Transcriptional Repressor Complex Research Articles

Ang-1 and VEGF: central regulators of angiogenesis.

Angiopoietin-1 (Ang-1) and Vascular Endothelial Growth Factor (VEGF) are central regulators of angiogenesis and are often inactivated in various cardiovascular diseases. VEGF forms complexes with ETS transcription factor family and exerts its action by downregulating multiple genes. Among the target genes of the VEGF-ETS complex, there are a significant number encoding key angiogenic regulators. Phosphorylation of the VEGF-ETS complex releases transcriptional repression on these angiogenic regulators, thereby promoting their expression. Ang-1 interacts with TEK, and this phosphorylation release can be modulated by the Ang-1-TEK signaling pathway. The Ang-1-TEK pathway participates in the transcriptional activation of VEGF genes. In summary, these elements constitute the Ang-1-TEK-VEGF signaling pathway. Additionally, Ang-1 is activated under hypoxic and inflammatory conditions, leading to an upregulation in the expression of TEK. Elevated TEK levels result in the formation of the VEGF-ETS complex, which, in turn, downregulates the expression of numerous angiogenic genes. Hence, the Ang-1-dependent transcriptional repression is indirect. Reduced expression of many target genes can lead to aberrant angiogenesis. A significant overlap exists between the target genes regulated by Ang-1-TEK-VEGF and those under the control of the Ang-1-TEK-TSP-1 signaling pathway. Mechanistically, this can be explained by the replacement of the VEGF-ETS complex with the TSP-1 transcriptional repression complex at the ETS sites on target gene promoters. Furthermore, VEGF possesses non-classical functions unrelated to ETS and DNA binding. Its supportive role in TSP-1 formation may be exerted through the VEGF-CRL5-VHL-HIF-1α-VH032-TGF-β-TSP-1 axis. This review assesses the regulatory mechanisms of the Ang-1-TEK-VEGF signaling pathway and explores its significant overlap with the Ang-1-TEK-TSP-1 signaling pathway.

Abstract ND05: The discovery of ARV-393, a potent, orally bioavailable BCL6 targeting PROTAC® for the treatment of Non-Hodgkin’s Lymphoma

Abstract Discovery of ARV-393, a potent, orally bioavailable BCL6 targeting PROTAC® for the treatment of Non-Hodgkin’s Lymphoma (NHL)Dan Sherman, Sheryl M. Gough, Lynn DeCarr, Sarah Eaton, Mark Bookbinder, Jennifer Pizzano, Elizabeth Bortolon, John Corradi, Rashaun Wilson, Daniel Rogoz, Ram Lingamaneni, Wei Zhang, Michelle Zhang, Xin Chen, Gan Wang, Hanqing Dong, Michael Berlin, Keith R. Hornberger, Lawrence Snyder, Achal Pashine and Ian Taylor. Arvinas, Inc., 5 Science Park, New Haven, CT, USA. Approximately 18,000 cases of Diffuse large B-cell lymphomas (DLBCL) are diagnosed worldwide per year making this cancer the most prevalent (30%) of the non-Hodgkin’s Lymphomas (NHL). Despite the success of R-CHOP treatment, approximately 40% of patients are refractory or relapse. While much progress has been made with CAR-Ts and bi-specific antibodies following frontline therapy in recent years, there remains a need for an orally bioavailable small-molecule that targets molecular drivers of NHL. The BCL6 transcription factor has long been known as an oncogenic driver of B-cell malignancy. The BCL6 gene has been found to be translocated and/or mutated resulting in deregulation of its expression, predominantly in DLBCL and Burkitt’s lymphoma (BL). The deregulated expression and scaffolding role of BCL6 in the transcriptional repressor complex make it an ideal target for the PROteolysis Targeting Chimera (PROTAC®) technology with one BCL6 degrader recently entering Phase 1 trials. We undertook a medicinal chemistry campaign to identify a BCL6-targeting PROTAC®. This effort identified ARV-393, an orally bioavailable PROTAC® that recruits BCL6 and the E3 ligase cereblon to rapidly degrade BCL6 via the cell’s natural ubiquitin proteasome system. ARV-393 is potent, demonstrating DC50 and GI50 values <1 nM in numerous DLBCL and BL cell lines. This agent demonstrates deep BCL6 degradation in vitro and in vivo, resulting in excellent TGI in several tumor xenograft models supporting a first-in-human trial in 1H 2024. We will present the chemical structure of ARV-393 and selected pre-clinical data. Citation Format: Dan Sherman. The discovery of ARV-393, a potent, orally bioavailable BCL6 targeting PROTAC® for the treatment of Non-Hodgkin’s Lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND05.

