Histone Acetyltransferase Complex Research Articles

Abstract SY04-03: Identification of SIRT1 as a novel therapeutic target in T-ALL

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy that affects both children and adults. Still, 20%-50% of patients show primary resistance or relapse after treatment, and ultimately die from their disease. Aberrant NOTCH1 signaling has a major role in the pathogenesis of TALL, as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. In this context, small-molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH1 activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH1 signaling, are being tested in clinical trials for the treatment of relapsed and refractory T-ALL. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by limited and delayed therapeutic response to these drugs, underscoring the need to identify novel therapeutic targets and to develop more effective drugs for the treatment of this disease. We previously demonstrated the importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs). Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation. Indeed, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in TALL. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Moreover, through the integration of GSI-washout experiments, epigenetic profiling and CRISPR/Cas9-induced experiments, we have identified a distal enhancer of Sirt1 that is bound and controlled by NOTCH1, which might help explain the broad upregulation of Sirt1 observed in T-ALL patients. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with EX-527 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemias leads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo. Mechanistically, acetyl-proteomics analyses revealed that acute deletion of Sirt1 consistently leads to hyperacetylation of Kat7 and Brd1, which are both part of a histone acetyltransferase complex. Indeed, Sirt1 loss results in global epigenetic changes including decreased levels of H4K12ac, which is a Kat7-target mark. Moreover, gene expression profiling analyses upon Sirt1 loss in leukemia in vivo revealed broad transcriptional changes. Gene-set enrichment analyses revealed that the transcriptional signature upon Sirt1 loss significantly correlates with the one obtained upon Kat7 loss, overall suggesting that Sirt1 loss leads to hyperacetylation of Kat7, which might be less active. Finally, our gene expression analyses also revealed a marked block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, glutaminolysis and TCA, with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal an oncogenic role for Sirt1 in T-ALL generation and progression, demonstrate that Sirt1 contributes to mediate resistance to anti-NOTCH1 therapies, identify a novel Notch1-Sirt1-Kat7 link and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. Citation Format: Daniel Herranz. Identification of SIRT1 as a novel therapeutic target in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY04-03.

Ontology Specific Alternative Splicing Changes in Alzheimer's Disease.

Alternative splicing (AS) is a common phenomenon and correlates with aging and aging-related disorders including Alzheimer’s disease (AD). We aimed to systematically characterize AS changes in the cerebral cortex of 9-month-old APP/PS1 mice. The GSE132177 dataset was downloaded from GEO and ENA databases, aligned to the GRCm39 reference genome from ENSEMBL via STAR. Alternative 3′SS (A3SS), alternative 5′SS (A5SS), skipped exon (SE), retained intron (RI), and mutually exclusive exons (MXE) AS events were evaluated using rMATS, rmats2sashimiplot, and maser. Differential genes or transcripts were analyzed using the limma R package. Gene ontology analysis was performed with the clusterProfiler R package. A total of 60,705 raw counts of AS were identified, and 113 significant AS events were finally selected in accordance with the selection criteria: 1) average coverage >10 and 2) delta percent spliced in (ΔPSI) >0.1. SE was the most abundant AS event (61.95%), and RI was the second most abundant AS type (13.27%), followed by A3SS (12.39%), thereafter A5SS and MXE comprised of 12.39%. Interestingly, genes that experienced SE were enriched in histone acetyltransferase (HAT) complex, while genes spliced by RI were enriched in autophagy and those which experienced A3SS were enriched in methyltransferase activity revealed by GO analysis. In conclusion, we revealed ontology specific AS changes in AD. Our analysis provides novel pathological mechanisms of AD.

Beyond Moco Biosynthesis-Moonlighting Roles of MoaE and MOCS2.

Molybdenum cofactor (Moco) biosynthesis requires iron, copper, and ATP. The Moco-containing enzyme sulfite oxidase catalyzes terminal oxidation in oxidative cysteine catabolism, and another Moco-containing enzyme, xanthine dehydrogenase, functions in purine catabolism. Thus, molybdenum enzymes participate in metabolic pathways that are essential for cellular detoxication and energy dynamics. Studies of the Moco biosynthetic enzymes MoaE (in the Ada2a-containing (ATAC) histone acetyltransferase complex) and MOCS2 have revealed that Moco biosynthesis and molybdenum enzymes align to regulate signaling and metabolism via control of transcription and translation. Disruption of these functions is involved in the onset of dementia and neurodegenerative disease. This review provides an overview of the roles of MoaE and MOCS2 in normal cellular processes and neurodegenerative disease, as well as directions for future research.

