Michael Topper

Inhibitors of DNA Methylation, Histone Deacetylation, and Histone Demethylation: A Perfect Combination for Cancer Therapy

Epigenetic silencing and inappropriate activation of gene expression are frequent events during the initiation and progression of cancer. These events involve a complex interplay between the hypermethylation of CpG dinucleotides within gene promoter and enhancer regions, the recruitment of transcriptional corepressors and the deacetylation and/or methylation of histone tails. These epigenetic regulators act in concert to block transcription or interfere with the maintenance of chromatin boundary regions. However, DNA/histone methylation and histone acetylation states are reversible, enzyme-mediated processes and as such, have emerged as promising targets for cancer therapy. This review will focus on the potential benefits and synergistic/additive effects of combining DNA-demethylating agents and histone deacetylase inhibitors or lysine-specific demethylase inhibitors together in epigenetic therapy for solid tumors and will highlight what is known regarding the mechanisms of action that contribute to the antitumor response.

Epigenetic therapy activates type I interferon signaling in murine ovarian cancer to reduce immunosuppression and tumor burden

Significance Therapies that activate the host immune system have shown tremendous promise for a variety of solid tumors. However, in most cancer types, fewer than half of patients respond to these immunotherapies. We propose epigenetic therapy as a mechanism to sensitize tumors to immune checkpoint therapy. We have shown that inhibiting DNA methylation triggers a viral defense pathway in tumors. Here we show that epigenetic therapy in a mouse model of ovarian cancer increases the numbers of activated immune cells, and that this is dependent on the interferon antiviral response. The combination of epigenetic therapy and immune checkpoint blockade leads to the greatest reduction in tumor burden and increase in survival, and may hold the greatest promise for patients. Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

Reply to Haffner et al.: DNA hypomethylation renders tumors more immunogenic

In the letter by Haffner et al. (1), they report that seminoma cell-intrinsic DNA hypomethylation is associated with endogenous retroviral expression, an IFN response, and lymphocytic infiltration. Their data complement and support our recent therapeutic study in a mouse model of ovarian cancer (2) and support our observations that low doses of the DNA methyltransferase (DNMT) inhibitor azacytidine (AZA), in combination with the histone deacetylase inhibitor, givinostat, activate type 1 IFN signaling in ovarian (2) and lung (3) cancer cells to increase numbers and activation of immune cells in the tumor microenvironment and to increase sensitivity to the … [↵][1]5To whom correspondence may be addressed. Email: zahnoci{at}jhmi.edu or sbaylin{at}jhmi.edu. [1]: #xref-corresp-1-1

Abstract LB-098: Sequential azacitidine and histone deacetylase inhibition induces a potent antitumor response in Kras G12D mouse model of NSCLC

Combination epigenetic treatment as a therapeutic approach holds much promise. However, to unlock the true potential of these combinatorial paradigms, further optimization is required. We have elucidated in our current study, a treatment schedule combining Azacitidine with Histone Deacetylase Inhibition (HDACi) which considers both schedule and isoform specificity. We have deployed a sequential schedule with HDACi administered post Azacitidine in a chronic low dose manner. This schedule takes advantage of two key parameters altered by Azacitidine: dsRNA induced immune signature and MYC depletion. In our system, HDACis amplify an existing immune signature and the MYC depleted environment acts as a potent sensitizer. HDACi induced augmentation of immune gene transcription seems driven predominantly through the inhibition of HDAC1/2 and HDAC6 isoforms, with broad spectrum HDACi inhibitors demonstrating the greatest potency. While the combination treatment induced perturbation of proliferation is driven by inhibition of HDAC1/2/3 isoforms. Applying this concept to a genetically engineered mouse model of NSCLC we observe a potent anti-tumor response as evidenced by a reduction in tumor burden, progression and proliferation. These phenotypic parameters are correlated with modification of both immune and proliferative gene signatures as well as alteration of key immune populations in the tumor microenvironment. We propose that this combinatorial alteration of both immune and proliferative parameters holds the promise to be a more robust treatment for NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Michael Topper, Michelle Vaz, Katherine Chiappinelli, Christina DeStefano Shields, Alyssa Wenzel, Cynthia Zahnow, Pam Strissel, Reiner Strick, Stephen Baylin. Sequential azacitidine and histone deacetylase inhibition induces a potent antitumor response in Kras G12D mouse model of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-098. doi:10.1158/1538-7445.AM2017-LB-098

