Combination Of Histone Deacetylase Inhibitors Research Articles

DIPG-74. TARGETING TRANSCRIPTIONAL DYSREGULATION IN DMG THROUGH CDK9 AND HDAC INHIBITOR COMBINATION THERAPY

Abstract BACKGROUND With no successful treatment options available, diffuse midline glioma (DMG) are fatal brain tumors with a median survival of ∼12 months. DMG are characterized by transcriptional dysregulation induced by the H3K27M driver mutations. Since these histone mutations cannot yet be directly targeted pharmacologically, our goal is to identify druggable targets of aberrant transcription in DMG. Previous studies identified RNA polymerase 2 (Pol2)-targeting cyclin-dependent kinase 9 inhibitors (CDK9i) and histone deacetylase inhibitors (HDACi) as potential DMG therapeutics, and CDK9i and HDACi are under investigation in ongoing glioma clinical trials. This study aims to develop CDK9i+HDACi combination therapies for DMG. METHODS We determined the consequences of CDK9i+HDACi combination treatment in DMG using in vitro dose curve analyses, orthotopic xenograft models, and molecular profiling through immunofluorescence imaging, Western blotting, flow cytometry and RNA-seq analysis. RESULTS We find that CDK9i+HDACi combination therapy synergistically reduces DMG growth in vitro and alters DMG protein post-translational modifications associated with gene regulation by reducing phos-Pol2-Ser2 and increasing histone acetylation. Further RNA-seq analysis identified unique transcriptional changes induced by CDK9i+HDACi, including the downregulation of genes associated with mitochondrial function, cell division, and DNA repair. In line with these gene expression changes, additional studies suggest that CDK9i+HDACi treatment induces DNA damage and cell cycle defects. Finally, preliminary pre-clinical studies using an orthotopic DMG xenograft model reveal that CDK9i and HDACi treatment reduces DMG tumor growth as measured by bioluminescent imaging. CONCLUSIONS Together, these studies suggest that CDK9i and HDACi co-treatment alters DMG protein modifications and gene expression leading to cell cycle defects, DNA damage, and reduced tumor growth. In view of the proposed synergistic role of CDK9i and HDACi in DMG, additional ongoing studies are examining if this combination may represent a novel approach for the treatment of DMG.

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.

Synergistic cytotoxicity of histone deacetylase, poly-ADP ribose polymerase inhibitors and decitabine in breast and ovarian cancer cells: Therapeutic implications.

e15193 Background: Innovative strategies are needed for breast and ovarian cancer. We explored the synergistic cytotoxicity of histone deacetylase inhibitors (HDACi), poly(ADP-ribose) polymerase inhibitors (PARPi), and decitabine (DAC, D) in breast and ovarian cancer cells. The drugs affect epigenetic regulation and DNA repair, making them promising anticancer therapies. Methods: Breast (MDAMB231 and MCF-7) and ovarian (HEY-T30 and SKOV-3) cells were exposed to HDACi (panobinostat, Pano, P; vorinostat; Vori, V), PARPi (talazoparib, TLZ, T; olaparib, Ola, O) and DAC. Synergism was assessed by MTT and clonogenic assays. Changes in DNA damage response/repair markers were assessed with Western blotting. Results: The combinations of HDACi, PARPi, and DAC had synergistic effects in all cell lines, evidenced by combination index <1. Clonogenic assay confirmed sensitivity of all cell lines to the 3-drug combinations (Pano, TLZ, DAC; Pano, Ola, DAC; Vori, TLZ, DAC; Vori, Ola, DAC). Cell proliferation was inhibited by 50% - 70%, and Annexin V positivity was 51% - 58% in all cells exposed to the combinations. Western blots showed inhibited protein PARylation, cleaved caspase 3 and PARP1 (suggestive of apoptosis), and down-regulated c-MYC. The 3-drug combinations induced more DNA damage (increased phosphorylation of ƔH2AX) than the individual drugs, impaired DNA repair (decreased ATM, BRCA1, ATRX proteins; Artemis), and altered gene expression (reduced NuRD complex subunits). DNA damage caused by HDACis and DAC may confer increased cellular dependence on protein PARylation, increasing sensitivity to combined HDACi, PARPi, and DAC. The P values comparing the inhibitory effects of each drug combination with that of individual drugs (MTT assay) are shown in Table ( P ≤ .05, statistically significant). Conclusions: The results support exploring HDACi, PARPi, and DAC combinations to overcome drug resistance and improve patient outcomes, and carefully designed clinical trials are warranted in breast and ovarian cancer. [Table: see text]

