Deacetylase Inhibitors Research Articles

Insufficient Effective Time of Suberanilohydroxamic Acid, a Deacetylase Inhibitor, Treatment Promotes PC3 Cell Growth.

Castration-resistant prostate cancer (CRPC) contributes mostly to prostate cancer-specific mortality, and conventional castration therapy is almost ineffective, new therapies are needed. As a new potential anti-cancer drug, histone deacetylases (HDACs) inhibitors were demonstrated to be effective in inhibiting drug-resistance cancers in preclinical studies, but the results from clinical trials on CRPC patients were disappointing, and the reasons are unknown. In this study, we investigated the effect of suberanilohydroxamic acid (SAHA), a broad-spectrum pan-HDAC inhibitor, on proliferation, apoptosis, cell cycle progression in PC3 cells, and found that, unlike significant inhibiting effects at high-dose, low-dose SAHA significantly promoted PC3 cell growth. Further colony formation assay showed that the inhibitory effect of SAHA is also dependent on the treatment time, high-dose SAHA also exhibited promoting effect on PC3 cells when the treatment time was insufficient. However, this effect was not observed in another CRPC cell line, DU145, or another HDAC inhibitor, Trichostatin A (TSA). Our results indicate that, instead of inhibitory effect, SAHA would promote PC3 cell growth if the dose is low or the treatment time is insufficient, but this effect has not been observed in other CRPC cell line or HDAC inhibitors.

Eml1 promotes axonal growth by enhancing αTAT1-mediated microtubule acetylation

Microtubule stabilization is critical for axonal growth and regeneration, and many microtubule-associated proteins are involved in this process. In this study, we found that the knockdown of echinoderm microtubule-associated protein-like 1 (EML1) hindered axonal growth in cultured cortical and dorsal root ganglion neurons. We further revealed that EML1 facilitated the acetylation of microtubules and that the impairment of axonal growth due to EML1 inhibition could be restored by treatment with deacetylase inhibitors, suggesting that EML1 affected tubulin acetylation. Moreover, we verified an interaction between EML1 and the alpha-tubulin acetyltransferase 1, which is responsible for the acetylation of alpha-tubulin. We thus proposed that EML1 might regulate microtubule acetylation and stabilization via alpha-tubulin acetyltransferase 1 and then promote axon growth. Finally, we verified that the knockdown of EML1 in vivo also inhibited sciatic nerve regeneration. Our findings revealed a novel effect of EML1 on microtubule acetylation during axonal regeneration.

Endosymbiosis in trypanosomatids: The bacterium division depends on microtubule dynamism

Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.

First in Class Dual Non-ATP-Competitive Glycogen Synthase Kinase 3β/Histone Deacetylase Inhibitors as a Potential Therapeutic to Treat Alzheimer's Disease.

Despite recent FDA approvals, Alzheimer's disease (AD) still represents an unmet medical need. Among the different available therapeutic approaches, the development of multitarget molecules represents one of the most widely pursued. In this work, we present a second generation of dual ligands directed toward highly networked targets that are deeply involved in the development of the disease, namely, Histone Deacetylases (HDACs) and Glycogen Synthase Kinase 3β (GSK-3β). The synthesized compounds are highly potent GSK-3β, HDAC2, and HDAC6 inhibitors with IC50 values in the nanomolar range of concentrations. Among them, compound 4 inhibits histone H3 and tubulin acetylation at 0.1 μM concentration, blocks hyperphosphorylation of tau protein, and shows interesting immunomodulatory and neuroprotective properties. These features, together with its ability to cross the blood-brain barrier and its favorable physical-chemical properties, make compound 4 a promising hit for the development of innovative disease-modifying agents.