Transcriptional Regulation of ING5 and its Suppressive Effects on Gastric Cancer.

ING5 targets histone acetyltransferase or histone deacetylase complexes for local chromatin remodeling. Its transcriptional regulation and suppressive effects on gastric cancer remain elusive. Luciferase assay, EMSA, and ChIP were used to identify the cis-acting elements and trans-acting factors of the ING5 gene. We analyzed the effects of SAHA on the aggressive phenotypes of ING5 transfectants, and the effects of different ING5 mutants on aggressive phenotypes in SGC-7901 cells. Finally, we observed the effects of ING5 abrogation on gastric carcinogenesis. EMSA and ChIP showed that both SRF (−717 to −678 bp) and YY1 (−48 to 25bp) interacted with the promoter of ING5 and up-regulated ING5 expression in gastric cancer via SRF-YY1-ING5-p53 complex formation. ING5, SRF, and YY1 were overexpressed in gastric cancer, (P<0.05), and associated with worse prognosis of gastric cancer patients (P<0.05). ING5 had positive relationships with SRF and YY1 expression in gastric cancer (P<0.05). SAHA treatment caused early arrest at S phase in ING5 transfectants of SGC-7901 (P<0.05), and either 0.5 or 1.0 μM SAHA enhanced their migration and invasion (P<0.05). The wild-type and mutant ING5 transfectants showed lower viability and invasion than the control (P<0.05) with low CDC25, VEGF, and MMP-9 expression. Gastric spontaneous adenocarcinoma was observed in Atp4b-cre; ING5f/f, Pdx1-cre; ING5f/f, and K19-cre; ING5f/f mice. ING5 deletion increased the sensitivity of MNU-induced gastric carcinogenesis. ING5 mRNA might be a good marker of gastric carcinogenesis, and poor prognosis. ING5 expression was positively regulated by the interaction of SRF-YY1-ING5-p53 complex within the ING5 promoter from −50 bp upstream to the transcription start site. ING5 deletion might contribute to the tumorigenesis and histogenesis of gastric cancer.

Oncogenic ZMYND11-MBTD1 fusion protein anchors the NuA4/TIP60 histone acetyltransferase complex to the coding region of active genes.

A recurrent chromosomal translocation found in acute myeloid leukemia leads to an in-frame fusion of the transcription repressor ZMYND11 to MBTD1, a subunit of the NuA4/TIP60 histone acetyltransferase complex. To understand the abnormal molecular events that ZMYND11-MBTD1 expression can create, we perform a biochemical and functional characterization comparison to each individual fusion partner. ZMYND11-MBTD1 is stably incorporated into the endogenous NuA4/TIP60 complex, leading to its mislocalization on the body of genes normally bound by ZMYND11. This can be correlated to increased chromatin acetylation and altered gene transcription, most notably on the MYC oncogene, and alternative splicing. Importantly, ZMYND11-MBTD1 expression favors Myc-driven pluripotency during embryonic stem cell differentiation and self-renewal of hematopoietic stem/progenitor cells. Altogether, these results indicate that the ZMYND11-MBTD1 fusion functions primarily by mistargeting the NuA4/TIP60 complex to the body of genes, altering normal transcription of specific genes, likely driving oncogenesis in part through the Myc regulatory network.

Chromatin Regulators Ahc1p and Eaf3p Positively Influence Nitrogen Metabolism in Saccharomyces cerevisiae.

There is a complex regulatory network of nitrogen metabolism in Saccharomyces cerevisiae, and many details of this regulatory network have not been revealed. This study explored the global regulation of nitrogen metabolism in S. cerevisiae from an epigenetic perspective. Comparative transcriptome analysis of S. cerevisiae S288C treated with 30 nitrogen sources identified nine chromatin regulators (CRs) that responded significantly to different nitrogen sources. Functional analysis showed that among the CRs identified, Ahc1p and Eaf3p promoted the utilization of non-preferred nitrogen sources through global regulation of nitrogen metabolism. Ahc1p regulated nitrogen metabolism through amino acid transport, nitrogen catabolism repression (NCR), and the Ssy1p-Ptr3p-Ssy5p signaling sensor system. Eaf3p regulated nitrogen metabolism via amino acid transport and NCR. The regulatory mechanisms of the effects of Ahc1p and Eaf3p on nitrogen metabolism depended on the function of their histone acetyltransferase complex ADA and NuA4. These epigenetic findings provided new insights for a deeper understanding of the nitrogen metabolism regulatory network in S. cerevisiae.

Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis

Nucleophosmin (NPM1) is a multifunctional histone chaperone that can activate acetylation‐dependent transcription from chromatin templates in vitro. Acetylation of NPM1 by p300 has been shown to further enhance its transcription activation potential. Moreover, its total and acetylated pools are increased in oral squamous cell carcinoma. However, the role of NPM1 or its acetylated form (AcNPM1) in transcriptional regulation in cells and oral tumorigenesis is not fully elucidated. Using ChIP‐seq analyses, we provide the first genome‐wide profile of AcNPM1 and show that AcNPM1 is enriched at transcriptional regulatory elements. AcNPM1 co‐occupies marks of active transcription at promoters and DNase I hypersensitive sites at enhancers. In addition, using a high‐throughput protein interaction profiling approach, we show that NPM1 interacts with RNA Pol II, general transcription factors, mediator subunits, histone acetyltransferase complexes, and chromatin remodelers. NPM1 histone chaperone activity also contributes to its transcription activation potential. Further, NPM1 depletion leads to decreased AcNPM1 occupancy and reduced expression of genes required for proliferative, migratory and invasive potential of oral cancer cells. NPM1 depletion also abrogates the growth of orthotopic tumors in mice. Collectively, these results establish that AcNPM1 functions as a coactivator during during RNA polymerase II‐driven transcription and regulates the expression of genes that promote oral tumorigenesis.

The Non-Specific Lethal (NSL) Histone Acetyltransferase Complex Transcriptionally Regulates Yin Yang 1-Mediated Cell Proliferation in Human Cells.

The human males absent on the first (MOF)-containing non-specific lethal (NSL) histone acetyltransferase (HAT) complex acetylates histone H4 at lysine K5, K8, and K16. This complex shares several subunits with other epigenetic regulatory enzymes, which highlights the complexity of its intracellular function. However, the effect of the NSL HAT complex on the genome and target genes in human cells is still unclear. By using a CRISPR/Cas9-mediated NSL3-knockout 293T cell line and chromatin immunoprecipitation-sequencing (ChIP-Seq) approaches, we identified more than 100 genes as NSL HAT transcriptional targets, including several transcription factors, such as Yin Yang 1 (YY1) which are mainly involved in cell proliferation, biological adhesion, and metabolic processes. We found here that the ChIP-Seq peaks of MOF and NSL3 co-localized with H4K16ac, H3K4me2, and H3K4me3 at the transcriptional start site of YY1. In addition, both the mRNA and protein expression levels of YY1 were regulated by silencing or overexpressing NSL HAT. Interestingly, the expression levels of cell division cycle 6, a downstream target gene of YY1, were regulated by MOF or NSL3. In addition, the suppressed clonogenic ability of HepG2 cells caused by siNSL3 was reversed by overexpressing YY1, suggesting the involvement of YY1 in NSL HAT functioning. Additionally, de novo motif analysis of MOF and NSL3 targets indicated that the NSL HAT complex may recognize the specific DNA-binding sites in the promoter region of target genes in order to regulate their transcription.

Loss of OsEAF6, a Subunit of the Histone Acetyltransferase Complex, Causes Hybrid Breakdown in Intersubspecific Rice Crosses.

Gene duplication plays an important role in genetic diversification, adaptive evolution, and speciation. Understanding the mechanisms and effects of postzygotic isolation genes is important for further studies of speciation and crop breeding. The duplicate recessive genes hwe1 and hwe2 cause hybrid breakdown, characterized by poor vegetative growth and reproductive dysgenesis in intersubspecific crosses between Oryza sativa ssp. indica and japonica. Using a map-based cloning strategy, we found that HWE1 and HWE2 encode the Esa1-associated factor 6 (EAF6) protein, a component of histone acetyltransferase complexes. The indica hwe1 and japonica hwe2 alleles lacked functional EAF6, demonstrating that the double recessive homozygote causes hybrid breakdown. Morphological and physiological observations showed that weak plants with double recessive homozygotes had serious morphological defects with a wide range of effects on development and organs, leading to leaves with reduced chlorophyll content, flower and pistil malformation, and anomalies of gametogenesis. These findings suggest that EAF6 plays a pivotal role in the transcriptional regulation of essential genes during the vegetative and reproductive development of rice.

