Combination Of Epigenetic Drugs Research Articles

Optimization of Epigenetic Modifier Drug Combination for Synergistic Effect against Glioblastoma Multiform Cancer Cell Lines

Glioblastoma multiforme (GBM), is a frequent class of malignant brain tumors. Epigenetic therapy, especially with synergistic combinations is highly paid attention for aggressive solid tumors like GBM. Here, RSM optimization has been used to increase the efficient arrest of U87 and U251 cell lines due to synergistic effects. Cell lines were treated with SAHA, 5-Azacytidine, GSK-126, and PTC-209 individually and then RSM was used to find most effective combinations. Results showed that optimized combinations significantly reduce cell survival and induce cell cycle arrest and apoptosis in both cell lines. Expression of cyclin B1 and cyclin D1 were decreased while caspase3 increased expression.

Epigenetic Reprogramming By Histone Deacetylase Inhibitors Improves CAR-T Cell Functions

Background: The exhaustion and diminished persistence of CAR-T cells primarily affect clinical efficacy and long-term outcomes. Epigenetic is an innate mechanism in regulating the biological function CAR-T cells. We previously dissected comprehensive dynamic changes of chromatin accessibility and transcription factors during CAR-T cell exhaustion (Jiang P, et al. Leukemia. 2022 Nov;36(11):2656-2668). Therefore, combined application with epigenetic drugs have much therapeutic potential. Methods: We initially performed an extensive screening among 370 epigenetic drugs to select candidates that could resist exhaustion and maintain phenotype of memory subsets, and thoroughly verify their effects on CAR-T cells in vitro. Next, we conducted multi-omics analysis comprising RNA-seq, ATAC-seq, and CUT&Tag-seq to explore underlying regulative mechanisms of epigenetic manipulation by epi-drugs. In the end, we used preclinical B-ALL NSG mouse model to verify anti-tumor activities of drug-treated CAR-T cells in vivo. Results: Among those screened epigenetic drugs, we identified two histone deacetylase inhibitors (HDACi), Chidamide and M344, could best promote maintenance of central memory subsets and resistance to exhaustion of CAR-T cells, with enhanced anti-leukemia activities in vitro (Figure 1a-c). Chidamide and M344 inhibited enzymatic activity and expression of HDAC1, which elevated the acetylation level of histone H3, particularly at H3K27 in CD19 CAR-T cells. Genetic knock-down of HDAC1 could partially reflect the phenotypic mimicry by Chidamide and M344 on CAR-T cells. By combining multi-omics data from bulk RNA-seq, ATAC-seq, and H3K27ac-targeted CUT&Tag-seq in Chidamide-or M344-treated CAR-T cells, we identified distinct patterns of epigenetic reprogramming, and enrichment of genes associated with exhaustion, as well as regulation of cellular differentiation and formation of memory subsets. In addition, analysis of motif and interaction network among transcription factors revealed altered regulation of gene transcription (data not shown). Finally, either Chidamide-pretreated CAR-T cells or in vivo administration of Chidamide after CAR-T cells infusion had exhibited enhanced and persistent tumor clearance in preclinical B-ALL NSG mouse model (Figure 1d). Conclusion: we conducted comprehensive screening of 370 epigenetic drugs and identified that administration of HDACi, particularly Chidamide and M344, could induce significantly enhanced anti-tumor function with maintenance of memory phenotype and reduced exhaustion in CAR-T cells through epigenetic reprogramming and altered regulation of gene transcription, which provided with basis and therapeutic targets for the rational combination of CAR-T cell therapy and epigenetic drugs in patients with hematological malignancies. Figure legends: Figure1. Screening of 370 epigenetic drugs identified M344 and Chidamide that enhanced maintenance of memory phenotype, resistance to exhaustion, and ability of tumor clearance in CAR-T cells. (a) The scatterplot of screening 370 compounds based on relative CD45RO +CD62L + proportion of central memory in treated CAR-T cell, showing of compared with DMSO control group by flow cytometry. Red indicates an increase in the proportion of memory CAR-T cells, while blue represents a decrease. (b) Frequency of co-expression of exhaustion-related inhibitory receptors (LAG-3, TIM-3, and PD-1) in DMSO or HDACi-treated groups. Data presented as mean ± SEM of independent donors (n=3 or more). (c) Flow cytometric analysis of central memory and effector memory subsets in CAR-T cells in each group. Data presented as mean ± SEM of independent donors (n=3 or more). (d) Bioluminescent imaging of tumor clearance in B-ALL NSG mouse. ‘X’ represents dead subject (end point). Survival of B-ALL NSG mice i.v. infused with T cells or treated CAR-T cells. Statistical analysis by Mantel-Cox test. Data presented as pooled from independent experiments (n=2).

Bone Marrow Microenvironment-Induced Chemoprotection in KMT2A Rearranged Pediatric AML Is Overcome by Azacitidine–Panobinostat Combination

Advances in therapies of pediatric acute myeloid leukemia (AML) have been minimal in recent decades. Although 82% of patients will have an initial remission after intensive therapy, approximately 40% will relapse. KMT2A is the most common chromosomal translocation in AML and has a poor prognosis resulting in high relapse rates and low chemotherapy efficacy. Novel targeted approaches are needed to increase sensitivity to chemotherapy. Recent studies have shown how interactions within the bone marrow (BM) microenvironment help AML cells evade chemotherapy and contribute to relapse by promoting leukemic blast survival. This study investigates how DNA hypomethylating agent azacitidine and histone deacetylase inhibitor panobinostat synergistically overcome BM niche-induced chemoprotection modulated by stromal, endothelial, and mesenchymal stem cells and the extracellular matrix (ECM). We show that direct contact between AML cells and BM components mediates chemoprotection. We demonstrate that azacitidine and panobinostat synergistically sensitize MV4;11 cells and KMT2A rearranged pediatric patient-derived xenograft lines to cytarabine in multicell coculture. Treatment with the epigenetic drug combination reduced leukemic cell association with multicell monolayer and ECM in vitro and increased mobilization of leukemic cells from the BM in vivo. Finally, we show that pretreatment with the epigenetic drug combination improves the efficacy of chemotherapy in vivo.