Abstract 7072: MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival

Abstract The MYC oncogenic transcription factor is acetylated by the p300 and GCN5 histone acetyltransferases. The significance of MYC acetylation and functions of specific acetylated lysine (K) residues have remained unclear. Here, we show that the major p300-acetylated K148(149) and K157(158) sites in human (or mouse) MYC and the main GCN5-acetylated K323 residue are reversibly acetylated in various malignant and non-malignant cells. Oncogenic overexpression of MYC enhances its acetylation and alters regulation of site-specific acetylation by proteasome and deacetylase inhibitors. Acetylation of MYC at different K residues differentially affects its stability in a cell type- dependent manner. Lysine-to-arginine substitutions indicate that although none of the acetylated K (AcK) residues is required for MYC stimulation of adherent cell proliferation, individual AcK sites have gene-specific functions controlling select MYC-regulated processes in cell adhesion, contact inhibition, apoptosis, and/or metabolism, and are required for the malignant cell transformation activity of MYC. Each AcK site is required for anchorage-independent growth of MYC-overexpressing cells in vitro, and both the AcK148(149) and AcK157(158) residues are also important for the tumorigenic activity of MYC-transformed cells in vivo. These AcK residues may also preferentially activate transcription via facilitating MYC binding to several cancer-associated target gene promoters and via enhancing RNAP II recruitment. With the knowledge that MYC lysine residues can be acetylated by HATs GCN5 and P300, some HATs and HAT associated cofactors (i.e. YEATS2 and PIN1) are important for MYC recruitment to DNA binding. By identifying MYC acetylation-dependent coregulators and their oncogenic function, we proposed to understand whether and how MYC acetylation residues cooperate with its cofactors and influence malignant transformation in breast cancer cells.Overall, this study will uncover the functions and mechanisms of MYC acetylation in regulation of malignant transformation and the impact on breast cancer initiation and progression. By analyzing deregulated target genes and MYC-relevant coactivators, the MYC AcK site-specific signaling pathways identified may offer new avenues for selective therapeutic targeting of MYC oncogenic activities. Citation Format: Jeffrey J. Pino, Matthew Hurd, Yifan Zhao, Kay Jang, Michael Allevato, Marina Vorontchikhina, Wataru Ichikawa, Ryan Gates, Emily Villalpando, Michael Hamilton, Francesco Failo, Songqin Pan, Yue Qi, Yu-wen Hung, Thomas Girke, David Ann, Victoria Seewaldt, Ernest Martinez. MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival [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 7072.

Histone Acetyltransferase and Deacetylase Inhibitors—New Aspects and Developments

Epigenetic processes modulate gene transcription and genomic stability, ensuring proper cell development and differentiation [...]

MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival.

The MYC oncogenic transcription factor is acetylated by the p300 and GCN5 histone acetyltransferases. The significance of MYC acetylation and the functions of specific acetylated lysine (AcK) residues have remained unclear. Here, we show that the major p300-acetylated K148(149) and K157(158) sites in human (or mouse) MYC and the main GCN5-acetylated K323 residue are reversibly acetylated in various malignant and nonmalignant cells. Oncogenic overexpression of MYC enhances its acetylation and alters the regulation of site-specific acetylation by proteasome and deacetylase inhibitors. Acetylation of MYC at different K residues differentially affects its stability in a cell type-dependent manner. Lysine-to-arginine substitutions indicate that although none of the AcK residues is required for MYC stimulation of adherent cell proliferation, individual AcK sites have gene-specific functions controlling select MYC-regulated processes in cell adhesion, contact inhibition, apoptosis, and/or metabolism and are required for the malignant cell transformation activity of MYC. Each AcK site is required for anchorage-independent growth of MYC-overexpressing cells in vitro, and both the AcK148(149) and AcK157(158) residues are also important for the tumorigenic activity of MYC transformed cells in vivo. The MYC AcK site-specific signaling pathways identified may offer new avenues for selective therapeutic targeting of MYC oncogenic activities.

Study on the activation of cell death mechanisms: in search of new therapeutic targets in glioblastoma multiforme

Malignant primary brain tumors remain among the most difficult cancers to treat, in particular, Glioblastoma Multiforme (GBM) is the deadliest brain tumor. The standard therapies currently used are not efficient enough in improving patients’ survival and quality of life. Cisplatin (CDDP), a platinum-based drug, has shown efficacy against different solid neoplasms, but it is also associated to different forms of off-target toxicity. To overcome the limitation in the use of CDDP in the treatment of GBM patients, fourth generation platinum compounds are been synthesized, one of them is the Pt(IV)Ac-POA, a prodrug with a medium-chain fatty acid as axial ligand, which acts as a histone 3 deacetylase inhibitor. Moreover, recently, the antioxidant effects of medicinal mushrooms have been shown to induce a lowering of the toxicity of chemotherapy drugs, inducing greater therapeutic efficiency, thus the combined therapy of chemotherapy and micotherapy could be helpful in the treatment of GBM reducing the adverse effects of the former thanks to phytotherapy’s antioxidant, anti-inflammatory, immunomodulatory and antitumoral activities. Here, through immunoblotting, ultrastructural and immunofluorescence analysis, we evaluated the contribution in the activation of different cell death pathway of Micotherapy U-Care, a medicinal blend supplement, used together with platinum-based compounds on human glioblastoma U251 cells.