The Arabidopsis NuA4 histone acetyltransferase complex is required for chlorophyll biosynthesis and photosynthesis.

Although two Enhancer of Polycomb-like proteins, EPL1A and EPL1B (EPL1A/B), are known to be conserved and characteristic subunits of the NuA4-type histone acetyltransferase complex in Arabidopsis thaliana, the biological function of EPL1A/B and the mechanism by which EPL1A/B function in the complex remain unknown. Here, we report that EPL1A/B are required for the histone acetyltransferase activity of the NuA4 complex on the nucleosomal histone H4 in vitro and for the enrichment of histone H4K5 acetylation at thousands of protein-coding genes in vivo. Our results suggest that EPL1A/B are required for linking the NuA4 catalytic subunits HISTONE ACETYLTRANSFERASE OF THE MYST FAMILY 1（HAM1) and HAM2 with accessory subunits in the NuA4 complex. EPL1A/B function redundantly in regulating plant development especially in chlorophyll biosynthesis and de-etiolation. The EPL1A/B-dependent transcription and H4K5Ac are enriched at genes involved in chlorophyll biosynthesis and photosynthesis. We also find that EAF6, another characteristic subunit of the NuA4 complex, contributes to de-etiolation. These results suggest that the Arabidopsis NuA4 complex components function as a whole to mediate histone acetylation and transcriptional activation specifically at light-responsive genes and are critical for photomorphogenesis.

MPTAC links alkylation damage signaling to sterol biosynthesis

Overproduction of reactive oxygen species (ROS) drives inflammation and mutagenesis. However, the role of the DNA damage response in immune responses remains largely unknown. Here we found that stabilization of the mismatch repair (MMR) protein MSH6 in response to alkylation damage requires interactions with the molybdopterin synthase associating complex (MPTAC) and Ada2a-containing histone acetyltransferase complex (ATAC). Furthermore, MSH6 promotes sterol biosynthesis via the mevalonate pathway in a MPTAC- and ATAC-dependent manner. MPTAC reduces the source of alkylating agents (ROS). Therefore, the association between MMR proteins, MPTAC, and ATAC promotes anti-inflammation response and reduces alkylating agents. The inflammatory responses measured by xanthine oxidase activity are elevated in Lymphoblastoid Cell Lines (LCLs) from some Fragile X-associated disorders (FXD) patients, suggesting that alkylating agents are increased in these FXD patients. However, MPTAC is disrupted in LCLs from some FXD patients. In LCLs from other FXD patients, interaction between MSH6 and ATAC was lost, destabilizing MSH6. Thus, impairment of MPTAC and ATAC may cause alkylation damage resistance in some FXD patients.

Interaction of transcription factor FoxO3 with histone acetyltransferase complex subunit TRRAP modulates gene expression and apoptosis

Forkhead box O (FoxO) transcription factors are conserved proteins involved in the regulation of life span and age-related diseases, such as diabetes and cancer. Stress stimuli or growth factor deprivation promotes nuclear localization and activation of FoxO proteins, which—depending on the cellular context—can lead to cell cycle arrest or apoptosis. In endothelial cells (ECs), they further regulate angiogenesis and may promote inflammation and vessel destabilization implicating a role of FoxOs in vascular diseases. In several cancers, FoxOs exert a tumor-suppressive function by regulating proliferation and survival. We and others have previously shown that FoxOs can regulate these processes via two different mechanisms: by direct binding to forkhead-responsive elements at the promoter of target genes or by a poorly understood alternative process that does not require direct DNA binding and regulates key targets in primary human ECs. Here, we performed an interaction study in ECs to identify new nuclear FoxO3 interaction partners that might contribute to FoxO-dependent gene regulation. Mass spectrometry analysis of FoxO3-interacting proteins revealed transformation/transcription domain–associated protein (TRRAP), a member of multiple histone acetyltransferase complexes, as a novel binding partner of FoxO family proteins. We demonstrate that TRRAP is required to support FoxO3 transactivation and FoxO3-dependent G1 arrest and apoptosis in ECs via transcriptional activation of the cyclin-dependent kinase inhibitor p27kip1 and the proapoptotic B-cell lymphoma 2 family member, BIM. Moreover, FoxO–TRRAP interaction could explain FoxO-induced alternative gene regulation via TRRAP-dependent recruitment to target promoters lacking forkhead-responsive element sequences.