Combining Epigenetic and Immunotherapy in Cancer: Molecular Mechanisms

BACKGROUND: Immunotherapy, particularly the idea of immune checkpoint blockage is currently draw much attention in cancer treatment. It has been approved as an adjuvant, however, it cannot be a single cancer treatment.CONTENT: The discovery of the basic ligand-receptor interactions between immune and cancer cells inside the tumor microenvironment has led to the current interest in immunotherapy, specifically immune checkpoint inhibition. Different ligands produced by cancer cells interact with immune cells' surface receptors, activating inhibitory pathways, such programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1), that cause immune cells to become immunologically tolerant. On the other side, epigenetic modulators also play a critical role in enhancing the tumor microenvironment and regaining immunological recognition and immunogenicity. Some findings showed that such immune suppression can be reversed through various mechanisms involving antigens pathways, immune genetic, and epigenetic pathways. These findings have created a very encouraging foundation for research on the combination of epigenetic and immunotherapeutic drugs as cancer treatments.SUMMARY: The effectiveness of this suggested paradigm can only be demonstrated by clinical studies. Epigenetic treatment might replace immune checkpoint therapy as a powerful new cancer care technique that is generally well tolerated and should be proven with adequate clinical trials.KEYWORDS: epigenetics, immunotherapy, PTM, DNMT, HDAC, immune check point

Azacitidine and Panobinostat Overcome Bone Marrow Microenvironment-Induced Chemoprotection in KMT2A-rearranged Pediatric AML

KMT2A is the most common gene rearrangement in pediatric acute leukemias and is difficult to treat with a five-year overall survival rate of less than 50%, owing to frequent relapse and poor response to chemotherapy. Recent studies have investigated how interactions within the bone marrow microenvironment help AML cells evade chemotherapy and contribute to relapse by promoting the survival of leukemic blasts. Our group has previously identified that epigenetic drug combination of DNA hypomethylating agent azacitidine and histone deacetylase inhibitor panobinostat reduces cell adhesion and promotes mobilization of leukemic cells to peripheral blood (Weber et al., 132:2637, Blood, 2018). In this study, we used a multi cell coculture of pediatric AML cells with bone marrow stromal cells, endothelial cells and pediatric mesenchymal stem cells to better mimic the bone marrow microenvironment. We observed that the chemoprotection index in multi-cell coculture was much higher than individual cell type (Figure 1A). While 1.5 uM cytarabine reduced MV4;11 cell viability by 50%, there was less than 1% reduction in cell viability in multi-cell coculture. We further investigated if the bone marrow microenvironment induced chemoprotection was mediated by soluble factors, or via adhesive interactions between adherent cell layer and/or the extracellular matrix (ECM). Conditioned media failed to afford chemoprotection, while separation of AML cells from multi-cell layer via Transwells or culturing AML cells in Matrigel-coated dishes showed partial chemoprotection. These results indicated that direct cell-cell or cell-ECM contact was necessary for the chemoprotection. Pre-treatment with azacitidine and panobinostat sensitized five distinct patient-derived xenograft (PDX) lines of KMT2A-rearranged AML to both cytarabine and daunorubicin in multi-cell coculture. These data indicate that azacitidine and panobinostat combination can synergistically overcome chemoprotection mediated by stromal, endothelial and mesenchymal stem cells. We also observed improved survival with pre-treatment of azacitidine panobinostat (2.5 mg/Kg each, Qd5) followed by chemotherapy consisting of cytarabine (50 mg/Kg, Qd5) and daunorubcin (1.5 mg/Kg, Qd3) in a PDX model of KMT2A-rearranged AML (Figure 1B, **P<0.05). To determine if these epigenetic modifers alter the expression of cell adhesion molecules, cells treated with azacitidine panobinostat combination were subjected to a high throughput flow cytometry assay (Lyoplate, Human cell surface marker screening panel). This analysis showed reduced cell surface expression of integrins, ICAMs and chemokine receptors, indicating that epigenetic drug combination likely causes mobilization and chemosensitization via downregulation of cell adhesion molecules which not only mediate adhesive interactions but also promote cell survival. In summary, our data show azacitidine and panobinostat can overcome bone marrow microenvironment induced chemoprotection in vitro and in vivo in KMT2A rearranged AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Selinexor Prolongs Survival in Patient-Derived Xenograft Models of Down Syndrome Myeloid Leukemia