Abstract 6055: COOTO-Me (10e) acts as a molecular glue to target NCOR/HDAC3 degradation

Abstract Histone deacetylase 3 (HDAC3) is overexpressed in cancer cells and functions as transcription repression complex with nuclear receptor corepressor (NCOR) to inhibit gene transcription. The pan-HDAC inhibitors have been approved for T-cell lymphoma with evident side effects. The selective HDAC3 inhibitors needs to be developed. Proteolysis-targeting chimera (PROTAC) approach is taken to design HDAC3 degraders with unsolved bioavailability obstacle. We screened small molecules with HDAC3 degradation ability and found two small molecules derived from glycyrrhetinic acid, methyl 2-cyano-3-oxo-18β-olean-1,9(11), 12-trien-30-oate (COOTO, 10e) and methyl 2-cyano-3,12-dioxo-18β-olean-1,9(11)-dien-30-oate (CDODO-Me, 10d), selectively decreased HDAC3 protein with increased acetylation of Histone 3. We explored the mechanism of HDAC3 degradation and found that 10e interrupted the interaction of HDAC3 with NCOR and induced degradation of both HDAC3 and NCOR protein. Biotin labeled 10e shows binding to both HDAC3 and NCOR directly as well as to E3 ligases SIAH2 and ITCH. Mass spectrometry analysis profiling reveals covalent interactions of 10e with multiple cysteine sites of SIAH2 in the binding regions for NCOR and HDAC3. Silencing SIAH2 blocks degradation of HDAC3. N-Acetylcysteine and GSH block the binding of 10e to HDAC3, NCOR and SIAH2. In addition, 10e decreased the levels of c-FLIP protein and increased the levels of NOXA protein leading to apoptosis induction. These data suggest that 10e functions as a molecular glue to attract SIAH2 to HDAC3 and NCOR to cause degradation through covalent binding. Citation Format: Ping Gong, Min Huang, Xin Li, Haoling Wang, Samuel Waxman, Linxiang Zhao, Yongkui Jing. COOTO-Me (10e) acts as a molecular glue to target NCOR/HDAC3 degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6055.

Set7/9 aggravates ischemic brain injury via enhancing glutamine metabolism in a blocking Sirt5 manner.

The aberrant expression of methyltransferase Set7/9 plays a role in various diseases. However, the contribution of Set7/9 in ischemic stroke remains unclear. Here, we show ischemic injury results in a rapid elevation of Set7/9, which is accompanied by the downregulation of Sirt5, a deacetylase reported to protect against injury. Proteomic analysis identifies the decrease of chromobox homolog 1 (Cbx1) in knockdown Set7/9 neurons. Mechanistically, Set7/9 promotes the binding of Cbx1 to H3K9me2/3 and forms a transcription repressor complex at the Sirt5 promoter, ultimately repressing Sirt5 transcription. Thus, the deacetylation of Sirt5 substrate, glutaminase, which catalyzes the hydrolysis of glutamine to glutamate and ammonia, is decreased, promoting glutaminase expression and triggering excitotoxicity. Blocking Set7/9 eliminates H3K9me2/3 from the Sirt5 promoter and normalizes Sirt5 expression and Set7/9 knockout efficiently ameliorates brain ischemic injury by reducing the accumulation of ammonia and glutamate in a Sirt5-dependent manner. Collectively, the Set7/9-Sirt5 axis may be a promising epigenetic therapeutic target.

In Vitro Determination of Temperature-Dependent DNA Binding of the Evening Complex Using Electrophoretic Mobility Shift Assays.