Phase I trial of chidamide, an oral HDAC inhibitor, in combination with oral etoposide in patients with refractory/recurrent neuroblastoma.

10034 Background: Chidamide, an oral subtype-selective histone deacetylase (HDAC) inhibitor, has been shown to be effective in adult patients with hematological tumors. However, there is no data on its safety and efficacy in pediatric cancer patients. Combination of HDAC inhibitors and etoposide have shown synergistic anti-tumor effects in preclinical studies of neuroblastoma. We conducted a phase I trial in pediatric patients with recurrent or refractory neuroblastoma to determine the maximum tolerable dose (MTD) of chidamide combined with oral etoposide treatment. Methods: Daily oral etoposide 35 mg/m2/dose was administered on days 1–21 in combination with escalating doses of chidamide (14 and 17 mg/m2/d) twice weekly in each 28-day cycle using the standard 3 + 3 design. Patients with response or stable disease after two cycles would maintain treatment until disease progression or unacceptable toxicity occurred. Chidamide pharmacokinetic testing was performed. Results: 31 patients were enrolled in this study. The median age was 7 years (range 3 – 18). 14 patients were included in the dose escalation phase (Ia), 17 patients in the expansion cohort (Ib). The MTD of chidamide was 14 mg/m2/d, with dose limiting neutropenia and thrombocytopenia observed at 17 mg/m2/d. Median number of cycles completed was 2.5 (range 1–11). A total of 19 patients (61.3%) experienced treatment-related grade 3/4 hematologic toxicity, and no grade 3/4 non-hematological toxicity was observed. No objective responses were seen. Conclusions: In children with refractory/recurrent neuroblastoma, chidamide is well-tolerated at 14 mg/m2/d twice weekly when combining with oral etoposide 35 mg/m2/d daily. Prolonged disease stability achieved in patients with minimal residual diseases deserves further investigation. Clinical trial information: NCT05338541 .

Abstract PO2-06-08: Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like Breast Cancer

Abstract Introduction: Basal-like breast cancer (BLBC) represents an aggressive subtype of triple-negative breast cancer (TNBC) that exhibits a high risk of recurrence and resistance to treatment. Histone deacetylase (HDAC) inhibitors (HDACis), including panobinostat and romidepsin, have obtained approval for the treatment of hematopoietic malignancies and demonstrated effectiveness in TNBC cells. We recently found that panobinostat and romidepsin potently induced TNBC cell growth inhibition and apoptosis via downregulation of HER3 through suppression of forkhead box protein A1 (FOXA1), a pioneering transcription factor. Nonetheless, the underlying mechanism through which HDACis suppress FOXA1, thereby inhibiting HER3 expression and its downstream signaling in FOXA1/HER3 co-expressing TNBC remains elusive. 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 FOXA1 was observed in BLBC specimens and cell lines tested. FOXA1 was co-expressed with HER3 and transcriptionally activated the HER3 expression in BLBC cells. HER3 formed heterodimers with epidermal growth factor receptor (EGFR), activating downstream signaling pathways in BLBC cells. Treatment with panobinostat and romidepsin resulted in growth inhibition and apoptosis in BLBC cells by downregulating FOXA1 expression and inhibiting HER3 signaling. Notably, the combination of HDACis with an EGFR inhibitor (gefitinib) or an anti-HER3 antibody synergistically enhanced the anti-survival effects on BLBC cells. Additionally, gene expression profiling datasets revealed a significant positive correlation between FOXA1 expression and the transcription factors GATA-Binding Protein 2/3 (GATA2/3). Patients with high GATA2/3-FOXA1 expression exhibited worse Relapse-Free Survival (RFS) compared to those with low expressions in BLBC via Kaplan-Meier analysis. Further investigations showed that GATA2/3 directly activated FOXA1 transcription by binding to its promoter. Moreover, ectopic expression of GATA2/3 not only restored FOXA1 expression downregulated by HDACis but also attenuated the anti-survival effects of HDACis on BLBC cells. Conclusion: HDACis exhibit potent inhibitory effects on BLBC cells via downregulation of GATA2/3-mediated repression of FOXA1 gene transcription, which in turn suppresses HER3 expression and signaling. Our findings indicate that epigenetic targeting of the GATA2/3-FOXA1-HER3 axis may be an effective therapeutic strategy for the eradication of BLBC tumors. Keywords: FOXA1, GATA2/3, HER3, HDAC inhibitors, Basal-like breast cancer Citation Format: Bolin Liu, Congcong Tan, Hui Lyu. Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-06-08.

