Histone Deacetylase Inhibitor Vorinostat Research Articles

EXTH-27. POLYCOMB OVERACTIVITY DRIVES TUMORIGENESIS IN AGGRESSIVE CUSHING’S DISEASE

Abstract INTRODUCTION Aggressive Cushing’s disease (CD) tumors (Ki67 > 3%) exhibit rapid growth, poor postsurgical outcomes, and resistance to conventional therapies. Polycomb group (PcG) proteins are canonical epigenetic silencers that can drive tumor aggressiveness. We asked if PcG underlies rare but fatal pituitary tumor progression. METHODS We performed paired multiome analysis (single nucleus RNAseq and ATACseq) in 5 aggressive CD adenomas, and adjacent normal gland. We verified protein expression with multiplexed immunohistochemistry (mIHC) and PcG activity by H3K27me3 ChIPseq. Aggressive murine pituitary tumor cells ATT20 were treated using PcG inhibitors (DZNep, tazemetostat), HDAC inhibitor (vorinostat), or siRNA (Ezh2, Suz12). PcG binding to the POMC promoter was assessed by reverse chromatin immunoprecipitation (rChIP). We treated one patient off-label with oral vorinostat 400mg daily for 6 days prior to radiation. RESULTS PcG loci (EZH2 and EED) were more accessible in aggressive CD adenoma versus normal gland corticotrophs, and EZH2, SUZ12 and EED mRNA was robustly overexpressed. mIHC confirmed SUZ12, EZH2 and H3K27me3 overexpression, and ChIPseq confirmed polycomb overactivity in aggressive CD adenomas. Polycomb inhibition by DZNep, vorinostat, or siRNAs led to proliferative arrest and Pomc suppression in ATT20 cells (P < 0.05 versus controls). Vorinostat enriched Suz12 at the Pomc promoter (P < 0.01), confirming direct silencing. In a patient with aggressive CD and hypercortisolemia despite two prior surgeries, a single course of vorinostat decreased cortisol (24mcg/dL to 7.5mcg/dL) and ACTH (63.7pg/mL to 44.9pg/mL) prior to radiation therapy initiation. CONCLUSIONS PcG hyperactivity is associated with a more aggressive CD phenotype. We report here the therapeutic potential of HDAC inhibition against aggressive CD adenomas in a first-in-human application.

Discovery of Janus Kinase and Histone Deacetylase Dual Inhibitors as a New Strategy to Treat Psoriasis.

Psoriasis is a common, chronic, recurrent, and inflammatory skin disease, which causes physical and psychological problems in patients and lacks effective and economic therapeutics. Herein, we designed Janus kinase (JAK) and histone deacetylase (HDAC) dual inhibitors as a new strategy for the treatment of psoriasis. In particular, compound 11i was identified with excellent inhibitory activity toward JAKs (JAK2 IC50 = 0.49 nM) and HDACs (HDAC6 IC50 = 12 nM). Moreover, it exhibited potent activities in inhibiting the proliferation of TNF-α-induced HaCAT cells and the production of nitric oxide. Importantly, compound 11i significantly ameliorated psoriasis-like skin lesions in an imiquimod-induced murine model with low toxicity, which was superior to JAK inhibitor momelotinib, HDAC inhibitor vorinostat, and their combination. This work provided a proof-of-concept for JAK/HDAC dual inhibitors as a promising strategy for the treatment of psoriasis.