Integrated proteome and acetylome analyses provide novel insights into early somatic embryogenesis of Dimocarpus longan

Longan (Dimocarpus longan) is a precious subtropical fruit with high nutritional value. The somatic embryogenesis (SE) affects the quality and yield of fruit. Apart from clonal propagation, SE has extensive applications in genetic improvement and mutation. Thus, understanding the molecular basis of embryogenesis in longan will help to develop strategies for mass production of quality planting material. Lysine acetylation (Kac) plays an important role in diverse cellular processes, but limited knowledge is available regarding acetylation modifications in plant early SE. In this study, the proteome and acetylome of longan embryogenic callus (ECs) and globular embryos (GEs) were investigated. In total, 7232 proteins and 14,597 Kac sites were identified, and this resulted in the discovery of 1178 differentially expressed proteins and 669 differentially expressed acetylated proteins. KEGG and GO analysis showed that glucose metabolism, carbon metabolism, fatty acid degradation, and oxidative phosphorylation pathways were influenced by Kac modification. Furthermore, sodium butyrate (Sb, a deacetylase inhibitor) led to reduced the proliferation and delayed the differentiation of ECs by regulating the homeostasis of reactive oxygen species (ROS) andindole-3-acetic acid (IAA). Our study provides a comprehensive proteomic and acetylomic analysis to aid in understanding the molecular mechanisms involved in early SE, representing a potential tool for genetic improvement of longan.

Synergic Induction of Autophagic Cell Death in Anaplastic Thyroid Carcinoma

Anaplastic thyroid carcinoma (ATC) has poor prognosis, high mortality rate and lack of effective therapy. A synergic combination of PD-L1 antibody together with cell death promoting substances like deacetylase inhibitors (DACi) and multi-kinase inhibitors (MKI) could sensitize ATC cells and promote decay by autophagic cell death. The PD-L1-inhibitor atezolizumab synergized with panobinostat (DACi) and sorafenib (MKI) leading to significant reduction of the viability, measured by real time luminescence, of three different patient-derived primary ATC cells, of C643 cells and follicular epithelial thyroid cells too. Solo administration of these compounds caused a significant over-expression of autophagy transcripts; meanwhile autophagy proteins were almost not detectable after the single administration of panobinostat, thus supporting a massive autophagy degradation process. Instead, the administration of atezolizumab caused an accumulation of autophagy proteins and the cleavage of the active caspases 8 and 3. Interestingly, only panobinostat and atezolizumab were able to exacerbate the autophagy process by increasing the synthesis, the maturation and final fusion with the lysosomes of the autophagosome vesicles. Despite ATC cells could be sensitized by atezolizumab via the cleavage of the caspases, no reduction of cell proliferation or promotion of cell death was observed. The apoptosis assay evidenced the ability of panobinostat alone and in combination with atezolizumab to induce the phosphatidil serine exposure (early apoptosis) and further the secondary necrosis. Instead, sorafenib was only able to cause necrosis. The increase of caspases activity induced by atezolizumab, the apoptosis and autophagy processes promoted by panobinostat synergize thus promoting cell death in well-established and primary anaplastic thyroid cancer cells. The combined therapy could represent a future clinical application for the treatment of such lethal and untreatable solid cancer.

HDAC19 recruits ERF4 to the MYB5a promoter and diminishes anthocyanin accumulation during grape ripening.

DNA acetylation alters the expression of responsive genes during plant development. In grapes (Vitis vinifera), however, little is known about this regulatory mechanism. In the present study, 'Kyoho' grapes treated with trichostatin A (TSA, a deacetylase inhibitor) were used for transcriptome sequencing and quantitative proteomics analysis. We observed that acetylation was associated with anthocyanin accumulation and gene expression. Acetylation positively regulated phenylalanine metabolism and flavonoid biosynthesis pathways. Using omics analysis, we detected an increase in the levels of the AP2/EREBP transcription factor family after TSA treatment, indicating its association with acetylation-deacetylation dynamics in grapes. Furthermore, ethylene response factor 4 (ERF4) physically interacted with VvHDAC19, a histone deacetylase, which synergistically reduced the expression of target genes involved in anthocyanin biosynthesis owing to the binding of VvERF4 to the GCC-box cis-regulatory element in the VvMYB5a promoter. VvHDAC19 and VvERF4 also controlled anthocyanin biosynthesis and accumulation by regulating acetylation levels of histones H3 and H4. Therefore, alterations in histone modification can significantly regulate the expression of genes involved in anthocyanin biosynthesis and affect grape ripening.

Ginsenoside Rg3 inhibits renal cell carcinoma cell migration, invasion, colony formation, and tube formation and enhances apoptosis through promoting the DNA demethylation and histone acetylation.

