Proteasome-dependent truncation of the negative heterochromatin regulator Epe1 mediates antifungal resistance

Epe1 histone demethylase restricts H3K9-methylation-dependent heterochromatin, preventing it from spreading over, and silencing, gene-containing regions in fission yeast. External stress induces an adaptive response allowing heterochromatin island formation that confers resistance on surviving wild-type lineages. Here we investigate the mechanism by which Epe1 is regulated in response to stress. Exposure to caffeine or antifungals results in Epe1 ubiquitylation and proteasome-dependent removal of the N-terminal 150 residues from Epe1, generating truncated tEpe1 which accumulates in the cytoplasm. Constitutive tEpe1 expression increases H3K9 methylation over several chromosomal regions, reducing expression of underlying genes and enhancing resistance. Reciprocally, constitutive non- cleavable Epe1 expression decreases resistance. tEpe1-mediated resistance requires a functional JmjC demethylase domain. Moreover, caffeine-induced Epe1-to-tEpe1 cleavage is dependent on an intact cell-integrity MAP kinase stress signalling pathway, mutations in which alter resistance. Thus, environmental changes provoke a mechanism that curtails the function of this key epigenetic modifier, allowing heterochromatin to reprogram gene expression, thereby bestowing resistance to some cells within a population. H3K9me-heterochromatin components are conserved in human and crop plant fungal pathogens for which a limited number of antifungals exist. Our findings reveal how transient heterochromatin-dependent antifungal resistant epimutations develop and thus inform on how they might be countered.

Mouse Models of Frequently Mutated Genes in Acute Myeloid Leukemia

Acute myeloid leukemia is a clinically and biologically heterogeneous blood cancer with variable prognosis and response to conventional therapies. Comprehensive sequencing enabled the discovery of recurrent mutations and chromosomal aberrations in AML. Mouse models are essential to study the biological function of these genes and to identify relevant drug targets. This comprehensive review describes the evidence currently available from mouse models for the leukemogenic function of mutations in seven functional gene groups: cell signaling genes, epigenetic modifier genes, nucleophosmin 1 (NPM1), transcription factors, tumor suppressors, spliceosome genes, and cohesin complex genes. Additionally, we provide a synergy map of frequently cooperating mutations in AML development and correlate prognosis of these mutations with leukemogenicity in mouse models to better understand the co-dependence of mutations in AML.

Venetoclax and Epigenetic Modifiers: Promising Novel Combinations for the Treatment of Multiple Myeloma