Combination treatment with histone deacetylase and carbonic anhydrase 9 inhibitors shows therapeutic potential in experimental diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) remains a significant therapeutic challenge due to the lack of effective and safe treatment options. This study explores the potential of combining histone deacetylase (HDAC) and carbonic anhydrase 9 (CA9) inhibitors in treating DIPG. Analysis of RNA sequencing data and tumor tissue from patient samples for the expression of the carbonic anhydrase family and hypoxia signaling pathway activity revealed clinical relevance for targeting CA9 in DIPG. A synergy screen was conducted using CA9 inhibitor SLC-0111 and HDAC inhibitors panobinostat, vorinostat, entinostat, and pyroxamide. The combination of SLC-0111 and pyroxamide demonstrated the highest synergy and was selected for further analysis. Combining SLC-0111 and pyroxamide effectively inhibited DIPG cell proliferation, reduced cell migration and invasion potential, and enhanced histone acetylation, leading to decreased cell population in S Phase. Additionally, the combination therapy induced a greater reduction in intracellular pH than either agent alone. Data from this study suggest that the combination of SLC-0111 and pyroxamide holds promise for treating experimental DIPG, and further investigation of this combination therapy in preclinical models is warranted to evaluate its potential as a viable treatment for DIPG.

Elevated choline drives KLF5-dominated transcriptional reprogramming to facilitate liver cancer progression.

An increase in the total choline-containing compound content is a common characteristic of cancer cells, and aberrant choline metabolism in cancer is closely associated with malignant progression. However, the potential role of choline-induced global transcriptional changes in cancer cells remains unclear. In this study, we reveal that an elevated choline content facilitates hepatocellular carcinoma (HCC) cell proliferation by reprogramming Krüppel-like factor 5 (KLF5)-dominated core transcriptional regulatory circuitry (CRC). Mechanistically, choline administration leads to elevated S-adenosylmethionine (SAM) levels, inducing the formation of H3K4me1 within the super-enhancer (SE) region of KLF5 and activating its transcription. KLF5, as a key transcription factor (TF) of CRC established by choline, further transactivates downstream genes to facilitate HCC cell cycle progression. Additionally, KLF5 can increase the expression of choline kinase-α (CHKA) and CTP:phosphocholine cytidylyltransferase (CCT) resulting in a positive feedback loop to promote HCC cell proliferation. Notably, the histone deacetylase inhibitor (HDACi) vorinostat (SAHA) significantly suppressed KLF5 expression and liver tumor growth in mice, leading to a prolonged lifespan. In conclusion, these findings highlight the epigenetic regulatory mechanism of the SE-driven key regulatory factor KLF5 conducted by choline metabolism in HCC and suggest a potential therapeutic strategy for HCC patients with high choline content.

Simultaneous inhibition of FLT3 and HDAC by novel 6-ethylpyrazine-2-Carboxamide derivatives provides therapeutic advantages in acute myelocytic leukemia

Synergetic inhibition of FMS-like tyrosine kinase 3 (FLT3) and histone deacetylase (HDAC) by small molecule chimera presents a promising therapeutic approach for acute myeloid leukemia (AML) with FLT3 mutations. In this study, we first observed that the combined use of FLT3 inhibitor gilteritinib and HDAC inhibitor vorinostat increased the survival rate of leukemia xenograft mouse model. Then, we employed a pharmacophore fusion strategy to develop a novel series of FLT3/HDAC dual inhibitors. Among them, compound 25h demonstrated superior inhibitory activity against both FLT3 and HDAC. In particular, compound 25h exhibited enhanced anti-proliferation activity against MOLM-13 cells in comparison to gilteritinib, vorinostat, and their combination, while maintaining reduced cytotoxicity towards normal cells. Mechanistically, the heightened anti-tumor effect of compound 25h was attributed to its more potent regulation of intracellular pathways, notably phosphorylation of ERK, compared to single drug and combination treatments. Furthermore, compound 25h demonstrated superior anti-tumor efficacy in the MOLM-13 xenograft model compared to combination therapy, along with reduced in vivo toxicity. To conclude, we have identified a novel FLT3/HDAC dual inhibitor that could serve as a potential candidate for the treatment of AML.

Synergistic Cytotoxicity of Histone Deacetylase and Poly-ADP Ribose Polymerase Inhibitors and Decitabine in Breast and Ovarian Cancer Cells: Implications for Novel Therapeutic Combinations.