Expression of Immunotherapy Target PRAME in Cancer Correlates with Histone H3 Acetylation and Is Unrelated to Expression of Methylating (DMNT3A/3B) and Demethylating (TET1) Enzymes.

Background/Objectives: Preferentially expressed antigen in melanoma (PRAME), a member of the cancer testis antigen family, is a promising target for cancer immunotherapy. Understanding the epigenetic mechanisms involved in the regulation of PRAME expression might be crucial for optimizing anti-PRAME treatments. Methods: Three malignancies of different lineages (sinonasal melanoma, testicular seminoma, and synovial sarcoma), in which immunohistochemical (IHC) reactivity for PRAME is a common yet variable feature, were studied. The expression of PRAME, ten-eleven translocation demethylase 1 (TET1), and DNA methyltransferase (DNMT) 3A and 3B were evaluated using immunohistochemistry. Moreover, the expression of two epigenetic marks, 5-hydroxymethylcytosine (5hmC) and histone 3 acetylation (H3ac), was tested. Results: All PRAME-positive tumors expressed medium-to-high levels of H3ac but differed considerably with respect to other markers. In seminomas, PRAME expression correlated with TET1, but in melanomas and synovial sarcomas, it correlated with both DNMTs and DNMT3A, respectively. Conclusions: PRAME expression was not determined by a balance between the global expression of DNA methylating/demethylating enzymes. However, histone acetylation may be one of the epigenetic mechanisms involved in PRAME regulation. Thus, the therapeutic combination of histone deacetylase inhibitors and PRAME immunotherapy merits further investigation.

Modulation of tumor microenvironment by targeting histone acetylation in bladder cancer

Alterations in the epigenetic machinery in both tumor and immune cells contribute to bladder cancer (BC) development, constituting a promising target as an alternative therapeutic option. Here, we have explored the effects of a novel histone deacetylase (HDAC) inhibitor CM-1758, alone or in combination with immune checkpoint inhibitors (ICI) in BC. We determined the antitumor effects of CM-1758 in various BC cell lines together with the induction of broad transcriptional changes, with focus on the epigenetic regulation of PD-L1. Using an immunocompetent syngeneic mouse model of metastatic BC, we studied the effects of CM-1758 alone or in combination with anti-PD-L1 not only on tumor cells, but also in the tumor microenvironment. In vitro, we found that CM-1758 has cytotoxic and cytostatic effects either by inducing apoptosis or cell cycle arrest in BC cells at low micromolar levels. PD-L1 is epigenetically regulated by histone acetylation marks and is induced after treatment with CM-1758. We also observed that treatment with CM-1758 led to an important delay in tumor growth and a higher CD8 + T cell tumor infiltration. Moreover, anti-PD-L1 alone or in combination with CM-1758 reprogramed macrophage differentiation towards a M1-like polarization state and increased of pro-inflammatory cytokines systemically, yielding potential further antitumor effects. Our results suggest the possibility of combining HDAC inhibitors with immunotherapies for the management of advanced metastatic BC.

A novel selenium analog of HDACi-based twin drug induces apoptosis and cell cycle arrest via CDC25A to improve prostate cancer therapy.