Multiple Myeloma (MM) is a haematological malignancy characterised by clonal proliferation of plasma cells within the bone marrow (Landgren and Weiss, 2009). Unfortunately, despite the major improvements to the treatment of MM within the last decade, it remains an incurable disease. Therefore, novel innovative combinations of less toxic therapies are warranted, especially for elderly patients with relapsed/refractory disease. The anti-apoptotic BCL-2 family of proteins (BCL-2, BCL-XL and MCL-1) are critical regulators of the intrinsic apoptotic pathway and determine the survival of human MM cells (Letai et al., 2004, Del Gaizo Moore et al., 2008). Recently, Venetoclax, a selective BCL-2 inhibitor, was FDA approved for the treatment of chronic lymphocytic leukaemia (CLL) and acute mylogenous leukemia (AML), in combination with demethylating agents (Roberts et al., 2016, DiNardo et al., 2018, DiNardo et al., 2019) It is known that some MM patients with t(11;14) have a good response to combination treatment with venetoclax, however certain patients who do not have t(11;14) also respond to venetoclax. Therefore, a biomarker for response is urgently required in MM, as it has heterogeneous anti-apoptotic dependencies. In AML, venetoclax is combined with the epigenetic modifier 5-azacytidine. Highlighting, that screening for epigenetic modifier's, maybe a useful approach to identify synergistic combination of treatments with venetoclax in MM. Methods: To assess anti-apoptotic protein dependence in MM cell lines (JJN3, RPMI-8226, KMS18, MM1S, U266) and primary patient samples, BH3 profiling was used. Briefly, cells are exposed to a series of BH3-only peptides (20-23 mer in length) following gentle permeabilisation of cell membrane with low concentrations of digitonin. The loss of mitochondrial potential (JC-1) or the release of cytochrome c (cytochrome-c-FITC antibody) was assessed by plate reader or by flow cytometry. Cell death was assessed by Annexin V/ propidium iodide staining by flow cytometry. Using primary patient samples, CD138 + cells were isolated using the Miltenyi MAC sorter. For the epigenetic screen cell viability was assessed by CellTiter-Glo® and death was then confirmed by Annexin V/Pi staining. Results BH3 profiling was used to assess anti-apoptotic dependence in a panel of five MM cell lines. It is a functional assay that interrogates BCL-2 protein interactions using synthetic BH3 peptides to measure the loss of mitochondrial membrane potential. The BH3 profiling was correlated to the response of the cell lines to a series of BH3 mimetics : venetoclax (selective BCL-2 inhibitor), ABT-263 (BCL-2, BCL-XL and BCL-W inhibitor), WEHI-539 (BCL-XL inhibitor) and AMG-176 (MCL-1 inhibitor). This data highlighted that BH3 profiling is a powerful tool for identifying anti-apoptotic dependnece in MM. It also showed and that there is a diverse anti-apoptotic dependence in MM cell lines and primary patient samples. Remarkably, one patient with plasma cell leukemia showed BCL-2 dependence by BH3 profiling, a t(11:14) translocation and a sustained in-vivo single agent response to venetoclax (Glavey et al., 2020).. Next, we performed an unbiased epigenetic modifier screen in two MM cell lines JJN3 (MCL-1 dependent cell lines) and KMS-18 (mixed anti-apoptotic dependence) to induce BCL-2 dependence and sensitivity to venetoclax. The screen included the following classes of epigenetic modifiers: histone deacetylase inhibitors, histone methyltransferase inhibitors, DNA methyltransferase inhibitors and histone acetylase inhibitors. Interestingly, two classes of epigenetic drugs were synergistic with venetoclax in three different MM cell lines (CI <0.8). The combination was also validated by dynamic BH3 profiling in cell lines MM patient samples ex-vivo. Further work is required, in the form of a clinical trial, to validate our hypothesis that this provides a highly effective novel combination therapy option for myeloma patients. Conclusion. BH3 profiling is a powerful tool to identify the anti-apoptotic dependence in MM cell lines and MM patient samples, which we can exploit pharmacologically to kill MM cells in a personalised medicine approach. Combining epigenetic modifiers with venetoclax induces BCL-2 dependence in MM and enhances response to treatment. Figure 1 Figure 1. Disclosures Flanagan: AbbVie: Research Funding. O'Dwyer: Bristol Myers Squibb: Research Funding; ONK Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Quinn: Takeda: Honoraria. Glavey: Janssen: Honoraria, Research Funding; Celgene and BMS company: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding. Ni Chonghaile: AbbVie: Research Funding.

Clonal Relapse Dynamics in Acute Myeloid Leukemia Following Allogeneic Hematopoietic Cell Transplantation