Electrophoretic mobility shift assays (EMSAs) of DNA-binding proteins and labeled DNA allow the qualitative and quantitative characterization of protein-DNA complex formation using native (nondenaturing) polyacrylamide or agarose gel electrophoresis. By varying the incubation temperature of the protein-DNA binding reaction and maintaining this temperature during electrophoresis, temperature-dependent protein-DNA interactions can be investigated. Here, we provide examples of the binding of a transcriptional repressor complex called the Evening Complex, comprising the DNA-binding protein LUX ARRYTHMO (LUX), the scaffold protein EARLY FLOWERING 3 (ELF3), and the adapter protein ELF4, to its cognate DNA and demonstrate direct detection and visualization of thermoresponsive binding in vitro. As negative controls we use the LUX DNA-binding domain and LUX full length protein, which do not exhibit temperature-dependent DNA binding.

Genome editing and molecular analyses of an Arabidopsis transcription factor, LATE FLOWERING.

Correct flower organ formation at the right timing is one of the most important strategies for plants to achieve reproductive success. Ectopic overexpression of LATE FLOWERING (LATE) is known to induce late flowering, partly through suppressing expression of the florigen-encoding gene FLOWERING LOCUS T (FT) in Arabidopsis. LATE is one of the C2H2 zinc finger transcription factors, and it has a canonical transcriptional repression domain called the ethylene-responsive element-binding factor-associated amphiphilic repression (EAR) motif at the end of its C terminus. Therefore, LATE is considered a transcriptional repressor, but its molecular function remains unclear. Our genome-edited late mutants exhibited no distinct phenotype, even in flowering, indicating the presence of redundancy from other factors. To reveal the molecular function of LATE and factors working with it, we investigated its transcriptional activity and interactions with other proteins. Transactivation activity assay showed that LATE possesses transcriptional repression ability, which appears to be attributable to both the EAR motif and other sequences. Yeast two-hybrid assay showed the EAR motif-mediated interaction of LATE with TOPLESS, a transcriptional corepressor. Moreover, LATE could also interact with CRABS CLAW (CRC), one of the most important regulators of floral meristem determinacy, through sequences in LATE other than the EAR motif. Our findings demonstrated the possibility that LATE can form a transcriptional repression complex with CRC for floral meristem determinacy.

The Tbx20-TLE interaction is essential for the maintenance of the second heart field.

T-box transcription factor 20 (Tbx20) plays a multifaceted role in cardiac morphogenesis and controls a broad gene regulatory network. However, the mechanism by which Tbx20 activates and represses target genes in a tissue-specific and temporal manner remains unclear. Studies show that Tbx20 directly interacts with the Transducin-like Enhancer of Split (TLE) family of proteins to mediate transcriptional repression. However, a function for the Tbx20-TLE transcriptional repression complex during heart development has yet to be established. We created a mouse model with a two amino acid substitution in the Tbx20 EH1 domain, thereby disrupting the Tbx20-TLE interaction. Disruption of this interaction impaired crucial morphogenic events, including cardiac looping and chamber formation. Transcriptional profiling of Tbx20EH1Mut hearts and analysis of putative direct targets revealed misexpression of the retinoic acid pathway and cardiac progenitor genes. Further, we show that altered cardiac progenitor development and function contribute to the severe cardiac defects in our model. Our studies indicate that TLE-mediated repression is a primary mechanism by which Tbx20 controls gene expression.

CSE1L is a negative regulator of the RB-DREAM pathway in p53 wild-type NSCLC and can be targeted using an HDAC1/2 inhibitor