Background: Selinexor, an inhibitor of the nuclear exportin protein XPO1, has been shown to promote cell cycle arrest and induce apoptosis in acute myeloid leukemia (AML) cells. By inhibiting protein export from the nucleus to the cytoplasm, Selinexor causes nuclear accumulation of proteins including tumor suppressors and translation initiation factor eIF4E, resulting in reduced translation of anti-apoptotic proteins BCL2 and MCL1. Venetoclax, a BCL2 inhibitor, has been considered to synergize with Selinexor in preclinical studies of multiple myeloma (Nguyen et a., Blood, 138:2270, 2021), lymphoma and AML (Fischer et al., Blood Adv, 4:586, 2020). A clinical trial evaluating the combination of Selinexor and venetoclax in relapsed/refractory pediatric AML is currently underway (NCT04898894). However, children with Down syndrome, who have a 500-fold increased risk of developing acute leukemia are excluded from this trial. Methods: The viability of mouse passaged Down syndrome myeloid leukemia (DS-ML) samples following exposure to Selinexor and/or venetoclax was evaluated ex vivo by flow cytometry using annexin V and propidium iodide staining. BLISS synergy scores were calculated using SynergyFinder. For the PDX models, 2-3 million cells were injected intravenously in NSG-SGM3 mice. The percentage of human cells in mouse peripheral blood was determined at periodic intervals by flow cytometry. Mice were randomly recruited to treatment groups (n=5 each) when the percentage of human CD45+ cells were first detected in blood. Selinexor (10 mg/Kg) and venetoclax (100 mg/Kg) were administered by oral gavage for 2 weeks and 4 weeks respectively. Mice were carefully monitored and euthanized at the first instance of pre-determined experimental endpoints. Kaplan-Meier survival estimates were generated using GraphPad Prism. Results: We have generated and extensively characterized patient-derived xenograft models of DS-ML, and used for preclinical evaluation of epigenetic drug combination (azacitidine and panobinostat) (Barwe et al., Blood, 134:2683, 2019), and a combination of azacitidine and venetoclax (Barwe et al., Blood, 138:3452, 2021). Ex vivo studies showed a dose-dependent decrease in the viability of mouse passaged DS-ML cells treated with Selinexor or venetoclax. The estimated EC50 concentrations for Selinexor were 0.31 uM (NTPL-60), 0.53 uM (NTPL-386), and 1.01 uM (15384-029). Venetoclax reduced cell viability at EC50 concentrations ranging from 0.37 uM to 1.54 uM. A combination of these drugs was either additive (NTPL-60), mildly synergistic (NTPL-386) or antagonistic (15384-029) (Figure 1A). We tested the efficacy of Selinexor and venetoclax alone or in combination in vivo in three distinct DS-ML PDX models. Selinexor treatment significantly increased median survival in all three models, while venetoclax was effective in two of three models (Figure 1B, *P<0.05, **P<0.01). However, unlike prior reports in multiple myeloma and AML, addition of venetoclax to the treatment regimen did not provide any survival advantage compared to Selinexor alone. This effect was also observed in the percentage of human CD45+ in blood over time, where human CD45+ blood counts were significantly higher in the combination than Selinexor treatment alone, indicating that Selinexor monotherapy was much more efficient in reducing leukemia burden. Conclusion: We evaluated a combination of Selinexor and venetoclax in DS-ML PDX models ex vivo and in vivo. The combination did not show significant synergy in any of the models ex vivo. Selinexor showed significant improvement in median survival in each of the models and performed better than venetoclax alone. Selinexor venetoclax combination was not beneficial than Selinexor alone in DS-ML PDX models. Studies are in progress to identify the determinants of combinatorial response to Selinexor and venetoclax in DS-ML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-cell analysis of a high-grade serous ovarian cancer cell line reveals transcriptomic changes and cell subpopulations sensitive to epigenetic combination treatment.

Ovarian cancer (OC) is a lethal gynecological malignancy with a five-year survival rate of only 46%. Development of resistance to platinum-based chemotherapy is a common cause of high mortality rates among OC patients. Tumor and transcriptomic heterogeneity are drivers of platinum resistance in OC. Platinum-based chemotherapy enriches for ovarian cancer stem cells (OCSCs) that are chemoresistant and contribute to disease recurrence and relapse. Studies examining the effect of different treatments on subpopulations of HGSOC cell lines are limited. Having previously demonstrated that combined treatment with an enhancer of zeste homolog 2 inhibitor (EZH2i) and a RAC1 GTPase inhibitor (RAC1i) inhibited survival of OCSCs, we investigated EZH2i and RAC1i combination effects on HGSOC heterogeneity using single cell RNA sequencing. We demonstrated that RAC1i reduced expression of stemness and early secretory marker genes, increased expression of an intermediate secretory marker gene and induced inflammatory gene expression. Importantly, RAC1i alone and in combination with EZH2i significantly reduced oxidative phosphorylation and upregulated Sirtuin signaling pathways. Altogether, we demonstrated that combining a RAC1i with an EZH2i promoted differentiation of subpopulations of HGSOC cells, supporting the future development of epigenetic drug combinations as therapeutic approaches in OC.

Epigenetic drugs induce the potency of classic chemotherapy, suppress post-treatment re-growth of breast cancer, but preserve the wound healing ability of stem cells

ABSTRACT Epigenetic therapy augments neoadjuvant chemotherapy (NACT) in breast cancer and may aid post-surgical wound healing affected by NACT. Our study investigates: (1) The cytotoxicity of classic paclitaxel chemotherapy on triple negative breast cancer (TNBC) independently and in combination with epigenetic drugs. (2) The sustainable inhibition of breast cancer regrowth following paclitaxel and epigenetic therapies. (3) The effects of paclitaxel with and without epigenetic therapy on the post-treatment viability and wound healing potential of adipose stem cells (ASCs). Cytotoxicity assays were performed on TNBC and ASCs. Cells were treated and recovered in drug-free medium. Cell viability was measured via cell counts and MTT assays. W -ound healing was tested with scratch assays. The combination of epigenetic drugs shows increased toxicity against TNBC cells compared to standard chemotherapy alone. Moreover, the combination of paclitaxel with epigenetic treatments causes cancer toxicity that is sustainable to TNBC cells after the drugs’ removal with minimal effect on ASCs wound healing ability. The use of epigenetic drugs in addition to standard chemotherapy is cytotoxic to TNBC cells and prevents post-treatment recovery of TNBC while maintaining ASC wound healing ability. This strategy may be useful in maximizing post-surgical wound healing following NACT in TNBC.