This study explored the effect and mechanism of Rg3 on renal cell carcinoma (RCC) progression. RCC cells were treated with different concentrations of Rg3, 5-Aza-dc (a methyltransferase inhibitor) or TSA (a deacetylase inhibitor). Rg3-induced cytotoxicity, migration, invasion, colony formation, tube formation and apoptosis of RCC cells were evaluated by CCK-8, wound healing, Transwell, colony formation, tube formation and flow cytometry assays, respectively. Methylation and expressions of p53, p21 and p16, and expressions of methylation-related genes and histone deacetylases and histone acetylation-related genes (H3 (acetyl K14), H3 (acetyl K9), H4 (acetyl K12), H4 (acetyl K5) and H4 (acetyl K16)) were analysed by qRT-PCR and western blot. Rg3 dose-dependently decreased the viability, inhibited migration, invasion, colony formation and tube formation, and enhanced apoptosis of RCC cells. Rg3 enhanced the demethylation levels and expressions of p53, p21 and p16 as well as the expressions of histone acetylation-related genes, but repressed the expressions of methylation-related genes and histone deacetylases. Rg3 had the same effect as 5-Aza-dc and TSA did on the above-mentioned cellular changes. Rg3 restrains RCC cell migration, invasion, colony formation and tube formation, yet enhances apoptosis through promoting demethylation of p53, p21 and p16, and histone acetylation.

CUDC907, a dual phosphoinositide-3 kinase/histone deacetylase inhibitor, promotes apoptosis of NF2 Schwannoma cells.

Neurofibromatosis Type 2 (NF2) is a rare tumor disorder caused by pathogenic variants of the merlin tumor suppressor encoded by NF2. Patients develop vestibular schwannomas (VS), peripheral schwannomas, meningiomas, and ependymomas. There are no approved drug therapies for NF2. Previous work identified phosphoinositide-3 kinase (PI3K) as a druggable target. Here we screened PI3K pathway inhibitors for efficacy in reducing viability of human schwannoma cells. The lead compound, CUDC907, a dual histone deacetylase (HDAC)/PI3K inhibitor, was further evaluated for its effects on isolated and nerve-grafted schwannoma model cells, and primary VS cells. CUDC907 (3 nM IG50) reduced human merlin deficient Schwann cell (MD-SC) viability and was 5–100 fold selective for MD over WT-SCs. CUDC907 (10 nM) promoted cell cycle arrest and caspase-3/7 activation within 24 h in human MD-SCs. Western blots confirmed a dose-dependent increase in acetylated lysine and decreases in pAKT and YAP. CUDC907 decreased tumor growth rate by 44% in a 14-day treatment regimen, modulated phospho-target levels, and decreased YAP levels. In five primary VS, CUDC907 decreased viability, induced caspase-3/7 cleavage, and reduced YAP levels. Its efficacy correlated with basal phospho-HDAC2 levels. CUDC907 has cytotoxic activity in NF2 schwannoma models and primary VS cells and is a candidate for clinical trials.

P1240: ANTI-PD-1-ANTIBODY (TISLELIZUMAB) COMBINED WITH DEACETYLASE INHIBITOR (CHIDAMIDE), LENALIDOMIDE AND ETOPOSIDE FOR THE TREATMENT OF REFRACTORY/RELAPSED EXTRANODAL NATURAL KILLER/T CELL LYMPHOMA, NASAL