Abstract 4019: Inhibiting DNA methylation causes an interferon response in cancer cells via endogenous retroviruses and recruits immune cells to the tumor microenvironment to sensitize to immune therapy

Therapies that activate the host immune system have shown tremendous promise for a wide variety of solid tumors, with patients exhibiting vigorous and durable responses. However, in most cancer types, fewer than half of patients respond to these immune therapies. We propose epigenetic therapy as a mechanism to sensitize these patients. DNA methyltransferase inhibitors (DNMTis) upregulate immune attraction, including the interferon response, in solid tumors. We have shown that in human epithelial ovarian cancer cells, DNMTis upregulate viral defense by cytosolic sensing of double-stranded RNA (dsRNA), triggering a Type I Interferon response and apoptosis. Demethylation and expression of bidirectionally transcribed endogenous retroviruses (ERVs) is a major component of the dsRNA that activates the response. Our work showed that treatment with the DNMTi 5-azacytidine (Aza) sensitizes mouse melanoma cells to subsequent anti-CTLA4 therapy, likely through activation of the interferon response and subsequent signaling to host immune cells. Our current work aims to verify this hypothesis. In addition, we observe that adding histone deacetylase inhibitors (HDACis) to DNMTis can augment the upregulation of specific ERVs and the resulting downstream interferon response in human cancer cell lines. Specifically, the ERV-K family as well as the Fc2 and ERV-9 families are increased by DNMTi treatment but further augmented by HDACi treatment, while HDACis alone have minimal effects on the ERVs and the downstream interferon response. We tested the hypothesis that epigenetic drugs sensitize to immune therapy by recruiting host immune cells in an immunocompetent mouse model of serous ovarian cancer. Treatment of this model with DNMTi and HDACi results in increased recruitment of (CD3+) T cells, including tumor-killing T Effector cells, to the tumor. This epigenetic therapy causes increased activation of CD8 T cells and natural killer cells, an increase in helper T cells, and a reduction in myeloid derived suppressor cells and macrophages. We observed upregulation of the immune checkpoint ligand PD-L1 on tumor cells by DNMTis and hypothesized that treatment of this mouse model with the above drug combination plus an antibody to the PD-L1 receptor (anti-PD-1) could reduce tumor burden. This combination does indeed significantly reduce tumor burden and increase survival. We thus define a major mechanism for how DNMTis and HDACis may induce cancer cells to increase attraction and activation of immune cells and sensitize patients to immunotherapy. Citation Format: Katherine B. Chiappinelli, Meredith L. Stone, Michael J. Topper, Lauren Murphy, Pamela L. Strissel, Reiner Strick, Cynthia A. Zahnow, Stephen B. Baylin. Inhibiting DNA methylation causes an interferon response in cancer cells via endogenous retroviruses and recruits immune cells to the tumor microenvironment to sensitize to immune therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4019.

Abstract 4653: Combination Azacitidine and histone deacetylase inhibition induces a multi factorial synergistic anti-tumor response in non-small cell lung cancer (NSCLC)