Introduction The 2-year survival for AML patients relapsing after allogeneic hematopoietic cell transplantation (alloHCT) is <20%, independent of the choice of relapse-treatment. Relapse detection in its molecular state enables early interventions and possibly prevention of hematological recurrence of the disease. The role of measurable residual disease (MRD) monitoring for risk stratification has been described for pre and post-alloHCT MRD analyses. Yet, it remains unclear, if and by which lead-time NGS assessment can detect MRD before impending relapse. We hypothesize that the functional class of mutations determines the relapse kinetics in AML after alloHCT. Methods We identified mutations present at AML relapse after alloHCT by Illumina myeloid panel sequencing covering 48 AML associated genes. Peripheral whole blood samples were retrospectively collected before hematological relapse, with a minimum of one sample per patient at three months prior to relapse and if available, additional monthly samples. Amplicon-based NGS and bioinformatics error-correction were performed on those samples as described in Thol et al. 2018. Positive MRD was defined as MRD detectable above the limit of detection. In the last step, we performed polynomic curve interpolation to model relapse dynamics. Results MRD was assessed in 75 AML patients after alloHCT using 203 AML-related mutations present at the time of relapse, corresponding to a median of 2.7 trackable mutations per patient (range 1-7). In total, 305 MRD analyses were performed from peripheral blood (median 1.5 per mutation, range 1-5) prior to relapse. VAFs measured above the limit of detection (median LOD across all targets 0.0315) ranged from 0.0048-26% (median 1.3%). In 45 of 75 patients (60%), we detected MRD in at least one sample and one marker before relapse. Of those, 23 patients (51%) were MRD positive in all markers before relapse and 22 patients (49%) were MRD positive in some, but not all markers before relapse. The majority of MRD-positive patients (30 of 45) were first detected three or fewer months before relapse, whereas 15 (33%) of 45 patients were MRD positive more than 3 months before relapse. The median time to relapse from the first MRD-positive sample to relapse was 2.9 months (range 0.6-10.2). Among the 203 mutations found in relapse, 93 (46%) were detectable by MRD monitoring before relapse while the remaining 110 markers (54%) remained undetectable prior to relapse. Of note, 88 of those 110 markers (80%) were measured only once before relapse, indicating that frequent sampling increases the likelihood of MRD detection. Genes in which mutations were found mostly MRD-positive were TET2 (6 out of 6), ASXL2 (4 out of 5), SF3B1 (4 out of 5), and RUNX1 (7 out of 9). Mutations in WT1 (1 out of 13), NRAS (1 out of 8), FLT3-ITD (9 out of 29), and PTPN11 (1 out of 5) were among the most common MRD negative mutations before relapse. To assess clonal relapse dynamics, pre-relapse samples were assigned to the monthly interval that best matched the sampling time. If MRD was measured positive at one time point, all the following monthly intervals were considered MRD-positive, whether a sample was available for that interval or not. The fraction of positive samples from all samples per time point was plotted against time to relapse and the function was approximated by fifth-order polynomials. The percentage of patients being MRD positive increased markedly with shortened distance to relapse. Thus, 29% of patients were MRD positive at 3 months, 44% at 2 months and 66% 1 month prior to relapse. Summarized by functional gene classes, mutations in tumor suppressor genes and especially signaling genes showed a higher slope and thus a shorter lead-time to relapse than mutations in epigenetic modifier genes (Figure 2). Conclusion In summary, hematologic relapse can be detected in peripheral blood in 29, 44, and 66% of patients at 3, 2, and 1 months before relapse by NGS-MRD analysis, respectively. Mutations in epigenetic modifier genes show a higher fraction of MRD positivity before relapse than other mutations. In contrast, mutations in signaling genes show a shorter lead-time to relapse. Figure 1 Figure 1. Disclosures Ganser: Celgene: Honoraria; Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria. Thol: Abbvie: Honoraria; Astellas: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Jazz: Honoraria; BMS/Celgene: Honoraria, Research Funding. Heuser: BergenBio: Research Funding; Bayer Pharma AG: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding.

TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis

Mutations in the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). Here, we investigate associations between TET2 mutations and DNA methylation in whole blood in 305 elderly twins, 15 patients with CCUS and 18 healthy controls. We find that TET2 mutations are associated with DNA hypermethylation at enhancer sites in whole blood in CHIP and in both granulocytes and mononuclear cells in CCUS. These hypermethylated sites are associated with leukocyte function and immune response and ETS-related and C/EBP-related transcription factor motifs. While the majority of TET2-associated hypermethylation sites are shared between CHIP and in AML, we find a set of AML-specific hypermethylated loci at active enhancer elements in hematopoietic stem cells. In summary, we show that TET2 mutations is associated with hypermethylated enhancers involved in myeloid differentiation in both CHIP, CCUS and AML patients.