P53 represses transcription by activating p21 expression and promoting formation of RB1-E2F1 and RBL1/RBL2-DREAM transcription repressor complexes. The DREAM complex is composed of DP1, RB-family proteins RBL1 or RBL2 (p107/p130), E2F4/5, and MuvB. We recently reported RBL2-DREAM contributes to improved therapy responses in p53 wild-type NSCLC cells and improved outcomes in NSCLC patients whose tumors express wild-type p53. In the current study we identified CSE1L as a novel inhibitor of the RBL2-DREAM pathway and target to activate RBL2-DREAM in NSCLC cells. CSE1L is an oncoprotein that maintains repression of genes that can be reactivated by HDAC inhibitors. Mocetinostat is a HDAC inhibitor in clinical trials with selectivity against HDACs 1 and 2. Knockdown of CSE1L in NSCLC cells or treatment with mocetinostat increased p21, activated RB1 and RBL2, repressed DREAM target genes, and induced toxicity in a manner that required wild-type p53. Lastly, we found high levels of CSE1L and specific DREAM-target genes are candidate markers to identify p53 wild-type NSCLCs most responsive to mocetinostat. Thus, we identified CSE1L as a critical negative regulator of the RB-DREAM pathway in p53 wild-type NSCLC that can be indirectly targeted with HDAC1/2 inhibitors (mocetinostat) in current clinical trials. High expression of CSE1L and DREAM target genes could serve as a biomarker to identify p53 wild-type NSCLCs most responsive to this HDAC1/2 inhibitor.

P-216 The PBK-FOXM1 axis disrupts the DREAM transcriptional repressor complex, contributing to the growth and genomic instability of myeloma

P-216 The PBK-FOXM1 axis disrupts the DREAM transcriptional repressor complex, contributing to the growth and genomic instability of myeloma

Disruption of RCAN1.4 expression mediated by YY1/HDAC2 modulates chronic renal allograft interstitial fibrosis

Chronic allograft dysfunction (CAD) is a major factor that hinders kidney transplant survival in the long run. Epithelial–mesenchymal transition (EMT) has been confirmed to significantly contribute to interstitial fibrosis/tubular atrophy (IF/TA), which is the main histopathological feature of CAD. Aberrant expression of the regulator of calcineurin 1 (RCAN1), recognized as an endogenous inhibitor of the calcineurin phosphatase, has been shown to be extensively involved in various kidney diseases. However, it remains unclear how RCAN1.4 regulates IF/TA formation in CAD patients. Herein, an in vivo mouse renal transplantation model and an in vitro model of human renal tubular epithelial cells (HK-2) treated with tumor necrosis factor-α (TNF-α) were employed. Our results proved that RCAN1.4 expression was decreased in vivo and in vitro, in addition to the up-regulation of Yin Yang 1 (YY1), a transcription factor that has been reported to convey multiple functions in chronic kidney disease (CKD). Knocking in of RCAN1.4 efficiently attenuated chronic renal allograft interstitial fibrosis in vivo and inhibited TNF-α-induced EMT in vitro through regulating anti-oxidative stress and the calcineurin/nuclear factor of activated T cells cytoplasmic 1 (NFATc1) signaling pathway. In addition, suppression of YY1 mediated by shRNA or siRNA alleviated TNF-α-induced EMT through abolishing reactive species partly in an RCAN1.4-dependent manner. Notably, we confirmed that YY1 negatively regulated RCAN1.4 transcription by directly interacting with the RCAN1.4 promoter. In addition, histone deacetylase 2 (HDAC2) interacted with YY1 to form a multi-molecular complex, which was involved in TNF-α-induced RCAN1.4 transcriptional repression. Therefore, RCAN1.4 is suggested to be modulated by the YY1/HDAC2 transcription repressor complex in an epigenetic manner, which is a mediated nephroprotective effect partly through modulating O2⋅− generation and the calcineurin/NFATc1 signaling pathway. Thus, the YY1–RCAN1.4 axis constitutes an innovative target for IF/TA treatment in CAD patients.

Acetylation of inorganic pyrophosphatase by S-RNase signaling induces pollen tube tip swelling by repressing pectin methylesterase.

