Pragya Srivastava

Immunomodulatory action of SGI-110, a hypomethylating agent, in acute myeloid leukemia cells and xenografts

The mechanism of clinical action for the FDA approved hypomethylating drugs azacitidine and decitabine remains unresolved and in this context the potential immunomodulatory effect of these agents on leukemic cells is an area of active investigation. Induced expression of methylated Cancer Testis Antigen (CTA) genes has been demonstrated in leukemic cell lines following exposure to hypomethylating drugs in vitro. SGI-110 is a novel hypomethylating dinucleotide with prolonged in vivo exposure and clinical activity in patients with MDS and AML. We demonstrate that this agent, like decitabine, produces robust re-expression of the CTAs NY-ESO-1 and MAGE-A, both in vitro and in leukemia-bearing AML xenografts. Upregulation of these genes in vitro was sufficient to induce cytotoxicity by HLA-compatible CD8+ T-cells specific for NY-ESO-1, a well-recognized and immunogenic CTA. Additionally, exposure to SGI-110 enhances MHC class I and co-stimulatory molecule expression, potentially contributing to recognition of CTAs. SGI-110, like the parent compound decitabine, induces expression of CTAs and might modulate immune recognition of myeloid malignancy.

Neem oil limonoids induces p53-independent apoptosis and autophagy.

Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.

Immunomodulatory action of the DNA methyltransferase inhibitor SGI-110 in epithelial ovarian cancer cells and xenografts

We aimed to determine the effect of SGI-110 on methylation and expression of the cancer testis antigens (CTAs) NY-ESO-1 and MAGE-A in epithelial ovarian cancer (EOC) cells in vitro and in vivo and to establish the impact of SGI-110 on expression of major histocompatibility (MHC) class I and Intracellular Adhesion Molecule 1 (ICAM-1) on EOC cells, and on recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. We also tested the impact of combined SGI-110 and NY-ESO-1-specific CD8+ T-cells on tumor growth and/or murine survival in a xenograft setting. EOC cells were treated with SGI-110 in vitro at various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1 and MAGE-A methylation, mRNA, and protein expression were determined and compared to controls. SGI-110 treated EOC cells were evaluated for expression of immune-modulatory genes using flow cytometry, and were co-cultured with NY-ESO-1 specific T-cell clones to determine immune recognition. In vivo administration of SGI-110 and CD8+ T-cells was performed to determine anti-tumor effects on EOC xenografts. SGI-110 treatment induced hypomethylation and CTA gene expression in a dose dependent manner both in vitro and in vivo, at levels generally superior to azacitidine or decitabine. SGI-110 enhanced the expression of MHC I and ICAM-1, and enhanced recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. Sequential SGI-110 and antigen-specific CD8+ cell treatment restricted EOC tumor growth and enhanced survival in a xenograft setting. SGI-110 is an effective hypomethylating agent and immune modulator and, thus, an attractive candidate for combination with CTA-directed vaccines in EOC.

Induction of cancer testis antigen expression in circulating acute myeloid leukemia blasts following hypomethylating agent monotherapy

Cancer testis antigens (CTAs) are promising cancer associated antigens in solid tumors, but in acute myeloid leukemia, dense promoter methylation silences their expression. Leukemia cell lines exposed to HMAs induce expression of CTAs. We hypothesized that AML patients treated with standard of care decitabine (20mg/m2 per day for 10 days) would demonstrate induced expression of CTAs. Peripheral blood blasts serially isolated from AML patients treated with decitabine were evaluated for CTA gene expression and demethylation. Induction of NY-ESO-1 and MAGEA3/A6, were observed following decitabine. Re-expression of NY-ESO-1 and MAGEA3/A6 was associated with both promoter specific and global (LINE-1) hypomethylation. NY-ESO-1 and MAGEA3/A6 mRNA levels were increased irrespective of clinical response, suggesting that these antigens might be applicable even in patients who are not responsive to HMA therapy. Circulating blasts harvested after decitabine demonstrate induced NY-ESO-1 expression sufficient to activate NY-ESO-1 specific CD8+ T-cells. Induction of CTA expression sufficient for recognition by T-cells occurs in AML patients receiving decitabine. Vaccination against NY-ESO-1 in this patient population is feasible.