Epigenetic Drug Combination Alters Methylation Patterns in Patient-Derived Xenograft Models of KMT2A-Rearranged Pediatric AML Treated In Vivo

Pediatric acute myeloid leukemia (AML) patients possessing rearrangement of the KMT2A (previously known as MLL) gene on 11q23 constitute a subclass with a particularly poor prognosis. The five-year survival rate for these patients is only about 44% due to poor response to conventional chemotherapy and frequent early relapse. Aberrant epigenetic modifications play an important role in leukemogenesis in KMT2A-rearranged leukemia. Accordingly, several epigenome modifying drugs have been tested in preclinical studies of KMT2A-rearranged leukemia. Acknowledging the co-regulatory effects of DNA methylation and histone modifications in determining chromatin structure and governing gene expression, we combined DNA hypomethylating agent azacitidine with histone deacetylase inhibitor panobinostat in the hopes of achieving greater efficacy. We showed that this epigenetic drug combination was more efficacious than single agents using cell line derived xenograft models of pediatric AML (Gopalakrishnapillai et al., Leuk Res, 2017).We evaluated the efficacy of this epigenetic drug combination in patient-derived xenograft models of KMT2A rearranged pediatric AML and observed that similar to MV4;11 model, this combination induced complete remission in NTPL-146 model with KMT2A-MLLT1 fusion (Fig. 1A, P<0.001). We analyzed the methylome of AML cells harvested from xenografted mice treated with control, azacitidine, panobinostat, or a combination of the two. Methylation sensitive restriction endonucleases were utilized to fragment genomic DNA prior to library construction for next generation sequencing. GenPro software platform designed for highly quantitative, sensitive, and low error-rate detection of methylation at individual CpG sites was used.Methylation patterns between treatment groups were discriminated using an ordinate analysis technique of non-metric multidimensional scaling (NMDS) (Fig. 1B). CpG methylation profiles were compared among the four groups analyzed to isolate patterns conserved within groups while also differing between groups. The first two component axes were plotted to locate the individual sample points in a 2D plane. Samples from distinct PDX models undergoing similar treatment clustered together. The panobinostat-treated samples showed minimal differences compared to the control, while the azacitidine-treated samples clustered away. Interestingly, the samples treated with the combination, did not overlap with either treatment, indicating that although panobinostat alone showed minimal impact on methylation patterns, panobinostat together with azacitidine produced a distinct methylation pattern. Venn intersection sets of statistically significant differentially methylated CpG sites in the 3-way analyses derived from the control group comparisons showed 2086 CpG sites exclusively altered in the combination treatment (Fig. 1C).In order to determine the effect of the combination treatment on global methylation, the differences in methylation load (dML) per each CpG site between control and the combination treatment were summed across 1MB genome intervals and the distribution of these dML was plotted (Fig. 1D). There was a strong shift in methylation signal, with the majority of the intervals being hypomethylated in the treatment group compared to the control. Although global hypomethylation was observed in combination treatment, the most statistically significant CpG sites were hypermethylated in the combination treatment compared to the control as seen in the volcano plot in which log fold-change was plotted against the p-value (Fig. 1E). Circular ideogram presented with a mean subtraction of CpG methylation scores to calculate a summation methylation load score across chromosomal domains (Fig. 1F). The correlative association between top CpG sites is shown as arcs tracking the highest correlation for each CpG site. Gene labels indicate the positions of the top 60 CpG sites, with green and red indicating higher methylation in control and in combination treatment respectively.In conclusion, we have identified differential methylation patterns following in vivo treatment of KMT2A rearranged pediatric AML xenograft models with azacitidine and panobinostat combination compared to azacitidine alone. These methylation changes are likely to influence the increased survival seen in mice receiving combination treatment. [Display omitted] DisclosuresGopalakrishnapillai: Geron: Research Funding. Marsh: Genome Profiling LLC: Current Employment. Barwe: Prelude Therapeutics: Research Funding.

Combinatorial Inhibition of Galectin-9 and CHK1 Represent a Novel Treatment Strategy for T-Cell Acute Lymphoblastic Leukemia

Due to improvements in treatment strategies, the five-year event-free survival rate for pediatric patients with acute lymphoblastic leukemia (ALL) is 90%. However, patients with relapse and refractory disease fare much worse with 5-year overall survival rates of less than 50% in patients receiving chimeric antigen receptor T-cell therapy and fewer than 20% of patients surviving after receiving hematopoietic stem cell transplantation. These dismal outcomes for patients with relapse and refractory disease highlight the need for novel treatment regimens when current therapeutic options are exhausted.T-cell acute lymphoblastic leukemia (T-ALL) accounts for around 15% and 25% of ALL cases in pediatric and adult populations, respectively. This disease is driven by various molecular changes including alterations in the epigenome due, in part, to deregulated epigenetic machinery such as the polycomb repressive complex 2 (PRC2). Despite this observation, and ongoing clinical trials determining the utility of epigenetic drugs for treating various hematological malignancies, the role of the epigenome in T-ALL pathogenesis and the efficacy of epigenetic modifying drugs as treatments for this disease is heavily understudied.Galectins are members of s-type lectins which promote diverse biological processes including adhesion, signaling, and immunosuppression. Galectin-9 (Gal-9) is an emerging therapeutic target for solid cancers and hematological malignancies given that its presence is associated with poor outcomes for multiple cancers. In unpublished studies, we have found that Gal-9 is expressed on the surface of multiple human ALL subtypes with the highest basal surface expression found on T-ALL cells. To determine how this lectin impacts the function of human T-ALL cells, we treated leukemia cells with immunoglobulin control (Ig Ctrl) or anti-Gal-9 antibody (αGal-9Ab) and assessed the impact of treatment on cell cycle progression, DNA damage, and apoptosis.We used two αGal-9Ab clones for these experiments, a commercially available antibody and LYT-200 (a proprietary antibody in Phase I clinical trials for solid tumors from PureTech Health). Treatment with the commercially available antibody, but not Ctrl Ig, increased histone 3 trimethylation (H3K2me 3/H3K4me 3) with accompanying decreases in EZH2 and RING1A protein expression in human T-ALL cell lines. Antibody-induced epigenetic changes also promoted cell cycle progression (G2M transition), DNA damage, and extensive apoptosis (>90%) in multiple human T-ALL cell lines (n>6). Importantly, LYT-200 single-agent treatment also induced cell death in human T-ALL cells, demonstrating that blocking multiple epitopes on Gal-9 is sufficient to induce T-ALL cytotoxicity. These results highlight a previously unreported role for Galectin-9 in the epigenetic regulation and survival of human T-ALL cells.Given our observations that epigenome stability is critical for the survival of human T-ALL cells, we next sought to determine if the combination of αGal-9Ab treatment and epigenetic modifying drugs would further enhance the cytotoxicity of human T-ALL cells. We tested the combination of αGal-9Ab treatment and multiple drugs targeting either histone acetylation, methylation, or phosphorylation. Of these, we found that combining αGal-9Ab and GDC-0575 (a CHK1 inhibitor) resulted in extensive DNA damage and cytotoxicity (>98%). Mechanistically, we found αGal-9Ab treatment induces DNA damage in multiple human T-ALL lines, which leads to CHK1 activation. Given that GDC-0575 inhibits CHK1 activity, and CHK1 is a master regulator of the DNA damage response, we predict that the enhanced cytotoxicity of human T-ALL cells treated with the combination therapy results from the inability to effectively repair DNA damage induced by αGal-9Ab treatment. Our findings describe a previously unrecognized role for Gal-9 in T-ALL pathogenesis and demonstrates the cytotoxic effects αGal-9Ab treatment (including LYT-200) in preclinical models of human T-ALL. DisclosuresLee: PureTech Health: Research Funding. Filipovic: PureTech Health: Research Funding. Henry: PureTech Health: Research Funding.