Transcriptional Regulation of the Y-Linked Mammalian Testis-Determining Gene SRY

Mammalian male sex differentiation is triggered during embryogenesis by the activation of the Y-linked testis-determining gene <i>SRY</i>. Since insufficient or delayed expression of <i>SRY</i> results in XY gonadal sex reversal, accurate regulation of <i>SRY</i> is critical for male development in XY animals. In humans, dysregulation of <i>SRY</i> may cause disorders of sex development. Mouse <i>Sry</i> is the most intensively studied mammalian model of sex determination. <i>Sry</i> expression is controlled in a spatially and temporally stringent manner. Several transcription factors play a key role in sex determination as trans-acting factors for <i>Sry</i> expression. In addition, recent studies have shown that several epigenetic modifications of <i>Sry</i> are involved in sex determination as cis-acting factors for <i>Sry</i> expression. Herein, we review the current understanding of transcription factor- and epigenetic modifier-mediated regulation of <i>SRY</i>/<i>Sry</i> expression.

Precursor-Directed Biosynthesis of Aminofulvenes: New Chalanilines from Endophytic Fungus Chalara sp.

The plant endophyte Chalara sp. is able to biotransform the epigenetic modifier vorinostat to form unique, aniline-containing polyketides named chalanilines. Here, we sought to expand the chemical diversity of chalaniline A-type molecules by changing the aniline moiety in the precursor vorinostat. In total, twenty-three different vorinostat analogs were prepared via two-step synthesis, and nineteen were incorporated by the fungus into polyketides. The highest yielding substrates were selected for large-scale precursor-directed biosynthesis and five novel compounds, including two fluorinated chalanilines, were isolated, purified, and structurally characterized. Structure elucidation relied on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry. All compounds were tested for their bioactivity but were not active in antimicrobial or cell viability assays. Aminofulvene-containing natural products are rare, and this high-yielding, precursor-directed process allows for the diversification of this class of compounds.

A Role of Alkbh5 at the Early Stage of Cell Differentiation in C2C12 Cells

Objectives N6-methyladenosine(m6A) is one of the most abundant mRNA modifications, controlling mRNA metabolism including splicing, stabilization, and translation efficiency. Although it has been reported that m6A was involved in cellular differentiation process, its underlying molecular mechanism is not fully understood. The object of this study was to discover ALKBH5, a m6A demethylase, as a key regulator in early myogenesis and adipogenesis using C2C12 model. Methods To identify the epigenetic modifier, we compared publicly available GEO data sets from two independent adipogenic and myogenic differentiation models. M6A levels of mRNAs were measured by dot blot assays upon differentiation. C2C12 cells were maintained under myogenesis or adipogenesis media(±rosiglitazone) for 5–8 days. Alkbh5 knockdown using siRNA and overexpression using GFP-tagged cDNA plasmid were transfected into cells 2 days before confluence. Gene expression analysis was done by qRT-PCR, following RNA isolation and conversion into cDNA. Oil-red-O staining was conducted to detect lipid droplets. Myotube length and width were measured by giemsa staining. Results We found Alkbh5 as a common epigenetic modifier, expression of which was significantly different in both early myogenic and adipogenic cells, compared to undifferentiated cells. Consistently, m6A levels, measured by dot blots, decreased during myogenesis(day 2), while increased during adipogenesis(24h). To examine if modulation of Alkbh5 expression affected cell differentiation, we conducted both Alkbh5 knockdown and overexpression in C2C12 myoblasts, followed by differentiating stimulation into myotube and adipocyte by appropriate media. Knockdown of Alkbh5 facilitates lipid accumulation, while overexpression of Alkbh5 represents significant increase in myotube length and width. Expression of myogenesis and adipogenesis markers were consistent with the phenotypes. Conclusions These results suggested that Alkbh5 had an important role in determination of cell fates towards myogenesis over adipogenesis at the early point of cell differentiation. Funding Sources This study was supported by the National Research Foundation of Korea the Korean National Cancer Center. JYC was supported by Brain Korea Four Project(Education Research Center for 4IR-Based Health Care).