Breast and ovarian cancers pose significant therapeutic challenges. We explored the synergistic cytotoxicity of histone deacetylase inhibitors (HDACis), poly(ADP-ribose) polymerase inhibitors (PARPis), and decitabine in breast (MDA-MB-231 and MCF-7) and ovarian (HEY-T30 and SKOV-3) cancer cell lines that were exposed to HDACi (panobinostat or vorinostat), PARPi (talazoparib or olaparib), decitabine, or their combinations. HDACi, PARPi, and decitabine combinations had synergistic cytotoxicity (assessed by MTT and clonogenic assays) in all cell lines (combination index < 1). Clonogenic assays confirmed the sensitivity of breast and ovarian cancer cell lines to the three-drug combinations (panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine; vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine). Cell proliferation was inhibited by 48-70%, and Annexin V positivity was 42-59% in all cell lines exposed to the three-drug combinations. Western blot analysis showed protein PARylation inhibition, caspase 3 and PARP1 cleavage, and c-MYC down-regulation. The three-drug combinations induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs, impaired the DNA repair pathways, and altered the epigenetic regulation of gene expression. These results indicate that HDACi, PARPi, and decitabine combinations should be further explored in these tumor types. Further clinical validation is warranted to assess their safety and efficacy.

Dual targeting of histone deacetylases and MYC as potential treatment strategy for H3-K27M pediatric gliomas.

Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.

Dual targeting of histone deacetylases and MYC as potential treatment strategy for H3-K27M pediatric gliomas

Diffuse midline gliomas (DMGs) are aggressive and fatal pediatric tumors of the central nervous system that are highly resistant to treatments. Lysine to methionine substitution of residue 27 on histone H3 (H3-K27M) is a driver mutation in DMGs, reshaping the epigenetic landscape of these cells to promote tumorigenesis. H3-K27M gliomas are characterized by deregulation of histone acetylation and methylation pathways, as well as the oncogenic MYC pathway. In search of effective treatment, we examined the therapeutic potential of dual targeting of histone deacetylases (HDACs) and MYC in these tumors. Treatment of H3-K27M patient-derived cells with Sulfopin, an inhibitor shown to block MYC-driven tumors in vivo, in combination with the HDAC inhibitor Vorinostat, resulted in substantial decrease in cell viability. Moreover, transcriptome and epigenome profiling revealed synergistic effect of this drug combination in downregulation of prominent oncogenic pathways such as mTOR. Finally, in vivo studies of patient-derived orthotopic xenograft models showed significant tumor growth reduction in mice treated with the drug combination. These results highlight the combined treatment with PIN1 and HDAC inhibitors as a promising therapeutic approach for these aggressive tumors.

Synergistic cytotoxicity of histone deacetylase and poly-ADP ribose polymerase inhibitors and decitabine in pancreatic cancer cells: Implications for novel therapy.

Histone deacetylase inhibitors (HDACi) can modulate the acetylation status of proteins, influencing the genomic instability exhibited by cancer cells. Poly (ADP ribose) polymerase (PARP) inhibitors (PARPi) have a direct effect on protein poly (ADP-ribosyl)ation, which is important for DNA repair. Decitabine is a nucleoside cytidine analogue, which when phosphorylated gets incorporated into the growing DNA strand, inhibiting methylation and inducing DNA damage by inactivating and trapping DNA methyltransferase on the DNA, thereby activating transcriptionally silenced DNA loci. We explored various combinations of HDACi and PARPi +/- decitabine (hypomethylating agent) in pancreatic cancer cell lines BxPC-3 and PL45 (wild-type BRCA1 and BRCA2) and Capan-1 (mutated BRCA2). The combination of HDACi (panobinostat or vorinostat) with PARPi (talazoparib or olaparib) resulted in synergistic cytotoxicity in all cell lines tested. The addition of decitabine further increased the synergistic cytotoxicity noted with HDACi and PARPi, triggering apoptosis (evidenced by increased cleavage of caspase 3 and PARP1). The 3-drug combination treatments (vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine; panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine) induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs and impaired the DNA repair pathways (decreased levels of ATM, BRCA1, and ATRX proteins). The 3-drug combinations also altered the epigenetic regulation of gene expression (NuRD complex subunits, reduced levels). This is the first study to demonstrate synergistic interactions between the aforementioned agents in pancreatic cancer cell lines and provides preclinical data to design individualized therapeutic approaches with the potential to improve pancreatic cancer treatment outcomes.