Epigenetic Regulation of Immunotherapy Response in Triple-Negative Breast Cancer.

Simple SummaryTriple-negative breast cancer (TNBC) outcomes are improving since the implementation of immunotherapy. However, objective response rates are still limited to a select group of patients. This is partly due to TNBC intrinsic immune evasive mechanisms and the lack of proper tumor microenvironment immune system activation. Dynamic epigenetic modifications contribute to immune surveillance and immune escape in cancer and can be reverted through epigenetic drugs. This review summarizes the epigenetic changes in TNBC cells and their contribution to the cancer cell–immunity cycle. Furthermore, it also describes how epigenetic drugs may provide novel biomarkers for immunotherapy and enhance the immune response. This manuscript lists the current clinical trials using epigenetic drugs alone or combined with either immune checkpoint inhibitors or small molecules.Triple-negative breast cancer (TNBC) is defined by the absence of estrogen receptor and progesterone receptor and human epidermal growth factor receptor 2 (HER2) overexpression. This malignancy, representing 15–20% of breast cancers, is a clinical challenge due to the lack of targeted treatments, higher intrinsic aggressiveness, and worse outcomes than other breast cancer subtypes. Immune checkpoint inhibitors have shown promising efficacy for early-stage and advanced TNBC, but this seems limited to a subgroup of patients. Understanding the underlying mechanisms that determine immunotherapy efficiency is essential to identifying which TNBC patients will respond to immunotherapy-based treatments and help to develop new therapeutic strategies. Emerging evidence supports that epigenetic alterations, including aberrant chromatin architecture conformation and the modulation of gene regulatory elements, are critical mechanisms for immune escape. These alterations are particularly interesting since they can be reverted through the inhibition of epigenetic regulators. For that reason, several recent studies suggest that the combination of epigenetic drugs and immunotherapeutic agents can boost anticancer immune responses. In this review, we focused on the contribution of epigenetics to the crosstalk between immune and cancer cells, its relevance on immunotherapy response in TNBC, and the potential benefits of combined treatments.

Deacetylase Plus Bromodomain Inhibition Downregulates ERCC2 and Suppresses the Growth of Metastatic Colon Cancer Cells.

Simple SummaryIn cancer cells, the DNA repair response can be exploited as an ‘Achilles heel’ to trigger programmed cell death pathways and tumor elimination. Rather than involving ‘naked’ DNA, repair occurs in the context of histone and non-histone proteins in the vicinity of the damage. Drugs that target different epigenetic mechanisms can lead to the synergistic downregulation of critical DNA repair factors, including those associated with poor survival in colorectal cancer patients. Notably, normal colonic epithelial cells are more resistant than colon cancer cells to the epigenetic drug combinations. In the current investigation, cell-based assays and preclinical animal models reaffirmed the crosstalk between DNA repair and epigenetic regulatory mechanisms, and provided new avenues for precision oncology and cancer interception.There is growing evidence that DNA repair factors have clinical value for cancer treatment. Nucleotide excision repair (NER) proteins, including excision repair cross-complementation group 2 (ERCC2), play a critical role in maintaining genome integrity. Here, we examined ERCC2 expression following epigenetic combination drug treatment. Attention was drawn to ERCC2 for three reasons. First, from online databases, colorectal cancer (CRC) patients exhibited significantly reduced survival when ERCC2 was overexpressed in colon tumors. Second, ERCC2 was the most highly downregulated RNA transcript in human colon cancer cells, plus Ercc2 in rat tumors, after treatment with the histone deacetylase 3 (HDAC3) inhibitor sulforaphane (SFN) plus JQ1, which is an inhibitor of the bromodomain and extraterminal domain (BET) family. Third, as reported here, RNA-sequencing of polyposis in rat colon (Pirc) polyps following treatment of rats with JQ1 plus 6-methylsulfinylhexyl isothiocyanate (6-SFN) identified Ercc2 as the most highly downregulated gene. The current work also defined promising second-generation epigenetic drug combinations with enhanced synergy and efficacy, especially in metastasis-lineage colon cancer cells cultured as 3D spheroids and xenografts. This investigation adds to the growing interest in combination approaches that target epigenetic ‘readers’, ‘writers’, and ‘erasers’ that are deregulated in cancer and other pathologies, providing new avenues for precision oncology and cancer interception.

A Perspective of Epigenetic Regulation in Radiotherapy.