Background: Extranodal Natural Killer/T Cell Lymphoma (ENKTL) is a highly aggressive non-Hodgkin lymphoma (NHL), with higher incidence in Asia, which is related to Epstein-Barr virus (EBV) infection. Patients with r/r-ENKTL have a poor prognosis, and is lack of effective treatment. Anti-PD-1-Antibody plus chidamide was effective and well tolerated on r/r-ENKTL (SCENT Trial, 2020 ASH). Lenalidomide is an immunomodulatory agent, which has been shown to be effective in treatment of r/r-lymphoma with an acceptable safety. Etoposide is an important chemotherapeutic agent in the treatment of ENKTL. Here we present preliminary results of anti-PD-1 antibody combined with chidamide, lenalidomide and etoposide for the treatment of r/r-lymphoma. Aims: To explore the efficacy and safety of anti-PD-1-Antibody (Tislelizumab) combined with Deacetylase Inhibitor (Chidamide), Lenalidomide and Etoposide for the treatment of refractory/relapsed Extranodal Natural Killer/T Cell Lymphoma, Nasal Type (r/r-ENKTL). Methods: This was a prospective, single-arm, open-label, multi-center, phase II clinical trial. This trial enrolled eligible patients with histologically confirmed r/r-ENKTL failing from at least 1-line treatment; ECOG performance status ≤ 2; adequate organ function and bone marrow function; and at least one measurable or evaluable lesion. Patients received 6 cycles of Tislelizumab (200mg), Chidamide (20mg biw), Lenalidomide (25mg d1-10) and Etoposide (100mg/m2 d1-3), every 21 days cycle. Then received 10 cycles of Tislelizumab (200mg) maintenance treatment, every 21 days cycle. The primary endpoints were objective response rate (ORR) based on Lugano 2014 criteria, duration of response (DOR), progression-free survival (PFS), overall survival (OS) and disease control rate (DCR). Secondary endpoints were median survival time (MST) and safety. Adverse events (AE) were rated according to the NCI Common Terminology Criteria for Adverse Events (CTCAE) 5.0. This trial is registered at ClinicalTrials.gov (NCT04038411). Results: From July 2020 to March 2022, 23 patients were screened and 20 eligible patients were enrolled from 4 centers in China, with median age at 36.5 years (range 17-60), 16 (80.0%) male, 15 (75.0%) patients with stage Ⅲ-Ⅳ of disease at screening, 10 (50.0%) patients received ≧2 lines of prior systemic therapy. Of 19 response evaluable patients, 18 (94.7%) achieved response including 9 (50%) patients with CR. The median time to initial response was 6.0 weeks (6-12 weeks). The 12-month PFS rate was 86.8%. 19 patients reported treatment-related AEs (TRAEs). The most frequently observed TRAEs were anemia (15 patients,78.9%), neutropenia (14 patients, 73.6%), leukopenia(11 patients, 57.9%), nausea (10 patients, 52.6%), thrombocytopenia (9 patients, 50%). The most frequent Grade (G) ≥3 TRAEs were neutropenia (9 patients, 50%). Immune-related AEs were reported in 4 patients (21.1%) with G1 hypothyroidism. No death was related to the study. Image:Summary/Conclusion: Tislelizumab combined with chidamide, lenalidomide and etoposide regimen have a very high response rate in r/r-ENKTL for the first time, and the safety is under control. It is a promising therapeutic option for this population, although further investigation is warranted.

Abstract 3279: Dual inhibition of EZH2 and histone deacetylases for the treatment of lymphomas with epigenetic aberrations

Abstract Background: Lymphomas are characterized by aberrations in epigenetic proteins that contribute to establishing and maintaining the malignant phenotype. Gain of function mutations in EZH2 histone methyltransferase and inactivating mutations in CREBBP acetyltransferase occur in up to 30% of diffuse large B cell lymphomas (DLBCLs). Half of DLBCLs with mutated EZH2 have also mutated CREBBP. In vitro, mutated EZH2 DLBCLs have a lower sensitivity to HDAC inhibitors, HDACi (Mensah et al, 2021). Here, we explored the dual pharmacological inhibition of EZH2 and HDAC for an improved anti-lymphoma activity. Methods: Biochemical evaluation and characterization of target engagement were done using fluorescence polarization, thermal shift, surface plasmon resonance, isothermal titration calorimetry and microscale thermophoresis. Computer modelling was performed using available EZH2 and HDACs crystal structures. Anti-proliferative activity was assessed after 7 days (d) using MTT and live imaging in DLBCL cells (n = 5). For cell cycle analysis by flow cytometry, cells were treated, fixed then stained with 7-AAD. Results: We designed and synthesized 2 dual EZH2/HDAC inhibitors, MC4343 and MC4355, starting from the structures of EZH2 inhibitor tazemetostat (taz) and HDACi vorinostat. In biochemical assays, MC4343 and MC4355 had equal potencies towards EZH2 (0.032 nM) but different specificities towards class I and class II HDACs: MC4355 showed 7.5-fold greater inhibition of HDAC3 compared to MC4343 (0.38 µM and 2.85 µM, respectively) and more potently inhibited HDACs 6 and 8 (0.016, 0.17 µM and no activity, respectively). Computational modelling showed that the coordinative functional group of MC4343, but not of MC4355, caused steric clashes with several HDACs in increasing order: HDAC1 = HDAC3 > HDAC8 >> HDAC4 = HDAC6. These results closely mirrored those obtained from the biochemical analysis. Both compounds inhibited proliferation in DLBCL cell lines irrespective of EZH2 or CREBBP mutational status but EZH2 and/or CREBBP mutants were more sensitive. MC4355 (IC50 range = 0.17 - 1.68 µM; median = 0.2 µM) was more potent than MC4343 (IC50 range = 0.17-2.72 µM; median = 1.78 µM). This was confirmed by live imaging analyses. SUDHL4, with EZH2 Y666N, showed poorer sensitivity to both inhibitors compared to DLBCLs with EZH2 Y646N/S. MC4343 and MC4355 induced cell death and G1 arrest in a dose-dependent manner. Pfeiffer, KARPAS422, WSUDLCL2, most sensitive to taz alone (IC50 = 4, 16, 77 nM), were most sensitive to MC4355 (IC50 = 200, 174, 171 nM). Notably, dual inhibitor treatment of Toledo and SUDHL4 with low sensitivity to taz (IC50 = 9, 14 µM), partially rescued sensitivity (IC50 = 1.7, 1.6 µM). Conclusions: We designed and synthesized 2 novel dual EZH2/HDAC inhibitors, MC4343 and MC4355, with robust anti-proliferative effects in DLBCL. Our data show the efficacy of this novel class of epigenetic agents in lymphomas. Citation Format: Afua Adjeiwaa Mensah, Sergio Valente, Milos Matkovic, Giulio Sartori, Chiara Falzarano, Chiara Tarantelli, Luciano Cascione, Stefano A. Pileri, Andrea Cavalli, Antonello Mai, Francesco Bertoni. Dual inhibition of EZH2 and histone deacetylases for the treatment of lymphomas with epigenetic aberrations [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 3279.

