B Cell Receptor Signaling Drives APOBEC3 Expression Via Direct Enhancer Regulation in Chronic Lymphocytic Leukemia B Cells

Constitutively activated B cell receptor (BCR) signaling is a primary biological feature of chronic lymphocytic leukemia (CLL). The biological events controlled by BCR signaling in CLL are not fully understood and need investigation. To make inroads we obtained blood samples from CLL patients before and after Bruton's tyrosine kinase inhibitors (BTKi) treatment and used them to study BCR signaling regulated genes. Here, by analysis of the chromatin states and gene expression profiles of CLL B cells from patients before and after BTKi ibrutinib treatment, we show that BTKi treatment leads to a decreased expression of APOBEC3 family genes in an enhancer regulation dependent manner. BTKi treatment reduces enrichment of enhancer markers (H3K4me1, H3K27ac) and chromatin accessibility at putative APOBEC3 enhancers. CRISPR-Cas9 directed deletion or inhibition of the putative APOBEC3 enhancers leads to reduced APOBEC3 expression. We further find that transcription factor NFATc1 couples BCR signaling with the APOBEC3 enhancer activity to control APOBEC3 expression. Importantly, enhancer regulated APOBEC3 expression contributes to replication stress in malignant B cells. We also demonstrate a novel mechanism for BTKi suppression of APOBEC3 expression via direct enhancer regulation in a NFATc1 dependent manner, implicating BCR signaling as a potential regulator of leukemic genomic instability.

Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif

Transcriptional regulation by Wnt signalling is primarily thought to be accomplished by a complex of β-catenin and TCF family transcription factors (TFs). Although numerous studies have suggested that additional TFs play roles in regulating Wnt target genes, their mechanisms of action have not been investigated in detail. We characterised a Wnt-responsive element (WRE) downstream of the Wnt target gene Axin2 and found that TCFs and Caudal-related homeodomain (CDX) proteins were required for its activation. Using a new separation-of-function TCF mutant, we found that WRE activity requires the formation of a TCF/CDX complex. Our systematic mutagenesis of this enhancer identified other sequences essential for activation by Wnt signalling, including several copies of a novel CAG DNA motif. Computational and experimental evidence indicates that the TCF/CDX/CAG mode of regulation is prevalent in multiple WREs. Put together, our results demonstrate the complex nature of cis- and trans- interactions required for signal-dependent enhancer activity.

Lysine Demethylase KDM6A in Differentiation, Development, and Cancer

ABSTRACT Lysine demethylase 6A (KDM6A), also known as UTX, belongs to the KDM6 family of histone H3 lysine 27 (H3K27) demethylases, which also includes UTY and KDM6B (JMJD3). The KDM6A protein contains six tetratricopeptide repeat (TPR) domains and an enzymatic Jumonji C (JmjC) domain that catalyzes the removal of di- and trimethylation on H3K27. KDM6A physically associates with histone H3 lysine 4 monomethyltransferases MLL3 (KMT2C) and MLL4 (KMT2D). Since its identification as an H3K27 demethylase in 2007, studies have reported KDM6A’s critical roles in cell differentiation, development, and cancer. KDM6A is important for differentiation of embryonic stem cells and development of various tissues. Mutations of KDM6A cause Kabuki syndrome. KDM6A is frequently mutated in cancers and functions as a tumor suppressor. KDM6A is redundant with UTY and functions largely independently of its demethylase activity. It regulates gene expression, likely through the associated transcription factors and MLL3/4 on enhancers. However, KDM6A enzymatic activity is required in certain cellular contexts. Functional redundancy between H3K27 demethylase activities of KDM6A and KDM6B in vivo has yet to be determined. Further understanding of KDM6A functions and working mechanisms will provide more insights into enhancer regulation and may help generate novel therapeutic approaches to treat KDM6A-related diseases.

Enhancer regulation for induced WNT3A expression during neuronal regeneration

The treatment of traumatic brain injury (TBI) is limited by a lack of knowledge about the mechanisms underlying neuronal regeneration. WNT family members have been implicated in neurogenesis and aberrant WNT signaling has been associated with neurodegenerative diseases. The current study compared the expression of WNT genes during regeneration of injured cortical neurons. Recombinant WNT3A showed positive effect in promoting neuronal regeneration via in vitro and in vivo TBI models. Intranasal administration of WNT3A protein to TBI mice increased NeuN+ cells compared to control mice as well as retained motor function based on behavior analysis. Since TBI is known to reprogram the epigenome, chromatin immunoprecipitation-sequencing of histone H3K27ac and H3K4me3 was performed to address the transcriptional regulation of WNT3A during neuronal regeneration. We predicted, characterized and proposed that a histone H3K4me1-marked enhancer may undergo topological transformation to regulate the WNT3A gene expression.