Radiation therapy (RT) has been employed as a tumoricidal modality for more than 100 years and on 470,000 patients each year in the United States. The ionizing radiation causes genetic changes and results in cell death. However, since the biological mechanism of radiation remains unclear, there is a pressing need to understand this mechanism to improve the killing effect on tumors and reduce the side effects on normal cells. DNA break and epigenetic remodeling can be induced by radiotherapy. Hence the modulation of histone modification enzymes may tune the radiosensitivity of cancer cells. For instance, histone deacetylase (HDAC) inhibitors sensitize irradiated cancer cells by amplifying the DNA damage signaling and inhibiting double-strand DNA break repair to influence the irradiated cells’ survival. However, the combination of epigenetic drugs and radiotherapy has only been evaluated in several ongoing clinical trials for limited cancer types, partly due to a lack of knowledge on the potential mechanisms on how radiation induces epigenetic regulation and chromatin remodeling. Here, we review recent advances of radiotherapy and radiotherapy-induced epigenetic remodeling and introduce related technologies for epigenetic monitoring. Particularly, we exploit the application of fluorescence resonance energy transfer (FRET) biosensors to visualize dynamic epigenetic regulations in single living cells and tissue upon radiotherapy and drug treatment. We aim to bridge FRET biosensor, epigenetics, and radiotherapy, providing a perspective of using FRET to assess epigenetics and provide guidance for radiotherapy to improve cancer treatment. In the end, we discuss the feasibility of a combination of epigenetic drugs and radiotherapy as new approaches for cancer therapeutics.

LATE BREAKING NEWS E-POSTER PRESENTATION: LBP 4 Novel Epigenetic Small Molecule Approaches and Single-Cell Epigenetic Analysis for DMD

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Although DMD is a progressive degenerative disease, there is evidence for early, embryonic-stage defects in myogenesis and gene expression in DMD. By understanding how these defects initiate and contribute to DMD pathology, we may be better positioned to identify and utilize DMD therapies. Using single-cell RNAseq, we have identified one of the earliest known DMD phenotypes: a novel transcriptional trajectory of DMD human induced pluripotent stem cells (hiPSCs) undergoing myogenesis. Many of the dysregulated genes are involved in somitogenesis, advocating for a modified muscle development program in DMD cells. We are taking advantage of the zebrafish DMD model to identify conserved transcriptional dysregulation during embryonic development. We hypothesize that epigenetic drugs, small molecules that target chromatin modifications, can be used to ameliorate these early DMD transcriptional defects as well as improve DMD pathology. We performed a novel screen of an epigenetic small molecule library using DMD zebrafish and identified a novel combination of epigenetic drugs that rescues muscle degeneration in DMD zebrafish. To test the mechanisms of these new drugs, and to better characterize the epigenetic perturbations in DMD, we are testing whether epigenetic drugs correct the early transcriptional dysregulation observed in DMD hiPSCs and zebrafish embryos. Our studies support the combined use of zebrafish and hiPSCs as models for understanding the developmental epigenetic defects in DMD and for identifying new potential DMD therapies.

CD81 knockout promotes chemosensitivity and disrupts in vivo homing and engraftment in acute lymphoblastic leukemia

AbstractRelapse remains a major obstacle to achieving 100% overall survival rate in pediatric hematologic malignancies like acute lymphoblastic leukemia (ALL). Relapse often results from the development of chemoresistance. One of the mechanisms of chemoresistance involves ALL cell interactions with the bone marrow (BM) microenvironment, providing a sanctuary. This phenomenon is known as BM microenvironment-induced chemoprotection. Members of the transmembrane 4 superfamily (tetraspanins; TSPANs) are known to mediate microenvironmental interactions and have been extensively studied in solid tumors. Although the TSPAN family member CD81 is a minimal residual disease marker, its biological role in ALL is not well characterized. We show for the first time that CD81 knockout induces chemosensitivity, reduces cellular adhesion, and disrupts in vivo BM homing and engraftment in B-ALL. This chemosensitization is mediated through control of Bruton tyrosine kinase signaling and induction of p53-mediated cell death. We then show how CD81-related signaling can be disrupted by treatment with the epigenetic drug combination of DNA hypomethylating agent azacitidine (aza) and histone deacetylase inhibitor panobinostat (pano), which we previously used to sensitize ALL cells to chemotherapy under conditions that promote BM microenvironment-induced chemoprotection. Aza/pano-mediated modulation of CD81 surface expression is involved in decreasing BM load by promoting ALL cell mobilization from BM to peripheral blood and increasing response to chemotherapy in disseminated patient-derived xenograft models. This study identifies the novel role of CD81 in BM microenvironment-induced chemoprotection and delineates the mechanism by which aza/pano successfully sensitizes ALL cells via modulation of CD81.

Abstract S01-01: Therapeutic targeting of TMPRSS2 and ACE2 as a potential strategy to combat COVID-19