Abstract 2969: Histone deacetylase (HDAC) inhibitors exhibit antitumor activity in triple negative breast cancer via suppression of HER3 triggered signaling

Abstract Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high rate of recurrence and refractoriness due to lack of well-defined molecular targets. Human epidermal growth factor receptor 3 (HER3) is differentially expressed in TNBC cells. Numerous studies indicate that elevated expression of HER3, through its dimerization with another receptor, is a major cause of treatment failure in human cancers. To date, there is no FDA-approved HER3-targeted cancer therapy. HDAC inhibitors (HDACis) have been approved for the treatment of refractory or relapsed cutaneous T cell lymphomas and show potency in TNBC cells. However, it remains unclear whether the HDACis may alter HER3 expression to influence the downstream signaling in HER3-overexpressing (HER3high) TNBC. Methods: Colony formation, MTS and LIVE/DEAD cell staining assays were used to detect cell viability. Apoptosis was detected by flow cytometry assays. QRT-PCR, western blots and immunohistochemistry were performed to determine the expression and activation of genes and/or proteins. Co-immunoprecipitation was performed to assess the interaction between proteins. Lentivirus vectors containing cDNA or shRNAs were used to overexpress or knockdown gene expression. Chromatin immunoprecipitation-quantitative PCR and dual luciferase reporter assays were performed to elucidate the regulatory role of gene transcription. Results: Elevated expression of HER3 were observed in approximately half of the TNBC specimens and cell lines tested. HER3 was co-expressed and formed heterodimers with epidermal growth factor receptor (EGFR) to activate the PI-3K/Akt signaling in HER3high-TNBC cells. Panobinostat and romidepsin induced growth inhibition and apoptosis in HER3high-TNBC cells via downregulating HER3 expression and inhibiting PI-3K/Akt signaling. Significantly, HDACis in combination with an EGFR inhibitor (gefitinib) or an Akt inhibitor (Akti1/2) synergistically enhanced the anti-survival effects on HER3high-TNBC cells. Besides, analyses of gene expression profiling datasets revealed a significantly positive correlation between expression of HER3 and transcription factor forkhead box A1 (FOXA1). Further studies discovered that FOXA1 activated HER3 expression through directly binding to HER3 promoter. Moreover, ectopic expression of FOXA1 not only rescued the expression of HER3-downregulated by HDACis, but also attenuated these HDACis-mediated anti-survival effects on HER3high-TNBC cells. Conclusion: HDAC inhibitors exhibits potent inhibitory effects on HER3high-TNBC cells via downregulation of FOXA1-mediated repression of HER3 gene transcription. Our data suggest that epigenetic targeting of FOXA1-HER3/EGFR-PI-3K/Akt signaling axis may be an effective therapeutic strategy for eradication of HER3high-TNBC tumors. Citation Format: Congcong Tan, Hui Lyu, sanbao Ruan, bolin Liu. Histone deacetylase (HDAC) inhibitors exhibit antitumor activity in triple negative breast cancer via suppression of HER3 triggered signaling [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 2969.