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Epigenetic therapy holds much promise as an emerging paradigm for the treatment of many disparate types of human cancer. However, the data in solid malignancies, particularly, would suggest further optimization is required to extract the true potential of these therapies. In our current study, we have deployed at low, clinically achievable doses, chronic combinatorial administration of the DNA methyltransferase inhibitor Azacitidine in conjunction with both benzamide and hydroxyamic acid based histone deacetylase inhibitors (HDACi) in NSCLC. We have developed our paradigms by sequentially employing chronic HDACi administration after Azacitidine. This strategy achieves durable alteration of the cell transcriptome with corresponding induction of both cell cycle arrest and apoptosis. These events appear driven by alterations, both transcriptionally and post transcriptionally of Myc and Myc regulatory proteins. Epigenetic treatment induced perturbation of Myc is facilitated by Wnt pathway inhibition through induction of Wnt antagonists, namely DKK proteins. Importantly, as part of this scenario of altering a Myc regulatory axis, there is a synergistic cytotoxic response mediated at least in part through downregulation of a critical effector, the Skp2 oncogene. Additionally, we observe significant induction of an interferon responsive viral defense gene signature as the result of epigenetic treatment. This gene signature has been shown to correlate with response to immune checkpoint blockade in the setting of metastatic melanoma. We propose that this combination of viral defense gene signature augmentation in conjunction with potent direct epithelial cancer cytotoxicity holds promise to be a more complete therapeutic paradigm for the treatment of NSCLC. Citation Format: Michael Topper, Christin Hanigan, Michelle Vaz, Katherine Chiappinelli, Julin Justin, Lauren Murphy, Cynthia Zahnow, Stephen Baylin. Combination Azacitidine and histone deacetylase inhibition induces a multi factorial synergistic anti-tumor response in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4653.

Abstract 4907: Epigenetic treatment of ovarian cancer cells increases immune cell recruitment to the tumor microenvironment: implications for response to immune checkpoint therapy

Immunosuppression and relapse represent two major clinical challenges in managing patients with ovarian high-grade serous carcinoma (HGSC). Strategies to increase the host immune response or decrease immunosuppression hold promise for clinical outcome in ovarian cancer patients. Blockade of inhibitory immune checkpoint pathways with antibodies directed at the PD-1/PD-L1 pathway has demonstrated encouraging anti-tumor activity in a number of solid tumors, including a small number of HGSC patients. We hypothesize that tumors which fail to respond to checkpoint inhibition are those in which the tumor cells do not provide a sufficient positive immune stimulus needed for recruitment of immune cells. Our studies suggest that the DNA demethylating agent, azacytidine (AZA), upregulates an immune signature (AIM, Aza induced immune genes) in cancer cells and that among carcinomas, ovarian HGSC appears most immunoresponsive to AZA. We hypothesize that AIM gene expression is elevated by epigenetic treatment with AZA and/or HDAC inhibitors and promotes the recruitment of tumor infiltrating immune cells to the tumor. Moreover, immune checkpoint inhibitors (anti- PD-1) may then lead to activation of these recruited immune cells and a tumor immune response. To test the hypothesis of whether AZA/HDACi treatment of tumor cells increases the recruitment of immune cells to the tumor microenvironment, the mouse ovarian cancer cell line, ID8, was treated ex vivo with AZA or HDACis and implanted into syngeneic C57Bl/6, immunocompetent mice. Ex vivo AZA treatment of the tumor cells led to increased recruitment of immune cells to the ascites, decreased tumor burden, and an increased sensitivity to anti-PD-1, while similar treatment with Entinostat as a single agent did not. To test the in vivo effects of these agents on both the tumor and microenvironment, mice with pre-established ID8 tumors were treated with AZA, Entinostat or Givinostat, and anti-PD-1 to test the drugs’ antitumorigenic and immunogenic effects in combination and as single agents. We found that combination therapy with AZA, HDACi, and anti-PD-1 led to the most significant decrease in tumor burden, increased survival of the mice, increased numbers of activated T cells and NK cells in the ascites fluid and a decreased number of myeloid derived suppressor cells. In summary, we have clearly demonstrated that tumor cells treated with AZA effectively recruit increased numbers of immune cells to the tumor ascites. HDACis and anti-PD1 are less effective in this regard when administered as single agents, but this effect is significantly increased when used in combination with a demethylating agent. These data have implications for improving the effectiveness of epigenetic therapy and interpreting outcomes from those trials which have used only a demethylating agent or an HDACi with checkpoint inhibition. Citation Format: Meredith L. Stone, Katherine B. Chiappinelli, Huili Li, Lauren Murphy, Michael Topper, Stephen Baylin, Cynthia Zahnow. Epigenetic treatment of ovarian cancer cells increases immune cell recruitment to the tumor microenvironment: implications for response to immune checkpoint therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4907.