Msl3 promotes germline stem cell differentiation in female Drosophila.

Gamete formation from germline stem cells (GSCs) is essential for sexual reproduction. However, the regulation of GSC differentiation is incompletely understood. Set2, which deposits H3K36me3 modifications, is required for GSC differentiation during Drosophila oogenesis. We discovered that the H3K36me3 reader Male-specific lethal 3 (Msl3) and histone acetyltransferase complex Ada2a-containing (ATAC) cooperate with Set2 to regulate GSC differentiation in female Drosophila. Msl3, acting independently of the rest of the male-specific lethal complex, promotes transcription of genes, including a germline-enriched ribosomal protein S19 paralog RpS19b. RpS19b upregulation is required for translation of RNA-binding Fox protein 1 (Rbfox1), a known meiotic cell cycle entry factor. Thus, Msl3 regulates GSC differentiation by modulating translation of a key factor that promotes transition to an oocyte fate.

Involvement of the zebrafish trrap gene in craniofacial development

Trrap (transformation/transcription domain-associated protein) is a component shared by several histone acetyltransferase (HAT) complexes and participates in transcriptional regulation and DNA repair; however, the developmental functions of Trrap in vertebrates are not fully understood. Recently, it has been reported that human patients with genetic mutations in the TRRAP gene show various symptoms, including facial dysmorphisms, microcephaly and global developmental delay. To investigate the physiological functions of Trrap, we established trrap gene-knockout zebrafish and examined loss-of-function phenotypes in the mutants. The trrap zebrafish mutants exhibited smaller eyes and heads than the wild-type zebrafish. The size of the ventral pharyngeal arches was reduced and the mineralization of teeth was impaired in the trrap mutants. Whole-mount in situ hybridization analysis revealed that dlx3 expression was narrowly restricted in the developing ventral pharyngeal arches, while dlx2b expression was diminished in the trrap mutants. These results suggest that trrap zebrafish mutants are useful model organisms for a human disorder associated with genetic mutations in the human TRRAP gene.

Beyond HAT Adaptor: TRRAP Liaisons with Sp1-Mediated Transcription.

The members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family play vital roles in multiple biological processes, including DNA damage response, metabolism, cell growth, mRNA decay, and transcription. TRRAP, as the only member lacking the enzymatic activity in this family, is an adaptor protein for several histone acetyltransferase (HAT) complexes and a scaffold protein for multiple transcription factors. TRRAP has been demonstrated to regulate various cellular functions in cell cycle progression, cell stemness maintenance and differentiation, as well as neural homeostasis. TRRAP is known to be an important orchestrator of many molecular machineries in gene transcription by modulating the activity of some key transcription factors, including E2F1, c-Myc, p53, and recently, Sp1. This review summarizes the biological and biochemical studies on the action mode of TRRAP together with the transcription factors, focusing on how TRRAP-HAT mediates the transactivation of Sp1-governing biological processes, including neurodegeneration.