Cancer therapy has moved from single agents to more mechanism-based targeted approaches. In recent years, the combination of HDAC inhibitors and other anticancer chemicals has produced exciting progress in cancer treatment. Herein, we developed a novel prodrug via the ligation of dichloroacetate to selenium-containing potent HDAC inhibitors. The effect and mechanism of this compound in the treatment of prostate cancer were also studied. Methods: The concerned prodrug SeSA-DCA was designed and synthesized under mild conditions. This compound's preclinical studies, including the pharmacokinetics, cell toxicity, and anti-tumor effect on prostate cancer cell lines, were thoroughly investigated, and its possible synergistic mechanism was also explored and discussed. Results: SeSA-DCA showed good stability in physiological conditions and could be rapidly decomposed into DCA and selenium analog of SAHA (SeSAHA) in the tumor microenvironment. CCK-8 experiments identified that SeSA-DCA could effectively inhibit the proliferation of a variety of tumor cell lines, especially in prostate cancer. In further studies, we found that SeSA-DCA could also inhibit the metastasis of prostate cancer cell lines and promote cell apoptosis. At the animal level, oral administration of SeSA-DCA led to significant tumor regression without obvious toxicity. Moreover, as a bimolecular coupling compound, SeSA-DCA exhibited vastly superior efficacy than the mixture with equimolar SeSAHA and DCA both in vitro and in vivo. Our findings provide an important theoretical basis for clinical prostate cancer treatment. Conclusions: Our in vivo and in vitro results showed that SeSA-DCA is a highly effective anti-tumor compound for PCa. It can effectively induce cell cycle arrest and growth suppression and inhibit the migration and metastasis of PCa cell lines compared with monotherapy. SeSA-DCA's ability to decrease the growth of xenografts is a little better than that of docetaxel without any apparent signs of toxicity. Our findings provide an important theoretical basis for clinical prostate cancer treatment.

Evaluation of the Synergistic Effect of the Combination of Doxorubicin and Vorinostat on Breast Tumor Cells Using the Loewe Model

The combination of two or more medications is increasingly more common in the development of new treatment guidelines for common diseases. Thus, the use of combinations of histone deacetylase inhibitors with chemotherapeutic agents is a current trend in solid tumor treatment. The aim of this study was to investigate an effective vorinostat (SAHA) to doxorubicin (DOX) ratio for the treatment of different subtypes of breast cancer. The survival of HCC-1954, SKBR-3, MCF-7, MCF-7/ADR, MDA-MB-231 cell lines was assessed under incubation conditions with 64 variants of SAHA and DOX combinations using the MTT assay. This made it possible to determine the effect of interactions of SAHA/DOX combinations (antagonistic, additive, synergistic), as well as calculate the SAHA/DOX synergy index using the Loewe additivity model. The effect of SAHA/DOX ratios with the highest synergistic index for each tumor cell line was confirmed using the Chou-Talalay method. It was shown that the SAHA/DOX combination exhibited the greatest synergism in relation to HCC-1954, MCF-7/ADR and SKBR-3 cell lines belonging to the HER2-positive subtype. The average value of SAHA/DOX ratio with the highest synergy against breast cancer cells was 30:1 (SAHA to DOX, respectively). The ability of SAHA/DOX combination to effectively trigger apoptosis was confirmed in the most sensitive to SAHA/DOX therapy HCC-1954 cells. Thus, the Loewe model made it possible to identify the drug combination with the highest synergistic anticancer effect, which was confirmed using Chou-Talalay method. The data obtained demonstrates great potential of SAHA/DOX combination (30:1) for the treatment of HER2-positive breast cancer.

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.