NY-ESO-1 Vaccination in Combination with Decitabine Induces Antigen-Specific T-lymphocyte Responses in Patients with Myelodysplastic Syndrome

Purpose: Treatment options are limited for patients with high-risk myelodysplastic syndrome (MDS). The azanucleosides, azacitidine and decitabine, are first-line therapy for MDS that induce promoter demethylation and gene expression of the highly immunogenic tumor antigen NY-ESO-1. We demonstrated that patients with acute myeloid leukemia (AML) receiving decitabine exhibit induction of NY-ESO-1 expression in circulating blasts. We hypothesized that vaccinating against NY-ESO-1 in patients with MDS receiving decitabine would capitalize upon induced NY-ESO-1 expression in malignant myeloid cells to provoke an NY-ESO-1–specific MDS-directed cytotoxic T-cell immune response. Experimental Design: In a phase I study, 9 patients with MDS received an HLA-unrestricted NY-ESO-1 vaccine (CDX-1401 + poly-ICLC) in a nonoverlapping schedule every four weeks with standard-dose decitabine. Results: Analysis of samples serially obtained from the 7 patients who reached the end of the study demonstrated induction of NY-ESO-1 expression in 7 of 7 patients and NY-ESO-1–specific CD4+ and CD8+ T-lymphocyte responses in 6 of 7 and 4 of 7 of the vaccinated patients, respectively. Myeloid cells expressing NY-ESO-1, isolated from a patient at different time points during decitabine therapy, were capable of activating a cytotoxic response from autologous NY-ESO-1–specific T lymphocytes. Vaccine responses were associated with a detectable population of CD141Hi conventional dendritic cells, which are critical for the uptake of NY-ESO-1 vaccine and have a recognized role in antitumor immune responses. Conclusions: These data indicate that vaccination against induced NY-ESO-1 expression can produce an antigen-specific immune response in a relatively nonimmunogenic myeloid cancer and highlight the potential for induced antigen-directed immunotherapy in a group of patients with limited options. Clin Cancer Res; 24(5); 1019–29. ©2017 AACR. See related commentary by Fuchs, p. 991

Pharmacological targeting of β-catenin in normal karyotype acute myeloid leukemia blasts

Over 11,000 people in the US are diagnosed with acute myeloid leukemia (AML) each year, and despite attempts to improve therapy, the overall 5-year survival remains 26%, largely due to a high rate of relapse. Defining the mechanisms responsible for persistence of the malignant clone is essential to

Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins.

Effect of Dietary Resveratrol in the Treatment of Cancer

Resveratrol (3,5,4′-trihydroxystilbene), a naturally occurring phytoalexin, is found in natural foods and food products such as grapes, peanuts, berries, and red wine. In recent years, resveratrol has gained considerable attention for its anticancer activities across multiple cancers. The anticancer effects of resveratrol are multifaceted and involve regulation of diverse cellular signaling, including apoptotic and non-apoptotic cell death, cell survival, and cell cycle progression. Anticancer activities of resveratrol are associated with engagement of AKT/PKB pathway, NF-κB pathway, death-receptor and TRAIL-induced apoptosis, and induction of p21/p27, as well as downregulation of cyclins and cyclin-dependent kinases. Resveratrol has been shown to have synergistic effects with radiation therapy. Resveratrol increases the therapeutic index of anticancer agents by sensitizing cancer cells to apoptosis. In preclinical studies, evidence gathered from mouse models suggests that resveratrol has beneficial effects against several types of human cancers and has been shown to inhibit or attenuate the initiation and progression of skin, prostate, colon, and breast cancers. Although resveratrol is widely considered to be well-tolerated in clinical trials for the treatment of human cancers, a limited number of studies have so far been conducted in human population because of poor bioavailability. These preliminary studies in human clinical trials have yet to recapitulate the physiological benefits seen in mouse models. This chapter provides a comprehensive description of resveratrol-mediated modulation of cellular signaling pathways, which may be exploited to develop further strategies for cancer therapy.