A Proof-of-Concept Study of Sequential Treatment with the HDAC Inhibitor Vorinostat following BRAF and MEK Inhibitors in BRAFV600-Mutated Melanoma.

The development of resistance limits the clinical benefit of BRAF and MEK inhibitors (BRAFi/MEKi) in BRAFV600-mutated melanoma. It has been shown that short-term treatment (14 days) with vorinostat was able to initiate apoptosis of resistant tumor cells. We aimed to assess the antitumor activity of sequential treatment with vorinostat following BRAFi/MEKi in patients with BRAFV600-mutated melanoma who progressed after initial response to BRAFi/MEKi. Patients with BRAFi/MEKi-resistant BRAFV600-mutated melanoma were treated with vorinostat 360 mg once daily for 14 days followed by BRAFi/MEKi. The primary endpoint was an objective response rate of progressive lesions of at least 30% according to Response Evaluation Criteria in Solid Tumors 1.1. Secondary endpoints included progression-free survival, overall survival, safety, pharmacokinetics of vorinostat, and translational molecular analyses using ctDNA and tumor biopsies. Of the 26 patients with progressive BRAFi/MEKi-resistant BRAFV600-mutated melanoma receiving treatment with vorinostat, 22 patients were evaluable for response. The objective response rate was 9%, with one complete response for 31.2 months and one partial response for 14.9 months. Median progression-free survival and overall survival were 1.4 and 5.4 months, respectively. Common adverse events were fatigue (23%) and nausea (19%). ctDNA analysis showed emerging secondary mutations in NRAS and MEK in eight patients at the time of BRAFi/MEKi resistance. Elimination of these mutations by vorinostat treatment was observed in three patients. Intermittent treatment with vorinostat in patients with BRAFi/MEKi-resistant BRAFV600-mutated melanoma is well tolerated. Although the primary endpoint of this study was not met, durable antitumor responses were observed in a minority of patients (9%).

THE ROLE OF THE VIRAL PROTEIN VPR IN THE EPIGENETIC REGULATION OF HIV-1 LATENCY

Abstract The primary obstacle to HIV-1 cure is the persistence of a long-lived latent reservoir, best characterized in memory CD4+ T cells. New research supports latency prevention as a therapeutic strategy. However, such an approach will require a better understanding of how HIV-1 establishes latency. We recently showed that histone deacetylases (HDACs) are critical for entry into latency. Whether HDACs interact with specific viral factors, particularly the HIV-1 accessory protein and coactivator Vpr, to regulate latency establishment is unknown. We therefore sought to determine how Vpr, both alone and in combination with the HDAC inhibitor vorinostat (VOR), regulates HIV-1 latency establishment. In a primary CD4+ T-cell model, we observed a p300-dependent synergy between Vpr and VOR in maintaining viral gene expression. We next assessed whether Vpr delivery during latent infection, a stage when viral proteins are not normally expressed, could enhance the ability of VOR alone to induce viral gene expression. We found that Vpr delivery reactivated latently infected CD4+ T cells to a greater extent than VOR treatment alone and that the combination of Vpr delivery and VOR treatment led to the highest level of reactivation. An improved understanding of how host and viral factors interact during HIV-1 latency establishment in CD4+ T cells will aid in the development of new therapies that prevent HIV-1 entry into the latent reservoir or that reverse latency in latently infected cells.