Codelivery of vorinostat and chloroquine by autophagy-inhibitory hollow ZrO2 nanoshells for synergistic combination chemotherapy

Autophagy induction is responsible for the chemoresistance of histone deacetylase (HDAC) inhibitors. The combination of HDAC inhibitors with autophagy inhibitors has been considered a new strategy for treating solid tumors. However, codelivery systems capable of transporting these two kinds of drugs with different physicochemical properties and the simultaneous release of drugs remain elusive. In this study, hollow zirconium dioxide (ZrO2) nanoshells were synthesized to encapsulate both the autophagy inhibitor chloroquine (CQ) and the HDAC inhibitor vorinostat (Vor) for effective combinational cancer therapy. The ZrO2 nanoshells showed high loading capacities for both CQ and Vor due to the high affinity of ZrO2 for the phosphate and hydroxamic acid groups of the drugs, and the drug-loaded ZrO2 nanoshells exhibited a controlled release profile. The intrinsic autophagy inhibitory effect of the ZrO2 nanoshells enhanced the effect of either the single drug (CQ or Vor) or the combination of these two drugs. The codelivery system enhanced the autophagy inhibition and hyperacetylation level in the cells compared with free drugs, resulting in a superior antitumor effect in 4T1 tumor-bearing mice. Taken together, the multifunctional ZrO2 nanoshells hold great potential for the codelivery of HDAC and autophagy inhibitors for solid tumors therapy.

Efficacy and safety of programmed cell death receptor 1 inhibition-based regimens in patients with pediatric malignancies: the real-world study in China.

Programmed death receptor 1 (PD-1) inhibition has shown durable response and mild adverse events (AEs) in adult malignancies. However, data on the clinical activity of PD-1 inhibition in pediatric patients are lacking. We comprehensively assessed the efficacy and safety of PD-1 inhibitor-based regimens for pediatric malignancies. We conducted a real-world, multi-institutional, retrospective analysis of pediatric malignancies treated with PD-1 inhibitor-based regimens. The primary endpoints were objective response rate (ORR) and progression-free survival (PFS). The secondary endpoints included disease control rate (DCR), duration of response (DOR), and AEs. The Kaplan-Meier method was used to calculate PFS and DOR. The National Cancer Institute Common Toxicity Criteria for AEs (version 5.0) were used to grade toxicity. A total of 93 and 109 patients were evaluated for efficacy and safety, respectively. For all efficacy-evaluable patients, PD-1 inhibitor monotherapy, combined chemotherapy, combined histone deacetylase inhibitor, and combined vascular endothelial growth factor receptor tyrosine kinase inhibitor cohorts, the ORR and DCR were 53.76%/81.72%, 56.67%/83.33%, 54.00%/80.00%, 100.00%/100.00%, and 12.50%/75.00%, respectively; the median PFS and DOR were 17.6/31.2 months, not achieved/not achieved, 14.9/31.2 months, 17.6/14.9 months, and 3.7/1.8 months, respectively; the incidence rate of AEs were 83.49%, 55.26%, 100.00%, 80.00%, and 100.00%, respectively. One patient in the PD-1 inhibitor-combined chemotherapy cohort discontinued treatment due to diabetic ketoacidosis. This largest retrospective analysis demonstrate that PD-1 inhibitor-based regimens are potentially effective and tolerable in pediatric malignancies. Our findings provide references for future clinical trials and practice of PD-1 inhibitors in pediatric cancer patients.

Abstract 6252: HDAC inhibition increases Ewing sarcoma sensitivity to chemotherapy