Abstract 681: SGI-110, a novel 5-aza-2'-deoxycytidine- containing dinucleotide, induces Cancer Germline (CG) antigen expression in Acute Myeloid Leukemia (AML) cells.

Introduction: CG antigens are genes whose normal expression is limited to the adult testis and embryonic ovary. Aberrant expression of these genes is reported in solid cancers where they are known to be immunogenic. CG targeted cancer vaccines are in clinical development for cancers with endogenous gene expression. The CG genes NY-ESO1 and MAGEA are silent in adult tissues due to CpG island hypermethylation. Treatment of non-expressing cancer cell lines in vitro with hypmomethylating drugs (HMAs) like Decitabine (DAC) and Azacytidine (AZA), both FDA approved for patients with myeloid cancers, can induce CG gene expression. We hypothesized that SGI-110 (Astex Pharmaceuticals, Inc.), a second generation hypomethylating agent with superior pharmacokinetics, would induce CG antigen genes in AML cells in vitro. Methods: HL60 and U937 cells were cultured in vitro using standard techniques and treated with phosphate buffered saline, 0.1, 1 or 5 μM SGI-110, 0.5μM DAC or 2μM AZA. Results are pooled from a minimum of three replicates. Viability by trypan blue exclusion and DNA, RNA and protein were obtained on day 5; DNA was bisulfite converted. Methylation (LINE-1, NY-ESO1, by pyrosequencing and MAGEA by methylation specific PCR), mRNA expression (by RT-PCR), and protein expression (by Western blot) were assessed. Results: Treatment with SGI-110 produced dose-dependent cytotoxicity. Viability at 0.1, 1 and 5μM SGI-110 doses was 47, 11, 8% in HL60 cells and 91, 41, 22% in U937 cells. Viability at the 1μM SGI-110 dose was comparable to 0.5μM DAC (11 and 38%, respectively) and less than 2μM AZA (51 and 30% respectively) in both cell lines. Baseline LINE-1 elements were 80% methylated in HL60 cells and 68% methylated in U937 cells. In both lines 1μM SGI-110 produced optimal LINE-1 demethylation, comparable to DAC. At baseline both NY-ESO1 and MAGEA3/6 promoters were densely hypermethylated in both HL60 and U937 and neither expressed mRNA; treatment with AZA, DAC and 1μM SGI-110 hypomethylated both genes and induced mRNA expression. No mRNA expression of CG genes was detected at the 0.1μM SGI-110 dose. NY-ESO1 and MAGEA protein expression were induced following 1 and 5μM doses of SGI-110 and DAC in both cell lines; in U937 cells AZA induced MAGEA but not NY-ESO1. Summary: SGI-110 induces global and gene specific hypomethylation as well as mRNA and protein expression of the CG genes NY-ESO1 and MAGEA3/6 in AML cells. This protein induction in vitro is at least as potent as that observed following AZA and DAC treatment. Induction of anti-MAGE specific T-cells has been reported in myeloid cancer patients receiving AZA + Vorinostat, suggesting that induced CG specific immunity may contribute to response to HMAs (Goodyear et al. Blood. 2010;116:1908). These data suggest that the combination of HMAs such as SGI-110 with CG vaccination may be of value in myeloid malignancy. Citation Format: Elizabeth A. Griffiths, Pragya Srivastava, Golda Collamat-Lai, Smitha R. James, Adam R. Karpf. SGI-110, a novel 5-aza-2’-deoxycytidine- containing dinucleotide, induces Cancer Germline (CG) antigen expression in Acute Myeloid Leukemia (AML) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 681. doi:10.1158/1538-7445.AM2013-681