HDACi vorinostat protects muscle from degeneration after acute rotator cuff injury in mice.

Rotator cuff (RC) injuries are characterized by tendon rupture, muscle atrophy, retraction, and fatty infiltration, which increase injury severity and jeopardize adequate tendon repair. Epigenetic drugs, such as histone deacetylase inhibitors (HDACis), possess the capacity to redefine the molecular signature of cells, and they may have the potential to inhibit the transformation of the fibro-adipogenic progenitors (FAPs) within the skeletal muscle into adipocyte-like cells, concurrently enhancing the myogenic potential of the satellite cells. HDACis were added to FAPs and satellite cell cultures isolated from mice. The HDACi vorinostat was additionally administered into a RC injury animal model. Histological analysis was carried out on the isolated supra- and infraspinatus muscles to assess vorinostat anti-muscle degeneration potential. Vorinostat, a HDACi compound, blocked the adipogenic transformation of muscle-associated FAPs in culture, promoting myogenic progression of the satellite cells. Furthermore, it protected muscle from degeneration after acute RC in mice in the earlier muscle degenerative stage after tenotomy. The HDACi vorinostat may be a candidate to prevent early muscular degeneration after RC injury.

Abstract 1850: From proteomic analysis to early investigations of combining inhibitors targeting PCNA and histone deacetylase

Abstract Proteomic changes were investigated in a neuroblastoma cell line, SK-N-AS, treated with a small molecule inhibitor, AOH1996, designed to specifically bind proliferating cell nuclear antigen (PCNA) in cancer. PCNA is crucial to DNA replication, DNA repair, transcription, cell cycle control, and chromatin organization through interactions with different proteins. Pathway analysis of differential protein expressions between treated and untreated cells revealed less G2/M DNA damage checkpoint regulation and decreased assembly of RNA polymerase complex. Phosphoproteomic analysis by PTM Signature Enrichment Analysis (PTM-SEA) identified signatures of kinase activities, signaling pathways, and perturbations. Specifically, the histone deacetylase inhibitors belinostat and vorinostat were identified as enriched perturbation signatures. Both inhibitors were tested in combination with AOH1996 in SK-N-AS as well as cell lines derived from breast, lung, ovarian, and pancreatic cancer, revealing drug synergies at specific dose combinations. Western blot analysis showed that belinostat decreased protein expression of PCNA, suggestive of mechanistic synergies with AOH1996+belinostat combination therapies. Our proteomic analysis reveals a therapeutic potential of combining AOH1996 and histone deacetylase inhibitors for treating cancer with possibly lower drug dose and toxicities compared to single drug treatment. Citation Format: Caroline M. Li, Krystine Garcia-Mansfield, Robert G. Lingeman, Long Gu, Robert J. Hickey, Patrick Pirrotte, Linda H. Malkas. From proteomic analysis to early investigations of combining inhibitors targeting PCNA and histone deacetylase [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 1850.

Abstract 2638: Sensitizing immunotherapy-refractory prostate cancer to immune checkpoint blockade therapy with optimized ketogenic diet regimens