An Oncogenic Enhancer Encodes Selective Selenium Dependency in AML

Deregulation of transcription is a hallmark of acute myeloid leukemia (AML) that drives oncogenic expression programs and presents opportunities for therapeutic targeting. We hypothesized that by integrating the active enhancer chromatin landscapes with pan-cancer enhancer and genetic dependency mapping, we would be able to identify targetable tumor-specific oncogenic enhancer regulation and gain mechanistic insights into the underlying basis of the regulation. Using this approach, we find that tumor-specific super enhancers statistically demarcate tumor-specific dependency (Fig. A). In addition to several well-known AML oncogenes (e.g. MYB, CDK6, FLI1…), we find SEPHS2 to have highly AML-specific enhancer regulation and genetic dependency (Fig. B). SEPHS2 is a key component of the selenoprotein production pathway and is required for the selenocysteine incorporation during protein translation (Fig. C). Across AML cell lines and primary patient samples, we observe a large AML-specific super enhancer at the SEPHS2 locus (Fig. D) that correlates with SEPHS2 genetic dependency (Fig. E) and includes a prominent binding site for AML transcription factors (Fig. D). We confirmed that the oncogenic transcription factor MYB strongly regulates SEPHS2 expression and that SEPHS2 expression correlates with poor prognosis in AML (not shown in this figure). Selenoproteins play an important role in mediating oxidative stress. We find that SEPHS2 knockout increases oxidative stress and that antioxidant treatment rescues viability effects of SEPHS2 knockout (Fig. F, G). Across murine and human in vivo AML models, genetic perturbation of selenoprotein production pathway genes strongly delays leukemogenesis (one example in Fig. H). Other cell lines (both cancerous and non-cancerous) are minimally affected by SEPHS2 knockout, confirming specificity in AML (Fig. E). As a druggable enzyme SEPHS2 merits strong consideration as a therapeutic target. In the interim, we hypothesized that selenium dietary restriction (Fig. I) may be able to phenocopy selenoprotein pathway inhibition with a lower regulatory burden. We show that mice tolerate selenium-depleted chow with no observable effects on body weight or hematopoiesis (one example in Fig. J). Confirming our hypothesis, AML transplanted into these mice exhibit a strong delay in leukemogenesis (Fig. K). Throughout the cancer biology field, there is broad interest in understanding how best to leverage pan-cancer genetic dependency data. We find that the integration of enhancer data adds another layer of specificity and helps provide mechanistic insight into the underlying basis of oncogenic deregulation and dependency. Our identification of AML-specific enhancer regulation of selenoprotein production - which has been minimally studied in this disease - validates our unbiased approach and points to selenoprotein production as a deregulated and therapeutically-actionable metabolic axis in AML. Figure Disclosures Lin: Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.

Chromatin landscapes reveal developmentally encoded transcriptional states that define human glioblastoma

Glioblastoma is an incurable brain cancer characterized by high genetic and pathological heterogeneity. Here, we mapped active chromatin landscapes with gene expression, whole exomes, copy number profiles, and DNA methylomes across 44 patient-derived glioblastoma stem cells (GSCs), 50 primary tumors, and 10 neural stem cells (NSCs) to identify essential super-enhancer (SE)–associated genes and the core transcription factors that establish SEs and maintain GSC identity. GSCs segregate into two groups dominated by distinct enhancer profiles and unique developmental core transcription factor regulatory programs. Group-specific transcription factors enforce GSC identity; they exhibit higher activity in glioblastomas versus NSCs, are associated with poor clinical outcomes, and are required for glioblastoma growth in vivo. Although transcription factors are commonly considered undruggable, group-specific enhancer regulation of the MAPK/ERK pathway predicts sensitivity to MEK inhibition. These data demonstrate that transcriptional identity can be leveraged to identify novel dependencies and therapeutic approaches.