Abstract PR12: Immunomodulatory effects of 5-Azacyditine in ovarian cancer cell lines

Epithelial ovarian cancer is a deadly disease due to late detection and lack of targeted therapies. Novel therapies for ovarian cancer are clearly needed and epigenetic agents have emerged as promising new therapies for ovarian cancer and other solid tumors. Clinical success observed for patients with lung cancer suggests that the actions of the DNA methyltransferase inhibitor 5-azacytidine (AZA) may stimulate the immune response and target immune cells to clear tumors. Ovarian cancer is a good candidate for immune therapy, as infiltrating lymphocytes predict longer time to recurrence. Therapies that upregulate the immune system have been shown to be effective in mouse and human ovarian cancers. To determine how epigenetic therapy affects ovarian cancer cells, we utilized genome-wide methylation and expression profiling on 17 ovarian cancer cell lines treated with low-dose AZA. We discovered upregulation of immunomodulatory pathways including viral defense, type I interferon signaling, antigen processing and presentation, and immune evasion via Gene Set Enrichment Analysis. In addition, we observed strong upregulation of cancer testis antigens and molecules that attract and activate natural killer cells. Endogenous retroviruses were also increased; increased transcription of these elements caused by reversal of DNA methylation may trigger the viral defense/ interferon response. This was a specific, not an off-target, effect of AZA, as a colon cancer cell line (DKO) genetically haploinsufficient for DNMT1 and lacking DNMT3b showed a similar upregulation of immune genes. Treatment with the chemotherapeutic agent carboplatin did not upregulate the same immune response genes. The cell lines that had the highest upregulation of immune genes after AZA treatment were often the best responders to AZA when grown as xenografts in NOD/SCID mice. We hypothesize that AZA activates the immune response in cancer cells, resulting in tumor cell killing. Validation of immune target genes showed that AZA treatment activates the interferon response, including transcription of interferon beta. Media transferred from AZA-treated cells to naive cells was sufficient to cause an interferon response in the target cells, as evidenced by increased levels of interferon-stimulated genes. FACS staining confirmed the upregulation of antigen-presenting MHC Class I molecules on the cell surface of AZA-treated ovarian cancer cells. Adding the histone deacetylase inhibitor MS275 (entinostat) to AZA treatment increased AZA-induced expression of interferon-stimulated genes IRF7 and IFI27. This combination treatment also caused an increase in the activating chromatin mark H3Ac at the IRF7 promoter. AZA increased transcript levels and cell surface expression of the PD-L1 molecule, ligand for PD-1 on lymphocytes and responsible for evasion of the host immune system by tumors, in ovarian cancer cell lines. We predict that AZA plus anti-PD-L1 (or PD-1) treatment, which has shown success in non-small cell lung cancer, might be effective for ovarian cancers. Future work will involve combination treatment of AZA and anti-PD-1 in immune competent ovarian cancer mouse models. The immune pathways upregulated by AZA were used to query hundreds of primary ovarian cancer samples from the Cancer Genome Atlas project (TCGA). Ovarian tumors classified clearly into “high” and “low” interferon gene expression subsets. The “high” interferon group was associated with higher levels of antigen presentation as well as an expression signature associated with better prognosis. The “low” interferon group indicates a group of solid tumors that could be targeted by AZA to upregulate levels of immunomodulatory pathways, and thus apoptosis or targeting by host immune cells. These preliminary results point to an “immune priming” role for the DNA methyltransferase inhibitor 5-azacytidine and could lead to clinical trials with combined AZA and immune therapies in epithelial ovarian cancer. This abstract is also presented as Poster B79. Citation Format: Katherine B. Chiappinelli, Ray-Whay Chiu Yen, Huili Li, Michael Topper, Cynthia A. Zahnow, Stephen B. Baylin. Immunomodulatory effects of 5-Azacyditine in ovarian cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR12.