Abstract The purpose of this study is to investigate strategies for enhancing immune checkpoint blockade (ICB) therapy in prostate cancer (PCa). To achieve this, we established a series of ICB-resistant PCa cell lines derived from a previously sensitive cell line, which originated from a murine model of metastatic PCa. Notably, we observed a reduction in major histocompatibility complex (MHC) class I protein levels in the resistant sublines. Our study demonstrated synergistic efficacy through the combination of anti-PD1 and anti-CTLA4 antibodies with histone deacetylase inhibitor (HDACi) vorinostat, a cyclic ketogenic diet (CKD), or β-hydroxybutyrate (BHB) supplementation. Importantly, these interventions significantly delayed PCa tumor progression. Furthermore, our analysis revealed that the effects of these interventions were contingent on both BHB and adaptive immunity. In-depth single-cell transcriptomic and proteomic profiling unveiled mechanisms involving cancer-cell-intrinsic changes (upregulated MHC class I molecules) and extrinsic factors, including CD8+ T cell chemoattraction, M1/M2 macrophage rebalancing, monocyte differentiation toward antigen-presenting cells, and reduced neutrophil presence. In conclusion, our findings provide valuable insights into the potential clinical utility of combining HDACi and optimized ketogenic diet regimens to enhance ICB therapy for PCa. Citation Format: Sean Murphy, Sharif Rahmy, Dailin Gan, Jun Li, Xuemin Lu, Xin Lu. Sensitizing immunotherapy-refractory prostate cancer to immune checkpoint blockade therapy with optimized ketogenic diet regimens [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 2638.

The Effects of Human Immunodeficiency Virus Type 1 (HIV-1) Antigen-Expanded Specific T-Cell Therapy and Vorinostat on Persistent HIV-1 Infection in People With HIV on Antiretroviral Therapy.

The histone deacetylase inhibitor vorinostat (VOR) can reverse human immunodeficiency virus type 1 (HIV-1) latency in vivo and allow T cells to clear infected cells in vitro. HIV-specific T cells (HXTCs) can be expanded ex vivo and have been safely administered to people with HIV (PWH) on antiretroviral therapy. Six PWH received infusions of 2 × 107 HXTCs/m² with VOR 400 mg, and 3 PWH received infusions of 10 × 107 HXTCs/m² with VOR. The frequency of persistent HIV by multiple assays including quantitative viral outgrowth assay (QVOA) of resting CD4+ T cells was measured before and after study therapy. VOR and HXTCs were safe, and biomarkers of serial VOR effect were detected, but enhanced antiviral activity in circulating cells was not evident. After 2 × 107 HXTCs/m² with VOR, 1 of 6 PWH exhibited a decrease in QVOA, and all 3 PWH exhibited such declines after 10 × 107 HXTCs/m² and VOR. However, most declines did not exceed the 6-fold threshold needed to definitively attribute decline to the study intervention. These modest effects provide support for the strategy of HIV latency reversal and reservoir clearance, but more effective interventions are needed to yield the profound depletion of persistent HIV likely to yield clinical benefit. Clinical Trials Registration. NCT03212989.

Androgen receptor blockade to activate NK cells and to upregulate the surface expression of HLA-E in prostate cancer cell lines.

