate is lysine specific demethylase 1 (LSD1). LSD1 is recruited by multiple FET fusions, including EWSR1::FLI1. LSD1 promotes EWSR1::FLI1 activity and treatment with the noncompetitive inhibitor SP-2509 blocks EWSR1::FLI1 transcriptional function. A similar molecule, seclidemstat (SP-2577), is currently in clinical trials for FET-rearranged sarcomas (NCT03600649). However, whether seclidemstat has pharmacological activity against FET fusions has not been demonstrated. Here, we evaluate the in vitro potency of seclidemstat against multiple FET-rearranged sarcoma cell lines, including Ewing sarcoma, desmoplastic small round cell tumor, clear cell sarcoma, and myxoid liposarcoma. We also define the transcriptomic effects of seclidemstat treatment and evaluated the activity of seclidemstat against FET fusion transcriptional regulation. Seclidemstat showed potent activity in cell viability assays across FET-rearranged sarcomas and disrupted the transcriptional function of all tested fusions. Though epigenetic and targeted inhibitors are unlikely to be effective as a single agents in the clinic, these data suggest seclidemstat remains a promising new treatment strategy for patients with FET-rearranged sarcomas.

Targeting natural splicing plasticity of APOBEC3B restricts its expression and mutagenic activity.

APOBEC3A (A3A) and APOBEC3B (A3B) enzymes drive APOBEC-mediated mutagenesis. Identification of factors affecting the activity of these enzymes could help modulate mutagenesis and associated clinical outcomes. Here, we show that canonical and alternatively spliced A3A and A3B isoforms produce corresponding mutagenic and non-mutagenic enzymes. Increased expression of the mutagenic A3B isoform predicted shorter progression-free survival in bladder cancer. We demonstrate that the production of mutagenic vs. non-mutagenic A3B protein isoforms was considerably affected by inclusion/skipping of exon 5 in A3B. Furthermore, exon 5 skipping, resulting in lower levels of mutagenic A3B enzyme, could be increased in vitro. Specifically, we showed the effects of treatment with an SF3B1 inhibitor affecting spliceosome interaction with a branch point site in intron 4, or with splice-switching oligonucleotides targeting exon 5 of A3B. Our results underscore the clinical role of A3B and implicate alternative splicing of A3B as a mechanism that could be targeted to restrict APOBEC-mediated mutagenesis.

Evaluating the Causal Link Between Malaria Infection and Endemic Burkitt Lymphoma in Northern Uganda: A Mendelian Randomization Study.

BACKGROUND: Plasmodium falciparum (Pf) malaria infection is suspected to cause endemic Burkitt Lymphoma (eBL), but the evidence remains unsettled. An inverse relationship between sickle cell trait (SCT) and eBL, which supports that between malaria and eBL, has been reported before, but in small studies with low power. We investigated this hypothesis in children in a population-based study in northern Uganda using Mendelian Randomization. METHODS: Malaria-related polymorphisms (SCT, IL10, IL1A, CD36, SEMA3C, and IFNAR1) were genotyped in 202 eBL cases and 624 controls enrolled during 2010-2015. We modeled associations between genotypes and eBL or malaria using logistic regression. FINDINGS: SCT was associated with decreased risk of eBL (adjusted odds ratio [OR] 0·37, 95% CI 0·21-0·66; p=0·0003). Decreased risk of eBL was associated with IL10 rs1800896-CT (OR 0·73, 95% CI 0·50-1·07) and -CC genotypes (OR 0·53, 95% CI 0·29-0·95, ptrend=0·019); IL1A rs2856838-AG (OR 0·56, 95% CI 0·39-0·81) and -AA genotype (OR 0·50, 95% CI 0·28-1·01, ptrend=0·0016); and SEMA3C rs4461841-CT or -CC genotypes (OR 0·57, 95% CI 0·35-0·93, p=0·0193). SCT and IL10 rs1800896, IL1A rs2856838, but not SEMA3C rs4461841, polymorphisms were associated with decreased risk of malaria in the controls. INTERPRETATION: Our results support a causal effect of malaria infection on eBL.