Abstract Purpose: The survival rate of those with Ewing sarcoma (ES) has seen very little improvement over the past several decades and remains dismal for those with recurrent or metastatic disease. Current treatment for Ewing sarcoma includes a combination of cytotoxic chemotherapeutic agents and local control with radiation and/or surgery. The purpose of this study is to identify currently available targeted agents which may be combined with chemotherapy for use in Ewing sarcoma to improve patient outcomes. Methods: Novel targeted approaches for the treatment of ES were identified using RNA sequencing performed on ES patient tumors with bioinformatic analysis of gene expression. HDAC inhibitors were tested on patient cell lines at escalating doses and cell viability was assessed with CellTiter-Glo 2.0 and analyzed with GraphPad Prism to determine the IC50. Cells were treated with Panobinostat for 12 and 24 hours and RNA sequencing was performed. Cell cycle analysis was performed using Incucyte® Cell Cycle Lentivirus Reagent and analyzed using an Incucyte® S3. At 24- and 48-hours western blot analysis was performed to evaluate the effect on cell cycle and apoptosis. Combination studies with standard chemotherapy were performed and viability was assessed with CellTiter-Glo 2.0. At 24 hours of treatment with a combination of doxorubicin and HDAC inhibition western blot analysis was performed to evaluate changes in DNA damage and repair. Results: RNA analysis identified HDAC2 as a potential therapeutic target. The pan-HDAC inhibitors, Panobinostat and Belinostat, and the HDAC1/2 inhibitor, Romidepsin, were cytotoxic to all Ewing sarcoma cell lines tested with clinically relevant IC50s. Mechanistically, pathways involved in cell cycle progression and DNA repair were repressed by Panobinostat as shown by RNA sequencing and western blotting (Cyclin D1, pRb, CHK1). Additionally, cell cycle analysis has shown a G1 arrest in response to HDAC inhibition. HDAC inhibitors significantly decrease cell viability when combined with standard chemotherapy (upfront and relapse therapy) compared to that of chemotherapy alone. When combined with doxorubicin, CHK1 and CHK2 were significantly decreased and pH2AX was significantly increased indicating an accumulation of DNA damage. Conclusion: This work highlights that combining HDAC inhibitors with standard of care chemotherapy may be an effective treatment and improve outcomes for Ewing sarcoma patients. Future studies of HDAC inhibitors combined with chemotherapy drugs will aim to determine efficacy in xenograft models. Citation Format: Kaitlyn H. Smith, Chloe Sholler, Divya Gandra, Kimberly McKinney, Karl Dykema, Abhinav Nagulapally, Erin Trovillion. HDAC inhibition increases Ewing sarcoma sensitivity to chemotherapy [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 6252.

Abstract 15671: Targeting Chromatin Structure: The Combined Inhibition of Histone Deacetylases and Bromodomain Proteins in Normalizing Persistently Activated Pulmonary Hypertensive Fibroblasts

Rationale: The multifactorial nature of pulmonary hypertension (PH) suggests epigenetic changes as potential determinants of vascular remodeling through regulating gene expression (both up-regulated and down-regulated genes) in vascular cells. Changes in the chromatin state (especially histone acetylation) of specific genes can lead to their repression or activation. We and others have demonstrated histone deacetylases inhibitors (HDACi) and histone acetylation readers inhibitors (BRDi) can prevent and ameliorate PH changes by normalizing pathological gene expression. However, the non-histone protein targets and the narrow therapeutic window of currently available HDACi limits their clinical utility. In this study, we evaluated the effect of combination treatments using a new HDACi (OKI-005, US patent) with high potency and selectivity for HDACs (targeting decreased genes) together with BRDi (targeting increased genes), in low doses. Methods and Results: Cultured human pulmonary artery fibroblasts derived from patients with IPAH (PH-Fibs) or from control donors (CO-Fibs) were used as in vitro model system. PH-Fibs exhibit increased pro-inflammatory genes (e.g. CXCL12), proliferation markers (e.g. Mki67) and anti-apoptosis genes (e.g. BCL2) as well as decreased anti-inflammatory genes (e.g. HMOX1), anti-proliferation genes (e.g. P21) and pro-apoptotic genes (e.g. PERP). In PH-Fibs, the increased expressed CXCL2 gene exhibited more opened chromatin structure and BRD4 binding (ChIP-assay), and BRDi (JQ1) can decrease its expression in a dose dependent trend. At the low doses, JQ1 (100 nM) alone is sufficient to normalize most of increased genes, while the new HDACi (OKI-005) was more effective in normalizing decreased genes in PH- Fibs. The combination of low dose of JQ1 (100 nM) and OKI-005 (64 nM) was sufficient to normalize both increased genes and decreased genes thus inhibiting the persistently activated PH-phenotype without deleterious effects on CO-Fibs. Conclusion: Combination of HDACi and BRDi in low doses is effective in normalizing the persistent activation of PH-Fibs without deleterious effects on control cells, offering promise for epigenetic-targeted therapies in PH.