CBIO-12. KAT5 ACTIVITY CONTROLS GLIOBLASTOMA CELL CYCLE DYNAMICS AND TUMOR GROWTH

Abstract Current standard of care therapy for glioblastoma (GB) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, non-cycling, quiescent-like states (G0 phase cells) are present in both normal tissue and tumors and play important roles in maintaining heterogeneity and cellular hierarchies. The presence of quiescent-like/G0 states therefore represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. However, it remains largely unclear to what degree or by what mechanisms tumor cells enter into or exit from quiescent-like states. To gain insight into how GB cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GB stem-like cells (GSCs) harboring a G0 reporter construct, which is stabilized when cells enter a G0-like state. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, including KAT5 itself. Remarkably, we show that knockout of KAT5 in vitro and in vivo dramatically increases G0 subpopulations in GSC cultures and GSC-induced tumors. Using genetically engineered GSC harboring KAT5 under the control of a Doxycyclin-titratable promoter, we establish that incrementally down regulating KAT5 activity is sufficient to slow cell cycle dynamics causing a build-up G0-like cells; and that partial inhibition of KAT5 leads to extended (mouse) patient survival. Further, in primary tumors, cell-based KAT5 activity assays revealed that high grade tumors harbor larger cell subpopulations with higher KAT5 activity than lower grade tumors. In summary, our results suggest that Tip60/KAT5 activity plays key roles in G0 ingress/egress for GBM tumors, may contribute to tumor progression, and may provide novel therapeutic opportunities.

Sirt1 Is a Novel Therapeutic Target in T-ALL

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy that affects both children and adults. Still, 20%-50% of patients show primary resistance or relapse after treatment, and ultimately die from their disease. Aberrant NOTCH1 signaling has a major role in the pathogenesis of T-ALL, as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. In this context, small-molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH1 activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH1 signaling, are being tested in clinical trials for the treatment of relapsed and refractory T-ALL. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by limited and delayed therapeutic response to these drugs, underscoring the need to identify novel therapeutic targets and to develop more effective drugs for the treatment of this disease. We previously demonstratedthe importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs). Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation. Indeed, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in T-ALL. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Moreover, through the integration of GSI-washout experiments, epigenetic profiling and CRISPR/Cas9-induced experiments, we have identified a distal enhancer of Sirt1 that is bound and controlled by NOTCH1, which might help explain the broad upregulation of Sirt1 observed in T-ALL patients. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with EX-527 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemiasleads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo. Mechanistically, acetyl-proteomics analyses revealed that acute deletion of Sirt1 consistently leads to hyperacetylation of Kat7 and Brd1, which are both part of a histone acetyltransferase complex. Indeed, Sirt1 loss results in global epigenetic changes including decreased levels of H4K12ac, which is a Kat7-target mark. Moreover, gene expression profiling analyses upon Sirt1 loss in leukemia in vivo revealed broad transcriptional changes. Gene-set enrichment analyses revealed that the transcriptional signature upon Sirt1 loss significantly correlates with the one obtained upon Kat7 loss, overall suggesting that Sirt1 loss leads to hyperacetylation of Kat7, which might be less active. Finally, our gene expression analyses also revealed a marked block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, glutaminolysis and TCA, with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal an oncogenic role for Sirt1 in T-ALL generation and progression, demonstrate that Sirt1 contributes to mediate resistance to anti-NOTCH1 therapies, identify a novel Notch1-Sirt1-Kat7 link and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. DisclosuresNo relevant conflicts of interest to declare.

ING Tumour Suppressors and ING Splice Variants as Coregulators of the Androgen Receptor Signalling in Prostate Cancer.

Prevention and overcoming castration resistance of prostate cancer (PC) remains one of the main unsolved problems in modern oncology. Hence, many studies are focused on the investigation of novel androgen receptor (AR) regulators that could serve as potential drug targets in disease therapy. Among such factors, inhibitor of growth (ING) proteins were identified. Some ING proteins act as AR transcriptional coregulators, indicating their relevance for PC research. The ING family consists of five protein-coding genes from ING1 to ING5 and pseudogene INGX. The ING genes were revealed through their sequence homology to the first identified ING1 from an in vivo screen. ING factors are a part of histone modification complexes. With the help of the conserved plant homeodomain (PHD) motif, ING factors bind to Histone 3 Lysine 4 (H3K4) methylation mark with a stronger affinity to the highest methylation grade H3K4me3 and recruit histone acetyltransferases (HAT) and histone deacetylases (HDAC) to chromatin. ING1 and ING2 are core subunits of mSIN3a-HDAC corepressor complexes, whereas ING3–5 interact with different HAT complexes that serve as coactivators. ING members belong to type II tumour suppressors and are frequently downregulated in many types of malignancies, including PC. As the family name indicates, ING proteins are able to inhibit cell growth and tumour development via regulation of cell cycle and cancer-relevant pathways such as apoptosis, cellular senescence, DNA repair, cell migration, invasion, and angiogenesis. Many ING splice variants that enhance the diversity of ING activity were discovered. However, it seems that the existence of multiple ING splice variants is underestimated, since alternative splice variants, such as the AR coregulators ING1 and ING3, counteract full-length ING and thus play an opposite functional role. These results open a novel prospective investigation direction in understanding ING factors biology in PC and other malignancies.