Self-incompatibility (SI) is a widespread genetically determined system in flowering plants that prevents self-fertilization to promote gene flow and limit inbreeding. S-RNase-based SI is characterized by the arrest of pollen tube growth through the pistil. Arrested pollen tubes show disrupted polarized growth and swollen tips, but the underlying molecular mechanism is largely unknown. Here, we demonstrate that the swelling at the tips of incompatible pollen tubes in pear (Pyrus bretschneideri [Pbr]) is mediated by the SI-induced acetylation of the soluble inorganic pyrophosphatase (PPA) PbrPPA5. Acetylation at Lys-42 of PbrPPA5 by the acetyltransferase GCN5-related N-acetyltransferase 1 (GNAT1) drives accumulation of PbrPPA5 in the nucleus, where it binds to the transcription factor PbrbZIP77, forming a transcriptional repression complex that inhibits the expression of the pectin methylesterase (PME) gene PbrPME44. The function of PbrPPA5 as a transcriptional repressor does not require its PPA activity. Downregulating PbrPME44 resulted in increased levels of methyl-esterified pectins in growing pollen tubes, leading to swelling at their tips. These observations suggest a mechanism for PbrPPA5-driven swelling at the tips of pollen tubes during the SI response. The targets of PbrPPA5 include genes encoding cell wall-modifying enzymes, which are essential for building a continuous sustainable mechanical structure for pollen tube growth.

Comprehensive analysis of REST corepressors (RCORs) in pan-cancer.

REST corepressors (RCORs) are the core component of the LSD1/CoREST/HDACs transcriptional repressor complex, which have been revealed differently expressed in various cancers, but the therapeutic and prognostic mechanisms in cancer are still poorly understood. In this study, we analyzed expression, prognostic value, molecular subtypes, genetic alteration, immunotherapy response and drug sensitivity of RCORs in pan-cancer. Clinical correlation, stemness index, immune infiltration and regulatory networks of RCORs in hepatocellular carcinoma (HCC) were detected through TCGA and GSCA database. In-vitro experiments were conducted to explore the role of RCOR1 in HCC cells. The expression of RCORs varied among different cancers, and have prognostic values in several cancers. Cancer subtypes were categorized according to the expression of RCORs with clinical information. RCORs were significantly correlated with immunotherapy response, MSI, drug sensitivity and genetic alteration in pan-cancer. In HCC, RCORs were considered as potential predictor of stemness and also had association with immune infiltration. The ceRNA-TF-kinase regulatory networks of RCORs were constructed. Besides, RCOR1 acts as an oncogene in HCC and promotes the proliferation of HCC cells by inhibiting cell cycle arrest and cell apoptosis. Taken together, our study revealed the potential molecular mechanisms of RCORs in pan-cancer, offering a benchmark for disease-related research.

Abstract A059: Epigenetic remodelling by class I HDAC inhibitors increases response to androgen signalling inhibitors in prostate cancer

Abstract Prostate cancer (PCa) is one of the most prevalent cancers in the male population with approximately 1 in 8 men diagnosed with the disease during their lifetime. The treatment options available varies depending on individual circumstances, with surgery or radiotherapy employed as first-line treatments for locally advanced PCa whilst hormone therapies are employed as the disease progresses toward castrate-resistant prostate cancer. Despite the advances in therapies and initial treatment effectiveness, tumours develop resistance and overcoming this resistance proves a major challenge for the treatment of the disease. Dysregulation of chromatin regulatory proteins and the epigenetic landscape are key factors contributing to the development, progression and persistence of PCa. Histone deacetylases (HDACs) are a family of chromatin-modifying proteins which assemble into various transcriptional repressor complexes and mediate gene repression through the removal of acetyl groups from histone tails, subsequently compacting the chromatin landscape impacting on cell proliferation, transcription, replication and DNA repair. Overexpression of HDACs in prostate cancer has been shown to shift the balance of acetylation, resulting in improper gene silencing. Therefore, we hypothesise that restoration of the acetylation homeostasis through HDAC inhibition is a potential therapy to overcome resistance in PCa. Here, we show that the class I HDAC inhibitor Entinostat as a single agent promotes anti-tumour effects in PCa by impairing cell proliferation and inducing apoptosis in a panel of PCa cell lines. Interestingly, we found that combination with the androgen signalling inhibitors (ASIs), Enzalutamide or Abiraterone, further enhances these anti-tumour effects. In vitro analysis of matched parental and Enzalutamide-/Abiraterone-resistant C4-2B and CWRR-1 cells further revealed an enhanced reduction in cell survival following combined Entinostat and ASIs treatments in the resistant models compared to parental. Genomic and transcriptomic analyses uncovered distinct molecular signatures of these resistant models. Treatment with Entinostat alone downregulated androgen receptor (AR) expression and combination with ASI further suppressed AR expression and downstream AR target genes. Taken together, these results demonstrate the potential for Entinostat to synergise with current standard-of-care therapies to target ASI resistance in PCa. In conclusion, this study provides strong evidence suggesting the effectiveness of class I HDAC inhibition by Entinostat in the treatment of castration-resistant prostate cancer and opens the door for further research into combinatorial therapies in targeting resistance. Citation Format: Rachel McCole, Ben McCullough, Judith M. Manley, Nicholas Forsythe, Simon S. McDade, Melissa LaBonte Wilson. Epigenetic remodelling by class I HDAC inhibitors increases response to androgen signalling inhibitors in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A059.