Abstract 5632: Combinatorial epigenetic targeting of PRC2 complexes upregulates MHC antigen presentation components in melanoma and gastrointestinal cancer cells

Abstract Low tumor immunogenicity is associated with immune escape, immunotherapy resistance, and poor patient survival [1-3]. Major histocompatibility complex (MHC) antigen presentation pathways (APPs) play a crucial role in promoting tumor T-cell recognition and initiating antitumoral immune responses. However, epigenetic silencing of APPs is a common mechanism in various cancers, including melanoma and gastrointestinal malignancies [3,4]. In the current investigation, mouse melanoma (B16/OVA) and human colon cancer (LOVO and HCT116), small intestine cancer (HUTU80), and normal colonic epithelial (CCD841) cell lines were treated with epigenetic drug candidates. As reported [5], a decrease in histone H3K27 methylation marks via polycomb repressive complex 2 (PRC2) inhibitors, as well as altered histone acetylation status from histone deacetylase 3 (HDAC3)-specific inhibition, increased the expression of APP components, based on immunoblotting and RT-qPCR experiments. Normal colonic epithelial cells showed resistance to the treatments, indicating cancer cell-specific effects of the test agents. In B16/OVA cells, flow cytometry and enzyme linked immunosorbent assays corroborated that HDAC3 and/or PRC2 inhibitor combinations increased cell surface occupancy of MHC-I complexes and increased CD8+ T-cell activation. These findings have implications for the clinical application of next-generation combinatorial epigenetic agents [6,7], targeted towards increased tumor immunogenicity and enhanced efficacy of new immunoepigenetic strategies. Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.

Abstract 5713: Alternative splicing of the chromatin modifier gene BAZ1A in colorectal cancer

Abstract Bromodomain Adjacent to Zinc finger Domain 1A (BAZ1A) is a non-catalytic subunit of the ATP-dependent chromatin assembly factor that regulates key cellular processes such as DNA replication, chromatin assembly, DNA repair, and transcriptional gene regulation (1). Defective DNA repair is a hallmark of cancer etiology, and BAZ1A is one of the deregulated proteins in colorectal cancer. The BAZ1A transcript exhibits full-length and truncated forms, generated via alternative splicing of exon-13. We observed that the histone deacetylase inhibitor sulforaphane (SFN) modulated alternative splicing of BAZ1A in human colon cancer cells by increasing the relative expression of the short vs. the long transcript. Cell Cycle and Apoptosis Regulator 2 (CCAR2) and Zinc finger protein 326 (ZIRD) are components of the DBIRD complex that integrates alternative splicing and the rate of transcript elongation (2). Knockdown of DBIRD members, including ZIRD and splicing factor hnRNPA1, downregulated expression of BAZ1A long form, whereas the BAZ1A short form was unaltered. Previous studies in SFN-treated colon cancer cells revealed that the BAZ1A bromodomain recognized a novel K97 acetylation site in CCAR2 (3). Co-immunoprecipitation analyses revealed that CCAR2 and BAZ1A interacted with each other in the presence and absence of SFN. On investigating the involvement of CCAR2 in BAZ1A alternative splicing, SFN treatment increased the expression of both BAZ1A long and short forms in CCAR2-/- HCT116 colon cancer cells. Although the total expression of BAZ1A was influenced by CCAR2 loss, alternative splicing of BAZ1A was independent of DBIRD complex-mediated regulation. Pursuing the role of p53 and p21 in BAZ1A alternative splicing, SFN treatment increased the expression of BAZ1A short form in p21-/- HCT116 cells, whereas this was impaired in p53-/- HCT116 cells. Overall, this investigation provided mechanistic insights into the regulation of alternative splicing in SFN-treated colon cancer cells, and BAZ1A functionalities that might be targeted via next-generation combinatorial epigenetic agents (4). Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.

Abstract 5701: HDAC inhibition decreases YAP1 expression but parodoxically activates TEAD transcription by the AP-1 pathway in breast cancer