Prognostic Value and Immunological Role of MORF4-Related Gene-Binding Protein in Human Cancers.

MORF4-related gene-binding protein (MRGBP) is the subunit of the NuA4 histone acetyltransferase complex which is involved in transcriptional activation of select genes principally by acetylation of nucleosomal histones H4 and H2A. Much of the research indicated an oncogenic role of MRGBP in the development of cancers. However, it is still unknown the role MRGBP plays in human cancers, which deserves further exploration. In this research, the expression profile, prognostic value of MRGBP, and the relationship between MRGBP and immune infiltration were explored in 33 types of cancer. The differences in MRGBP expression in tumor and normal tissues were explored using data from The Cancer Genome Atlas, Gene Expression Omnibus and ONCOMINE. Analysis of the association between MRGBP and prognosis using Kaplan-Meier survival curve and COX analysis. The data of Tumor mutational burden (TMB), microsatellite instability (MSI) from TCGA. The relationship Between MRGBP expression and immunity was analyzed using the ESTIMATE algorithm and CIBERSORT. Furthermore, we explored MRGBP expression and the relationship between MRGBP expression and macrophage infiltration using immunohistochemical analysis in lower grade glioma (LGG). Our results revealed that MRGBP was highly expressed in most cancer tissues compared with normal tissues. Tumors with increased MRGBP expression had a high clinicopathologic stage and poor prognosis. The expression of MRGBP was closely related to the TMB, MSI. We also found a significant negative correlation between MRGBP expression and stromal scores and immune scores in various types of cancer. Furthermore, MRGBP expression was associated with a variety of immune cells including B cells, NK cells, T cells, and macrophages. LGG and LIHC was selected as representative cancer types for further study, the results of immunohistochemistry indicated that the protein levels of MRGBP were significantly elevated in tumor tissues. Moreover, our LIHC data analysis showed that patients with high MRGBP expression were associated with short survival rates and MRGBP was a risk factor to determine OS. Immunohistochemistry also confirmed that M0 macrophage infiltration in the MRGBP-high group significantly increased. In conclusion, these results reveal that MRGBP can serve as a potential prognostic biomarker and it plays an important role in tumor immune infiltration in various tumors, especially in LGG and LIHC.

Antagonistic relationship of NuA4 with the non-homologous end-joining machinery at DNA damage sites.

The NuA4 histone acetyltransferase complex, apart from its known role in gene regulation, has also been directly implicated in the repair of DNA double-strand breaks (DSBs), favoring homologous recombination (HR) in S/G2 during the cell cycle. Here, we investigate the antagonistic relationship of NuA4 with non-homologous end joining (NHEJ) factors. We show that budding yeast Rad9, the 53BP1 ortholog, can inhibit NuA4 acetyltransferase activity when bound to chromatin in vitro. While we previously reported that NuA4 is recruited at DSBs during the S/G2 phase, we can also detect its recruitment in G1 when genes for Rad9 and NHEJ factors Yku80 and Nej1 are mutated. This is accompanied with the binding of single-strand DNA binding protein RPA and Rad52, indicating DNA end resection in G1 as well as recruitment of the HR machinery. This NuA4 recruitment to DSBs in G1 depends on Mre11-Rad50-Xrs2 (MRX) and Lcd1/Ddc2 and is linked to the hyper-resection phenotype of NHEJ mutants. It also implicates NuA4 in the resection-based single-strand annealing (SSA) repair pathway along Rad52. Interestingly, we identified two novel non-histone acetylation targets of NuA4, Nej1 and Yku80. Acetyl-mimicking mutant of Nej1 inhibits repair of DNA breaks by NHEJ, decreases its interaction with other core NHEJ factors such as Yku80 and Lif1 and favors end resection. Altogether, these results establish a strong reciprocal antagonistic regulatory function of NuA4 and NHEJ factors in repair pathway choice and suggests a role of NuA4 in alternative repair mechanisms in situations where some DNA-end resection can occur in G1.