A novel transcriptional repressor complex MYB22-TOPLESS-HDAC1 promotes rice resistance to brown planthopper by repressing F3'H expression.

The brown planthopper (BPH) is the most destructive pest of rice. The MYB transcription factors are vital for rice immunity, but most are activators. Although MYB22 positively regulates rice resistance to BPH and has an EAR motif associated with active repression, it remains unclear whether it is a transcriptional repressor affecting rice-BPH interaction. Genetic analyses revealed that MYB22 regulates rice resistance to BPH via its EAR motif. Several biochemical experiments (e.g. transient transcription assay, Y2H, LCA, and BiFC) indicated that MYB22 is a transcriptional repressor that interacts with the corepressor TOPLESS via its EAR motif and recruits HDAC1 to form a tripartite complex. Flavonoid-3'-hydroxylase (F3'H) is a flavonoid biosynthesis pathway-related gene that negatively regulates rice resistance to BPH. Based on a bioinformatics analysis and the results of EMSA and transient transcription assays, MYB22 can bind directly to the F3'H promoter and repress gene expression along with TOPLESS and HDAC1. We revealed a transcriptional regulatory mechanism influencing the rice-BPH interaction that differs from previously reported mechanisms. Specifically, MYB22-TOPLESS-HDAC1 is a novel transcriptional repressor complex with components that synergistically and positively regulate rice resistance to BPH through the transcriptional repression of F3'H.

A novel transcriptional repressor complex MYB22–TOPLESS–HDAC1 promotes rice resistance to brown planthopper by repressing F3′H expression

A novel transcriptional repressor complex MYB22–TOPLESS–HDAC1 promotes rice resistance to brown planthopper by repressing F3′H expression

Abstract 6011: Methylation profiling reveals the role of PRC2 in regulating DNA methylome changes in malignant peripheral nerve sheath tumors

Abstract Neurofibromatosis type 1 (NF1) syndrome is an autosomal dominant cancer predisposition syndrome defined by germline deletion of one NF1 allele. Somatic loss of heterozygosity in the remaining NF1 allele in Schwann lineage cells gives rise to benign tumors known as plexiform neurofibromas. These tumors have a high risk of developing into cancerous lesions known as malignant peripheral nerve sheath tumors (MPNST) through the sequential deletion of CDKN2A/B followed by recurrent mutations in SUZ12 or EED, constituting the inactivation of polycomb repressor complex 2 (PRC2). PRC2 is a transcriptional repressor complex involved in the silencing of several genes throughout cell development and differentiation. Loss of this complex leads to a depletion in trimethylation marks on Histone H3 Lysine 27 (H3K27me3) and the subsequent gain of acetylation marks on the same lysine residue (H3K27Ac). However, full range of epigenetic consequences of these events are currently not well characterized, especially with respect to MPNST formation and development. The Largaespada lab has developed Schwann cell lines engineered to harbor deletions in NF1 and either SUZ12 or EED and assayed the methylation status of 850,000 CpG sites using the EPIC array. The resulting methylation profiles reveal a large degree of change in promoter methylation between cell lines with and without PRC2 inactivation. These changes, however, appear to be inconsistent. To further understand this phenomenon, I have analyzed differential methylation patterns between lines with SUZ12/EED deletions on top of NF1 deletion compared to those with NF1 deletion alone. Pathway analysis of genes whose promoters are differentially methylated upon PRC2 reveals a bias towards developmental modules such as morphogenesis and neuronal differentiation, suggesting that DNA methylation is a mechanism by which PRC2 loss promotes stemness in Schwann lineage cells prior to MPNST formation. Additionally, differentially methylated regions were overlapped with functional non-coding elements in the genome to understand potential ripple effects in the epigenome. These findings underline the importance of DNA methylation in contributing to tumor formation and define unique functions by which PRC2 specifically leverages diverse methylation states to drive malignant transformation in Schwann lineage cells. Citation Format: Christopher M. Stehn, Minu Bhunia, Kyle B. Williams, Alex T. Larsson, David A. Largaespada. Methylation profiling reveals the role of PRC2 in regulating DNA methylome changes in malignant peripheral nerve sheath tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6011.