Abstract The Hippo pathway is an evolutionally conserved pathway that is crucial in numerous aspects of cell biology, including contact inhibition, cell density, and others. Hippo pathway is well reported to be dysregulated in multiple cancer types and plays important oncogenic roles in human cancers. In mammalian cells, YAP is regulated by a phosphorylated cascade mediated by the kinases MST1/2 and LATS1/2, known as the canonical Hippo pathway. These upstream kinases phosphorylate YAP/TAZ and leads to its cytosolic degradation. Perturbation of the Hippo pathway leads to YAP intranuclear translocation and downstream activation of target genes, inducing proliferative signals and thus initiating oncogenesis. Novel mechanism linking the hippo pathway to various oncogenic mechanisms are rapidly discovered and reported, further establishing the significance of this pathway and its role in cancer biology. Breast cancer is the top leading cause of cancer in women and poses a significant morbidity and economic burden on society. Hormone positive (defined as positivity of the estrogen receptor ER and/or the progesterone receptor PR) breast cancer composes 70% of breast cancer. Endocrine therapy, chemotherapy, and more recently, CDK4/6 inhibitors and PI3K inhibitors are currently approved strategies towards breast cancer. Histone deacetylase (HDAC) inhibitors have been FDA approved for several hematological cancers, and recently have shown inconsistent results in clinical trials for breast cancer. In our study, we demonstrate and characterize a link between HDAC inhibition and the Hippo pathway. We discover that HDAC inhibitors suppress YAP expression both transcriptionally and translationally. Paradoxically, we demonstrate that although YAP is downregulated, TEAD mediated transcription of downstream Hippo targets is activated. Mechanistically, HDAC inhibition leads to upregulation of the AP-1 transcriptional machinery that cooperates with TEAD to facilitate transcription. By RNA-seq, chromatin studies and CRISPR knockout studies, we confirm that HDAC inhibition have the ability to activate downstream of Hippo pathway, suggesting a possible mechanism for breast cancer resistance towards HDAC inhibitor. Co-targeting HDAC inhibition and AP-1 results in enhanced efficacy of HDAC inhibitors and may be a promising approach for development towards breast cancer. Citation Format: Ting-Yi Lin, Yi-Ru Tseng, Naikuan Wang, Ta-Chung Chao, Chun-Yu Liu, Ling-Ming Tseng, Jiun-I Lai. HDAC inhibition decreases YAP1 expression but parodoxically activates TEAD transcription by the AP-1 pathway in breast cancer [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 5701.

Abstract 2673: Combined inhibition of PI3K and HDAC1/2 as a novel treatment strategy for RAS/RAF wildtype colorectal cancer in a patient derived organoid model

Abstract Background: Treating colorectal cancer (CRC) using targeted therapies based on each patient’s individual mutational profile continues to gain interest. Oncogenic mutations of the PIK3CA gene leads to tumor initiation and progression. Copanlisib inhibits the PI3K pathway and has proven to be an effective treatment strategy for various cancers characterized by overactivation of the PI3K signaling cascade. However, resistance mechanisms to targeted therapies such as copanlisib are frequently encountered, and can be overcome through combination with another molecule such as romidepsin, a histone deacetylase (HDAC) inhibitor. Here we aim to identify a novel therapeutic regimen for improved treatment of the molecular subtypes of CRC that can be targeted with PI3K inhibition. Methods: Organoids were derived from colorectal cancer tumor tissue from two different biopsy sites from one patient with PIK3CA mutant rectal cancer (RC46A and RC46B), as well as from a patient with APC and TP53 mutant CRC (MTB74) under approved IRB protocols. All organoids were plated and allowed to mature for 24 to 48 hours before treatment and baseline images were obtained. The organoids were imaged again 48 hours later, and response was determined by measuring the change in organoid diameter using ImageJ. Additionally, organoids were collected for immunoblotting to examine alterations in the PI3K pathway as well as histone acetylation, and proteins involved in apoptosis. Results: Control organoids demonstrated increased median relative changes in diameter of +53.38%, +43.04% and +46.2% for RC46A, RC46B and MTB74, respectively. In comparison, the combination treatment group exhibited median relative changes for RC46A, RC46B and MTB74 as -29.19%, -46.46% and -100% (p<0.001). These results were supported with immunoblot analysis that revealed a synergistic increase in histone acetylation with the combination treatment. Single agent copanlisib treatment elicited alterations in PI3K signaling including downregulation of pS6, pAKT, p4EBP1 as expected. Lastly, an increase in cleaved PARP was observed in combination treated organoids which is indicative of increased apoptosis. Conclusions: As molecular diagnostic technology continues to advance, treating each individual cancer with a targeted therapy based on molecular profiling is becoming more feasible. Dual inhibition of the PI3K pathway and HDAC is effective in reducing cell growth and proliferation of CRC organoids characterized by PIK3CA mutations, as well as other molecular subtypes such as cancers with APC and TP53 mutations. This combination strategy shows promise for improving treatment response and reducing resistance in patients across these molecular subtypes. Citation Format: Alyssa K. DeZeeuw, Rebecca A. DeStefanis, Susan N. Payne, Autumn M. Olson, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Combined inhibition of PI3K and HDAC1/2 as a novel treatment strategy for RAS/RAF wildtype colorectal cancer in a patient derived organoid model [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 2673.