Histone deacetylase inhibitor use as a radiosensitizer in solid organ malignancies: a systematic review protocol.

The objective of this review is to evaluate the efficacy and adverse effects of histone deacetylase inhibitors (HDACi) in combination with radiotherapy for the treatment of solid organ malignancies. Histone deacetylase inhibitors are a diverse class of drugs that have shown promise as novel anti-cancer therapeutics via epigenetic modification and radiosensitization of neoplastic cells. The aim of HDACi in combination with radiotherapy is to reduce radiation dosage requirements, improve radiotherapy efficacy, and reduce treatment side effects. This review will consider studies utilizing HDACi in conjunction with radiotherapy in adult patients with solid organ malignancy. Sources to be included in this review include experimental and quasi-experimental study designs, analytical studies, and descriptive observational studies. A systematic review of effectiveness will be conducted in accordance with JBI methodology. A detailed search will be conducted via MEDLINE (Ovid), Embase (Ovid), and Scopus. A search of the Cochrane Central Register of Controlled Trials, the International Clinical Trials Registry Platform, and ClinicalTrials.gov will also be performed for relevant trials. Inclusion and exclusion criteria will be utilized to select studies, and papers selected for retrieval will be assessed for methodological validity using the JBI critical appraisal instruments. Evidence will be extracted from eligible studies and summarized using quantitative methods, where possible, including meta-analysis and assessment of heterogeneity. Where statistical pooling is not possible, the findings will be presented in diagrammatic or tabular form accompanied by a narrative summary. PROSPERO CRD42021293005.

Pediatric glioblastoma cells are sensitive to drugs that inhibit eIF2α dephosphorylation and its phosphomimetic S51D variant.

We found that pediatric glioblastoma (PED-GBM) cell lines from diffuse intrinsic pontine glioma (DIPG) carrying the H3K27M mutation or from diffuse hemispheric glioma expressing the H3G34R mutation are sensitive to the combination of vorinostat (a histone deacetylase inhibitor) and PARP-1 inhibitors. The combined treatment increased the phosphorylation of eIF2α (P-eIF2α) relative to each drug alone and enhanced the decrease in cell survival. To explore the role played by increased P-eIF2α in modulating PED-GBM survival and response to treatments, we employed brain-penetrating inhibitors of P-eIF2α dephosphorylation: salubrinal and raphin-1. These drugs increased P-eIF2α, DNA damage, and cell death, similarly affecting the sensitivity of DIPG cells and derived neurospheres to PARP-1 inhibitors. Interestingly, these drugs also decreased the level of eIF2Bϵ (the catalytic subunit of eIF2B) and increased its phosphorylation, thereby enhancing the effect of increased P-eIF2α. Transient transfection with the S51D phosphomimetic eIF2α variant recapitulated the effect of salubrinal and raphin-1 on PED-GBM survival and sensitivity to PARP-1 inhibitors. Importantly, either salubrinal or raphin-1 dramatically increased the sensitivity of DIPG cells to radiation, the main treatment modality of PED-GBM. Finally, PED-GBM was more sensitive than normal human astrocytes to salubrinal, raphin-1, and the treatment combinations described herein. Our results indicate that combinations of histone deacetylase inhibitors and PARP-1 inhibitors should be evaluated for their toxicity and efficacy in PED-GBM patients and point to drugs that increase P-eIF2α or modulate its downstream effectors as a novel means of treating PED-GBM.