Abstract The novel SARS-CoV-2 infection responsible for the COVID-19 pandemic is expected to have an adverse effect on the progression of multiple cancers, including prostate cancer, due to the ensuing cytokine storm associated oncogenic signaling. A better understanding of the host cell factors and their regulators will help identify potential therapies to block SARS-CoV-2 infection at an early stage and thereby prevent cancer progression. Host cell infection by SARS-CoV-2 requires the binding of the viral spike S protein to ACE2 receptor and priming by the serine protease TMPRSS2—encoded by a well-known androgen response gene and highly expressed in patients diagnosed with prostate cancer. Epidemiologic data showing increased severity and mortality of SARS-CoV-2 disease in men suggest a possible role for androgen in the transcriptional activation of ACE2 and TMPRSS2 in the lungs and other primary infection sites. Here, by performing in vivo castration in mice, RT-PCR, immunoblotting, Co-IP, and pseudovirus infection assays in multiple cell lines, we present evidence for the transcriptional regulation of TMPRSS2 and ACE2 by androgen, their endogenous interaction, as well as a novel combination of drugs in blocking viral infection. In adult male mice, castration led to a significant loss in the expression of ACE2 and TMPRSS2 at the transcript and protein levels in the lung, heart, and small intestine. Intriguingly, castrated mice displayed a substantial increase in ACE2 in the kidney, which could potentially be due to the low blood pressure resulting from androgen deprivation. Endogenous TMPRSS2 and ACE2 were found to be physically interacting, as observed by reciprocal immunoprecipitation followed by immunoblotting. Importantly, along with full-length zymogen form, a prominent novel small isoform of TMPRSS2 was found to be associated with ACE2 in lung and prostate cells. In an overexpression system, camostat—a serine protease inhibitor specific to TMPRSS2—inhibited the cleavage of spike S, suggesting a direct role of this serine protease in priming the viral spike S protein, a prerequisite for an active infection. Furthermore, a combination of camostat, antiandrogen, and epigenetic drugs at sublethal concentrations blocked SARS-CoV-2 pseudovirus infection in multiple cell types. Together, our preclinical data provide a strong rationale for clinical evaluation of TMRPSS2 inhibitors, antiandrogens, and epigenetic drug combinations along with antiviral drug remdesivir as early as clinically possible to prevent progression to pneumonia and multiorgan failure as a result of hyperinflammatory responses in COVID-19 patients. Citation Format: Qu Deng, Reyaz Ur Rasool, Ramakrishnan Natesan, Irfan A. Asangani. Therapeutic targeting of TMPRSS2 and ACE2 as a potential strategy to combat COVID-19 [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S01-01.

Modulation of CD81 By Epigenetic Drug Combination Sensitizes Acute Lymphoblastic Leukemia Via Decreased BTK Signaling

Background We previously discovered that the epigenetic drug combination - azacitidine (aza, DNMTi) and panobinostat (pano, HDACi) can sensitize ALL cells to chemotherapy (Quagliano et al., Leuk Res, 44:101, 2017). This sensitization occurred through a decrease in cellular adhesion and a modulation of the cell surface expression of the tetraspanin protein CD81 (Quagliano et al., Blood, 132:3957, 2018). This study aims to elucidate the molecular mechanism by which CD81 surface modulation by pre-treatment with aza/pano leads to chemosensitization. Methods CD81 knockout Nalm6 (CD81KO) cells were generated by CRISPR/Cas9 mutagenesis. IHC was performed using anti-human mitochondria antibody on femurs harvested from NSG-SGM3 mice at 72 h post transplantation with WT or CD81KO cells. For adhesion assay, ALL cells stained with VPD450 were co-cultured with Saos-2 cells for 24 h. Unbound cells were washed, and bound cells were collected for flow cytometry analysis. CD19 surface expression was determined after 48 h treatment with aza/pano (500 nM/1.5 nM) using flow cytometry. For western blot analysis, aza/pano treated cells were transferred on to Saos-2 monolayers and treated with Ara-C (30 nM) for 16 h. Results CD81KO cells not only had decreased homing and engraftment visualized by reduced ALL cell presence in bone marrow (Fig. 1A), but also had 45% reduction in cellular adhesion to osteoblasts compared to WT Nalm6 cells (Fig. 1B), indicating that CD81 downregulation results in reduced bone marrow interactions. To investigate the signaling effects downstream of aza/pano-mediated CD81 modulation, we first tested if CD19 was affected due to its prominent role in the formation of the B-cell co-receptor and because CD19 requires CD81 for proper membrane trafficking (Cherukuri et al., J Immunol, 172:370, 2004). CD19 surface expression was reduced in aza/pano-treated cells and completely knocked out in CD81KO cells (Fig. 1C). CD19 is known to prolong and amplify the activation of Bruton's tyrosine kinase (BTK), so we also analyzed the phosphorylation of BTK in aza/pano-treated cells and found it to be reduced by 63% (Fig. 1D). This reduction in BTK phosphorylation may be responsible for the decrease in cell adhesion following treatment with aza/pano because prior studies observed reduced adhesion following BTK inhibition (Herman et al., Clin Cancer Res, 21:4642, 2015). Taken together, these data suggest that aza/pano induced reduction in cell adhesion was mediated by downregulation of CD81 and BTK dephosphorylation. We studied how the expression of p53 and its target BCL2 associated X protein (BAX), which are known to be induced by Ara-C, were affected following pre-treatment with aza/pano. Cells with Ara-C treatment alone or the aza/pano pre-treatment both had minor induction of p53 expression compared to control, while aza/pano pre-treated cells that received Ara-C had further increase in p53 protein (1.7-fold increase compared to Ara-C alone, Fig. 1E). Bax mRNA and protein were increased 4.2-fold in aza/pano-treated cells following Ara-C treatment compared to Ara-C alone. Increased Bax expression following Ara-C treatment in aza/pano pre-treated cells was accompanied by a 3.9-fold increase in cleavage of caspase-3 compared to cells treated with Ara-C alone, which in turn causes cleavage of Poly-ADP Ribose Polymerase (PARP) (Fig. 1E). Both p53 and Bax protein levels were higher in CD81KO cells than in WT cells (Fig. 1E), suggesting that aza/pano-mediated reduction of CD81 is involved in inducing the expression of p53. CD81KO cells that were treated with Ara-C also had further increase in p53 and Bax expression compared to Ara-C treated WT cells (Fig. 1E), consistent with an increased sensitivity to chemotherapy. To confirm the role of BTK inhibition in the induction of p53, Nalm6 cells were treated with the BTK inhibitor fenebrutinib for 24 h (10 nM). Following treatment, expression of p53 was induced and Bax expression was 2.6-fold higher in treated cells compared to untreated cells (Fig. 1E). Taken together, these data suggest that modulation of the surface expression of CD81 and the phosphorylation of BTK by aza/pano induces sensitization via upregulation of p53 and overexpression of Bax. Conclusion We identify a novel mechanism by which aza/pano treatment induces chemosensitization by reducing cell adhesion via modulating CD81 surface expression, reducing BTK phosphorylation, and inducing p53. Figure 1 Disclosures No relevant conflicts of interest to declare.