Abstract 5973: The brain penetrant CDK4/6 Inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of NSCLC, CRC, and HER2-positive breast cancer

Abstract Cell cycle deregulation is a hallmark of cancer and the hyperactivation and overexpression of CDKs are often drivers of cancer pathogenesis. Cyclin-dependent kinase 4 and 6 (CDK4)/(CDK6) are critical mediators of cellular transition into S phase and important for the initiation, growth, and survival of many cancers. Activated CDK4/CDK6 complexes phosphorylate Rb1, reduce their binding affinities and release Rb1-containing transcription repressor complexes from E2F transcription factors, resulting in activation of E2F controlled cell cycle genes and progression of the cell cycle. At present three CDK4/CDK6 inhibitors are approved for the treatment of ER+/HER2- breast cancer, and are being explored in other cancer indications as well. Previously we described a novel brain penetrant CDK4/CDK6 inhibitor, PRT3645, that exhibits single digit nanomolar biochemical potency against CDK4/CDK6 and >2000-fold selectivity against CDK1, CDK2 and CDK9. PRT3645 inhibits cellular phosphorylation of Rb and exhibits a protein binding-adjusted cellular IC50 of <300 nM. PRT3645 exhibits favorable in-vitro safety pharmacology and ADME properties, including increased brain penetration, and demonstrates oral bioavailability across rodents, dog, and non-human primates. In addition to robust monotherapy activity observed in preclinical models of ER+/HER2- breast cancer, we explored the activity of PRT3645 in other tumor types as well as in combination with other targeted therapies. In NSCLC, PRT3645 treatment resulted in significant inhibition of cell lines that harbor activation of the RAS/MEK/ERK pathway in proliferation assays and demonstrated comparable high synergy scores when combined with clinically approved covalent KRAS G12C inhibitors. In-vivo, oral PRT3645 was well tolerated and induced anti-tumor efficacy in two KRAS G12C mutant xenograft models that harbor the CDKN2A (p16) deletion. Anti-tumor efficacy was further improved when PRT3645 was combined with KRAS/MEK inhibitors in xenograft models and the combination therapy was well tolerated. In addition, we explored combinations of PRT3645 with a brain penetrant receptor tyrosine kinase inhibitor (TKI), an approved treatment for patients with advanced HER2+ breast cancer, including patients with brain metastases. In a HER2+ orthotopic human breast cancer brain metastasis model, PRT3645 was highly efficacious in combination with HER2 kinase inhibition and enhanced median survival significantly. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species and brain penetrance. In preclinical studies, PRT3645 was highly efficacious when combined with KRAS/MEK inhibitors, and with a brain penetrant HER2 receptor TKI, both in-vitro and in-vivo. PRT3645 has advanced into Phase 1 clinical trials (NCT05538572). Citation Format: Yue Zou, Srijita Dhar, Kirsten Gallagher, Andrew Buesking, Sarah Pawley, Ryan Holmes, Xiaowei Wu, Katarina Rohlfing, Min Wang, Joseph Rager, Tom Emm, Stefan Ruepp, Miles Cowart, Jing Ni, Jean Zhao, Bruce Ruggeri, Andrew Combs, Kris Vaddi, Sandy Geeganage, Ashish Juvekar, Sang Hyun Lee, Peggy Scherle. The brain penetrant CDK4/6 Inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of NSCLC, CRC, and HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5973.