Histone Deacetylase Inhibitors Restore Cancer Cell Sensitivity towards T Lymphocytes Mediated Cytotoxicity in Pancreatic Cancer.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is an aggressive pancreatic cancer that is resistant to most treatments due to its tumour microenvironment. In search of an effective therapeutic agents that can overcome the tumour microenvironment, we analysed the PDAC patients genomic profilings and identified that patients with high cytotoxic T lymphocytes (CTL) killing activity were associated with better clinical outcomes. Through genomic and proteomic approaches, we identified potential small molecules that may restore CTL activity in PDAC. We validated the findings using two CTL-resistant PDAC cells and concluded that histone deacetylase inhibitors (givinostat and dacinostat) can reverse CTL sensitivity in CTL-resistant PDAC cells.Despite medical advancements, the prognosis of pancreatic ductal adenocarcinoma (PDAC) has not improved significantly over the past 50 years. By utilising the large-scale genomic datasets available from the Australia Pancreatic Cancer Project (PACA-AU) and The Cancer Genomic Atlas Project (TCGA-PAAD), we studied the immunophenotype of PDAC in silico and identified that tumours with high cytotoxic T lymphocytes (CTL) killing activity were associated with favourable clinical outcomes. Using the STRING protein–protein interaction network analysis, the identified differentially expressed genes with low CTL killing activity were associated with TWIST/IL-6R, HDAC5, and EOMES signalling. Following Connectivity Map analysis, we identified 44 small molecules that could restore CTL sensitivity in the PDAC cells. Further high-throughput chemical library screening identified 133 inhibitors that effectively target both parental and CTL-resistant PDAC cells in vitro. Since CTL-resistant PDAC had a higher expression of histone proteins and its acetylated proteins compared to its parental cells, we further investigated the impact of histone deacetylase inhibitors (HDACi) on CTL-mediated cytotoxicity in PDAC cells in vitro, namely SW1990 and BxPC3. Further analyses revealed that givinostat and dacinostat were the two most potent HDAC inhibitors that restored CTL sensitivity in SW1990 and BxPC3 CTL-resistant cells. Through our in silico and in vitro studies, we demonstrate the novel role of HDAC inhibition in restoring CTL resistance and that combinations of HDACi with CTL may represent a promising therapeutic strategy, warranting its further detailed molecular mechanistic studies and animal studies before embarking on the clinical evaluation of these novel combined PDAC treatments.

The Role of HDACs in the Response of Cancer Cells to Cellular Stress and the Potential for Therapeutic Intervention.

Throughout the process of carcinogenesis, cancer cells develop intricate networks to adapt to a variety of stressful conditions including DNA damage, nutrient deprivation, and hypoxia. These molecular networks encounter genomic instability and mutations coupled with changes in the gene expression programs due to genetic and epigenetic alterations. Histone deacetylases (HDACs) are important modulators of the epigenetic constitution of cancer cells. It has become increasingly known that HDACs have the capacity to regulate various cellular systems through the deacetylation of histone and bounteous nonhistone proteins that are rooted in complex pathways in cancer cells to evade death pathways and immune surveillance. Elucidation of the signaling pathways involved in the adaptive responses to cellular stress and the role of HDACs may lead to the development of novel therapeutic agents. In this article, we overview the dominant stress types including metabolic, oxidative, genotoxic, and proteotoxic stress imposed on cancer cells in the context of HDACs, which guide stress adaptation responses. Next, we expose a closer view on the therapeutic interventions and clinical trials that involve HDACs inhibitors, in addition to highlighting the impact of using HDAC inhibitors in combination with stress-inducing agents for the management of cancer and to overcome the resistance to current cancer therapy.

Discovery of BRAF/HDAC Dual Inhibitors Suppressing Proliferation of Human Colorectal Cancer Cells.

The combination of histone deacetylase inhibitor and BRAF inhibitor (BRAFi) has been shown to enhance the antineoplastic effect and reduce the progress of BRAFi resistance. In this study, a series of (thiazol-5-yl)pyrimidin-2-yl)amino)-N-hydroxyalkanamide derivatives were designed and synthesized as novel dual inhibitors of BRAF and HDACs using a pharmacophore hybrid strategy. In particular, compound 14b possessed potent activities against BRAF, HDAC1, and HDAC6 enzymes. It potently suppressed the proliferation of HT-29 cells harboring BRAFV600E mutation as well as HCT116 cells with wild-type BRAF. The dual inhibition against BRAF and HDAC downstream proteins was validated in both cells. Collectively, the results support 14b as a promising lead molecule for further development and a useful tool for studying the effects of BRAF/HDAC dual inhibitors.

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.