Abstract 2368: Domatinostat increases apoptosis, G2M cell-cycle arrest and immunogenicity of Merkel cell carcinoma

Abstract Merkel cell carcinoma (MCC) is a rare, highly aggressive skin cancer prevalent in elderly and immunocompromised patients. MCC is highly immunogenic as it is either associated with Merkel cell polyoma virus (MCPyV) integration or UV-associated mutations in non-viral cases. Indeed, immune checkpoint inhibitors (CPI) like PD-1/PD-L1 blocking antibodies exert a strong clinical activity; however, primary or secondary resistance often occurs. Domatinostat (4SC-202) is an orally available small molecule inhibitor targeting histone deacetylases (HDAC) class I currently in clinical evaluation to improve response to CPI (SENSITIZE, NCT03278665). In syngeneic tumor mouse models domatinostat treatment demonstrated immune-modulatory effects by increasing the intra-tumoral infiltration of cytotoxic CD8+ T cells (CTLs) and enhancing gene expression of a CPI response signature. Immune escape mechanisms described for MCC include low intra-tumoral levels of CTLs and reduced expression of antigen-presenting MHC class I molecules. Our previous data suggest that low MHC-I levels are reversible and involve epigenetic silencing of genes encoding the antigen-processing machinery (APM). Here, we present novel preclinical data about the efficacy and mode of action of domatinostat in MCC. Global gene expression by single cell RNA-seq revealed regulation of genes involved, among others, in apoptosis and antigen presentation. Importantly, domatinostat additionally inhibited proliferation of MCC cell lines by induction of a G2M cell-cycle arrest and apoptosis. Moreover, expression of MCPyV-encoded transforming early genes, particularly during the G1-phase, was inhibited by domatinostat. Thus, it exerts also a direct anti-tumoral effect. In viable cells, domatinostat increases their susceptibility to immune responses, as qPCR and immunoblot analysis confirmed the induction of APM and MHC-I expression by domatinostat. APM and MHC-I expression has been reported to be restored by an epigenetic drug combination (vorinostat plus mithramycin). In contrast, we now provide evidence that the single agent treatment with domatinostat alone is sufficient for increasing immunogenicity of MCC cells. In summary, domatinostat counteracts immune escape of MCC in many aspects suggesting a combination treatment of the HDACi domatinostat with CPI as a promising therapeutic strategy. Prospective clinical trials are needed to confirm this hypothesis. Citation Format: Lina Song, Anne Catherine Bretz, Jan Gravemeyer, Ivelina Spassova, Svetlana Hamm, Shakhlo Muminova, Rene Bartz, Juergen C. Becker. Domatinostat increases apoptosis, G2M cell-cycle arrest and immunogenicity of Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2368.

Regulation of OCT2 transcriptional repression by histone acetylation in renal cell carcinoma

ABSTRACTRenal cell carcinoma (RCC) is a common malignant tumour affecting the urinary system, and multidrug resistance is one of the major reasons why chemotherapy for this type of cancer often fails. Previous studies have shown that loss of the human organic cation transporter OCT2 is the main factor contributing to oxaliplatin resistance in RCC, and that DNA hypermethylation and histone methylation play important roles in the transcriptional repression of OCT2 in RCC. In this study, we found that histone acetylation also regulates OCT2 repression in RCC and elucidated the underlying mechanisms. In normal renal cells, HDAC7 combines with MYC at the OCT2 promoter, resulting in a decrease in free HDAC7, which in turn increases the levels of H3K18ac and H3K27ac at the OCT2 promotor and activates OCT2 expression. In RCC cells, however, the interaction between HDAC7 and MYC does not occur, which leads a high abundance of HDAC7 and low levels of H3K18ac and H3K27ac at the OCT2 promoter, thereby resulting in the inhibition of OCT2 transcription. We found that combined treatment using the DNA methylation inhibitor decitabine and the histone deacetylase inhibitor vorinostat significantly increased the expression of OCT2 in RCC cell lines, which sensitized these cells to oxaliplatin. We accordingly propose that the combination of anticancer agents and epigenetic drugs can provide a novel chemotherapeutic regimen.

Epigenetic treatment combinations to effectively target cisplatin-resistant germ cell tumors: past, present, and future considerations.

Type II germ cell tumors represent the most common solid malignancy in men aged 15-45years. Despite high cure rates of >90% over all stages, 10-15% of advanced patients develop treatment resistance and potentially succumb to their disease. Treatment of refractory germ cell tumors remains unsatisfactory, and new approaches are needed to further improve outcomes. With this narrative review, we highlight epigenetic mechanisms related to resistance to standard systemic treatment, which may act as promising targets for novel combined epigenetic treatment approaches. A comprehensive literature search of PubMed and MEDLINE was conducted to identify original and review articles on resistance mechanisms and/or epigenetic treatment of germ cell tumors invitro and invivo. Review articles were hand-searched to identify additional articles. Distinct epigenetic phenomena have been linked to chemotherapy resistance in germ cell tumors, among which DNA hypermethylation, histone acetylation, and bromodomain proteins appear as promising targets for therapeutic exploitation. Inhibitors of key regulators, for example DNA methyltransferases (e.g. decitabine, guadecitabine), histone deacetylases (e.g. romidepsin), and bromodomain proteins (e.g. JQ1) decreased cell viability, triggered apoptosis, and growth arrest. Additionally, these epigenetic drugs induced differentiation and led to loss of pluripotency and re-sensitization towards cisplatin in cell lines and animal models. Epigenetic treatments hold promise to (i) reduce the treatment burden of and (ii) overcome resistance to standard cisplatin-based chemotherapy. Combined approaches may enhance activity, while the ideal target and treatment combination of epigenetic drugs, either with another epigenetic agent or conventional cytotoxic agents need to be defined. Epigenetic (combination) treatment for germ cell tumors should be further explored in pre-clinical and clinical research for its potential to further improve germ cell tumor treatment.