Drug addiction unveils a repressive methylation ceiling in EZH2-mutant lymphoma.

Drug addiction, a phenomenon where cancer cells paradoxically depend on continuous drug treatment for survival, has uncovered cell signaling mechanisms and cancer codependencies. Here we discover mutations that confer drug addiction to inhibitors of the transcriptional repressor polycomb repressive complex 2 (PRC2) in diffuse large B-cell lymphoma. Drug addiction is mediated by hypermorphic mutations in the CXC domain of the catalytic subunit EZH2, which maintain H3K27me3 levels even in the presence of PRC2 inhibitors. Discontinuation of inhibitor treatment leads to overspreading of H3K27me3, surpassing a repressive methylation ceiling compatible with lymphoma cell survival. Exploiting this vulnerability, we show that inhibition of SETD2 similarly induces the spread of H3K27me3 and blocks lymphoma growth. Collectively, our findings demonstrate that constraints on chromatin landscapes can yield biphasic dependencies in epigenetic signaling in cancer cells. More broadly, we highlight how approaches to identify drug addiction mutations can be leveraged to discover cancer vulnerabilities.

The PRMT6/PARP1/CRL4B Complex Regulates the Circadian Clock and Promotes Breast Tumorigenesis.

Circadian rhythms, as physiological systems with self-regulatory functions in living organisms, are controlled by core clock genes and are involved in tumor development. The protein arginine methyltransferase 6 (PRMT6) serves as an oncogene ina myriadof solid tumors, including breast cancer. Hence, the primary aim of the current study is to investigate the molecular mechanisms by which the PRMT6 complex promotes breast cancer progression. The results show that PRMT6, poly(ADP-ribose) polymerase 1 (PARP1), and the cullin 4 B (CUL4B)-Ring E3 ligase (CRL4B) complex interact to form a transcription-repressive complex that co-occupies the core clock gene PER3 promoter. Moreover, genome-wide analysis of PRMT6/PARP1/CUL4B targets identifies a cohort of genes that is principally involved in circadian rhythms. This transcriptional-repression complex promotes the proliferation and metastasis of breast cancer by interfering with circadian rhythm oscillation. Meanwhile, the PARP1 inhibitor Olaparib enhances clock gene expression, thus, reducing breast carcinogenesis, indicating that PARP1 inhibitors have potential antitumor effects in high-PRMT6 expression breast cancer.

PbAP2-FG2 and PbAP2R-2 function together as a transcriptional repressor complex essential for Plasmodium female development.

Gametocyte development is a critical step in the life cycle of Plasmodium. Despite the number of studies on gametocyte development that have been conducted, the molecular mechanisms regulating this process remain to be fully understood. This study investigates the functional roles of two female-specific transcriptional regulators, PbAP2-FG2 and PbAP2R-2, in P. berghei. Knockout of pbap2-fg2 or pbap2r-2 impairs female gametocyte development, resulting in developmental arrest during ookinete development. ChIP-seq analyses of these two factors indicated their colocalization on the genome, suggesting that they function as a complex. These analyses also revealed that their target genes contained a variety of genes, including both male and female-enriched genes. Moreover, differential expression analyses showed that these target genes were upregulated through the disruption of pbap2-fg2 or pbap2r-2, indicating that these two factors function as a transcriptional repressor complex in female gametocytes. Formation of a complex between PbAP2-FG2 and PbAP2R-2 was confirmed by RIME, a method that combines ChIP and MS analysis. In addition, the analysis identified a chromatin regulator PbMORC as an interaction partner of PbAP2-FG2. Comparative target analysis between PbAP2-FG2 and PbAP2-G demonstrated a significant overlap between their target genes, suggesting that repression of early gametocyte genes activated by PbAP2-G is one of the key roles for this female transcriptional repressor complex. Our results indicate that the PbAP2-FG2-PbAP2R-2 complex-mediated repression of the target genes supports the female differentiation from early gametocytes.