190 Background: The blockade of the androgen receptor (AR) pathway is an effective treatment for prostate cancer (PCa), but most patients progress to castration-resistant prostate cancer (mCRPC). AR signaling modulates CD8+ T cytotoxic function, revealing the immune-modulatory function of the AR pathway. We have described the activation of NK cells by AR inhibitors and the potential synergistic effect with anti-NKG2a combination.1 Strategies to activate NK cells can be limited by the expression of HLA-E, a ligand of the inhibitory checkpoints on NK cells NKG2A. We investigated the mechanisms of AR-dependent modulation of HLA-E on tumor cells and the ADT-enhancing effect on patient-derived NK cells. Methods: PC cell lines were treated with second-generation androgen pathway inhibitors in vitro (enzalutamide 10uM, darolutamide 15 uM), and the expression of HLA-E was evaluated by flow cytometry. To evaluate the modulation of HLA-E by AR, the AR-negative cell lines (PC3 and DU145) were stably transduced with an inducible AR system. The pan HDAC inhibitors vorinostat (0.4 uM) and panobinostat (2.5 uM) were used to evaluate the regulation of HLA-E by epigenetics. We analyzed the activation status of paired peripheral blood NK cells isolated from patients with PCa prior to and post-androgen deprivation therapy (ADT). The patients (n=6) had a median time between collection of 26.3±2.3 days. Results: The AR-responsive LNCaP cell line displayed an increase in surface expression of HLA-E upon treatment with enzalutamide (Enza) or darolutamide (Daro) ([C]: 12.1±1.3%, Enza: 22.3±3.3%, Daro: 19.1±1.1%, p=0.004). AR blockade did not change the surface expression of HLA-E of AR negative PC3 ([C]: 10.5±2.7%, Daro: 13.1±2.3%, p=0.17) or DU145 cell lines ([C]: 3.5±1.7%, Daro: 2.5±0.6%, p=0.12). Transduction of AR in PC3 and DU145 lead to upregulation of HLA-E expression with AR blockade (PC3-AR+ [C]: 13.1±3.7%, Daro: 43.1±5.3%, DU145-AR+ [C]: 4.5±1.4%, Daro: 27.2±3.6%, p=0.001). The upregulation of HLA-E upon AR blockade was suppressed if cell lines were co-treated with vorinostat or panobinostat. Patient-derived peripheral blood NK cells displayed enhanced cytotoxic activity after ADT (expression of Granzyme B pre-ADT: 12.3±2.3%, post-ADT: 36.5±5.7% p=0.0017, Perforin pre-ADT: 3±0.6%, post-ADT: 26.5±3.7% p=0.001), and upregulation of the inhibitory checkpoint NKG2a (pre-ADT: 5.2±1.2%, post-ADT: 12.3±1.4%, p=0.04). Conclusions: Androgen inhibitors upregulate the expression of HLA-E in PCa cell lines by an AR-dependent mechanism regulated by epigenetics. ADT promotes peripheral blood patient-derived NK cell activation and upregulation of inhibitory NKG2A receptor. These findings support further investigative approaches targeting the HLA-E and NKG2A in mCRPC. 1. Schwermann, AACR 2023.

The FOXP3+ Pro-Inflammatory T Cell: A Potential Therapeutic Target in Crohn’s Disease

Background and aimsThe incidence of Crohn’s disease (CD) continues to increase worldwide. The contribution of CD4+ cell populations remains to be elucidated. Here, we aim to provide an in-depth transcriptional assessment of CD4+ T cells driving chronic inflammation in CD. MethodsWe performed single cell RNA-sequencing in CD4+ T cells isolated from ileal biopsies of CD patients compared to healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signalling networks. We overlapped our differentially expressed genes with publicly available microarray datasets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD TREGs and to test predicted therapeutics. ResultsWe identified five distinct FOXP3+ regulatory T (Treg) subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into inflammation-associated subtypes. These proinflammatory Tregs displayed a unique responsiveness to TNFα signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid co-culture system. As predicted in silico, the histone deacetylase inhibitor Vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3+ cells in vitro. ConclusionWe identified a novel, proinflammatory FOXP3+ T cell subpopulation in CD patients and developed a pipeline to specifically target these cells using the FDA-approved drug Vorinostat.

Staphylococcus aureus induces drug resistance in cancer T cells in Sézary syndrome

AbstractPatients with Sézary syndrome (SS), a leukemic variant of cutaneous T-cell lymphoma (CTCL), are prone to Staphylococcus aureus infections and have a poor prognosis due to treatment resistance. Here, we report that S aureus and staphylococcal enterotoxins (SE) induce drug resistance in malignant T cells against therapeutics commonly used in CTCL. Supernatant from patient-derived, SE-producing S aureus and recombinant SE significantly inhibit cell death induced by histone deacetylase (HDAC) inhibitor romidepsin in primary malignant T cells from patients with SS. Bacterial killing by engineered, bacteriophage-derived, S aureus–specific endolysin (XZ.700) abrogates the effect of S aureus supernatant. Similarly, mutations in major histocompatibility complex (MHC) class II binding sites of SE type A (SEA) and anti-SEA antibody block induction of resistance. Importantly, SE also triggers resistance to other HDAC inhibitors (vorinostat and resminostat) and chemotherapeutic drugs (doxorubicin and etoposide). Multimodal single-cell sequencing indicates T-cell receptor (TCR), NF-κB, and JAK/STAT signaling pathways (previously associated with drug resistance) as putative mediators of SE-induced drug resistance. In support, inhibition of TCR-signaling and Protein kinase C (upstream of NF-κB) counteracts SE-induced rescue from drug-induced cell death. Inversely, SE cannot rescue from cell death induced by the proteasome/NF-κB inhibitor bortezomib. Inhibition of JAK/STAT only blocks rescue in patients whose malignant T-cell survival is dependent on SE-induced cytokines, suggesting 2 distinct ways SE can induce drug resistance. In conclusion, we show that S aureus enterotoxins induce drug resistance in primary malignant T cells. These findings suggest that S aureus enterotoxins cause clinical treatment resistance in patients with SS, and antibacterial measures may improve the outcome of cancer-directed therapy in patients harboring S aureus.

Efficacy of HDAC Inhibitors in Driving Peroxisomal β-Oxidation and Immune Responses in Human Macrophages: Implications for Neuroinflammatory Disorders.

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by β-oxidation. Impaired VLCFA catabolism due to primary or secondary peroxisomal alterations is featured in neurodegenerative and neuroinflammatory disorders such as X-linked adrenoleukodystrophy and multiple sclerosis (MS). Here, we identified that healthy human macrophages upregulate the peroxisomal genes involved in β-oxidation during myelin phagocytosis and pro-inflammatory activation, and that this response is impaired in peripheral macrophages and phagocytes in brain white matter lesions in MS patients. The pharmacological targeting of VLCFA metabolism and peroxisomes in innate immune cells could be favorable in the context of neuroinflammation and neurodegeneration. We previously identified the epigenetic histone deacetylase (HDAC) inhibitors entinostat and vorinostat to enhance VLCFA degradation and pro-regenerative macrophage polarization. However, adverse side effects currently limit their use in chronic neuroinflammation. Here, we focused on tefinostat, a monocyte/macrophage-selective HDAC inhibitor that has shown reduced toxicity in clinical trials. By using a gene expression analysis, peroxisomal β-oxidation assay, and live imaging of primary human macrophages, we assessed the efficacy of tefinostat in modulating VLCFA metabolism, phagocytosis, chemotaxis, and immune function. Our results revealed the significant stimulation of VLCFA degradation with the upregulation of genes involved in peroxisomal β-oxidation and interference with immune cell recruitment; however, tefinostat was less potent than the class I HDAC-selective inhibitor entinostat in promoting a regenerative macrophage phenotype. Further research is needed to fully explore the potential of class I HDAC inhibition and downstream targets in the context of neuroinflammation.

HDAC inhibitors: Promising agents for leukemia treatment

The essential role of epigenetic modification in the pathogenesis of a series of cancers have gradually been recognized. Histone deacetylase (HDACs), as well-known epigenetic modulators, are responsible for DNA repair, cell proliferation, differentiation, apoptosis and angiogenesis. Studies have shown that aberrant expression of HDACs is found in many cancer types. Thus, inhibition of HDACs has provided a promising therapeutic approach alternative for these patients. Since HDAC inhibitor (HDACi) vorinostat was first approved by the Food and Drug Administration (FDA) for treating cutaneous T-cell lymphoma (CTCL) in 2006, the combination of HDAC inhibitors with other molecules such as chemotherapeutic drugs has drawn much attention in current cancer treatment, especially in hematological malignancies therapy. Up to now, there have been more than twenty HDAC inhibitors investigated in clinic trials with five approvals being achieved. Indeed, Histone deacetylase inhibitors promote or enhance several different anticancer mechanisms and therefore are in evidence as potential antileukemia agents. In this review, we will focus on possible mechanisms by how HDAC inhibitors exert therapeutic benefit and their clinical utility in leukemia.