Re-expression Of Tumor Suppressor Genes Research Articles

The DNA Methyltransferase Inhibitor 5-Aza-4'-thio-2'-Deoxycytidine Induces C>G Transversions and Acute Lymphoid Leukemia Development.

DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that decrease 5'-cytosine methylation. DNMTi are used clinically based on the hypothesis that cytosine demethylation will lead to re-expression of tumor suppressor genes. 5-Aza-4'-thio-2'-deoxycytidine (Aza-TdCyd or ATC) is a recently described thiol-substituted DNMTi that has been shown to have anti-tumor activity in solid tumor models. In this study, we investigated the therapeutic potential of ATC in a murine transplantation model of myelodysplastic syndrome. ATC treatment led to the transformation of transplanted wild-type bone marrow nucleated cells into lymphoid leukemia, and healthy mice treated with ATC also developed lymphoid leukemia. Whole-exome sequencing revealed 1,000 acquired mutations, almost all of which were C>G transversions in a specific 5'-NCG-3' context. These mutations involved dozens of genes involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Human cells treated in vitro with ATC showed 1,000 acquired C>G transversions in a similar context. Deletion of Dck, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C>G transversions. Taken together, these findings demonstrate a highly penetrant mutagenic and leukemogenic phenotype associated with ATC. Significance: Treatment with a DNA methyltransferase inhibitor generates a distinct mutation signature and triggers leukemic transformation, which has important implications for the research and clinical applications of these inhibitors.

Abstract 1484: 5-Aza-4'-thio-2'-deoxycytidine induces C to G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia

Abstract Myelodysplastic syndromes (MDS) are a diverse group of blood cancers characterized by peripheral blood cytopenias and dysplastic blood cell morphology. Up to 40% of patients with high risk MDS will transform to acute myeloid leukemia (AML), an aggressive blood cancer with significant morbidity and an overall 5-year survival of less than 35%. Both MDS and AML have been linked to hypermethylation of cytosine residues. Hypermethylation in the 5’ regulatory region can result in epigenetic downregulation of gene expression, and it has been speculated that hypermethylation of tumor suppressor genes can contribute to malignant transformation. DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that are used clinically to decrease 5’-cytosine methylation, with the aim of inducing the re-expression of tumor suppressor genes resulting in a halt of tumor progression. 5-Aza-4’-thio-2’-deoxycytidine (Aza-TdCyd or ATC) is a cytidine analog that incorporates an aza modification (nitrogen in place of carbon) of the cytosine base and a thioether modification of the deoxyribose sugar. ATC is a promising new DNMT1i that has demonstrated efficacy in preclinical studies of solid tumors. Based on these prior studies, we sought to characterize the potential therapeutic effect of ATC in MDS.Mice that express a NUP98::HOXD13 (NHD13) transgene develop an MDS that closely recapitulates all of the key features of human MDS, including pancytopenia, dysplastic morphology, and transformation to AML. We transplanted NHD13 and wild-type (WT) hematopoietic stem and progenitor cells into WT recipients, and used this model of MDS to investigate the therapeutic potential of ATC. We found that ATC treatment led to both T and B lineage lymphoid leukemia (ALL) originating from the NHD13 cells. Surprisingly, ATC treatment also led to T and B lineage ALL in the WT cells, and further experiments demonstrated that non-transplanted, healthy WT mice developed T and B cell ALL after treatment with ATC. In sum, all mice that received at least 8 cycles of ATC therapy at the 1.0 mg/kg dose developed leukemia. Whole exome sequencing of tumors revealed thousands of acquired mutations, almost all of which were C>G transversions in a previously unrecognized, specific 5’-NCG-3’ context. These mutations involved a number of genes well-known to be involved in human lymphoid leukemia, such as Notch1, Pten, Pax5, Trp53, and Nf1. Treatment of the human U937 and CEM cell lines in vitro showed thousands of acquired C>G transversions in a similar context. Taken together, these findings demonstrate that ATC can be a potent mutagen in human and mouse cells, both in vitro and in vivo. Citation Format: Ryan Matthew Bertoli, Yang Jo Chung, Michael Difilippantonio, Anthony Wokasch, Madison R. Marasco, Haley Klimaszewski, Susannah Garber, Yuelin Jack Zhu, Robert L. Walker, Dengchao Cao, James Doroshow, Paul Meltzer, Peter D. Aplan. 5-Aza-4'-thio-2'-deoxycytidine induces C to G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1484.

DNA methyltransferase inhibition promotes recruitment of myeloid-derived suppressor cells to the tumor microenvironment through induction of tumor cell-intrinsic interleukin-1

DNA methyltransferase (DNMT) inhibitors are used for treatment of certain hematological malignancies and exert anti-cancer activity through diverse mechanisms, including reexpression of tumor suppressor genes and anti-viral responses triggered by expression of endogenous retroviruses. Despite advances in the pharmacokinetic properties of DNMT inhibitors, the efficacy of these drugs in solid cancers remains low. Here, we show in cell lines and clinical and experimental tumors across multiple cancer types that DNMT inhibition induces the expression of interleukin-1 (IL-1), a cytokine with proinflammatory and protumorigenic properties. Specifically, this tumor-intrinsic IL-1 expression modulates the chemokine landscape of tumors and leads to the recruitment of monocytic myeloid-derived suppressor cells to the tumor microenvironment, processes that can be blocked by IL-1 antagonists. Molecular analysis demonstrates complex patterns of IL-1 and interferon activation and crosstalk in response to DNMT inhibition, which depend on the integrity of IRF- and NF-κB-mediated antiviral pathways and may determine the outcome of DNMT-inhibitor treatment. Together, our results show that DNMT inhibitors may negatively affect the microenvironment of a large subset of tumors and suggest that co-treatment with IL-1 antagonists may be a favorable combination for these patients.

Non-epigenetic induction of HEXIM1 by DNMT1 inhibitors and functional relevance

We have been studying the role of Hexamethylene bisacetamide (HMBA) Induced Protein 1 (HEXIM1) as a tumor suppressor whose expression is decreased in breast and prostate cancer. The anti-cancer actions of HEXIM1 in melanomas and AML have been reported by other groups. Previous studies have shown that 5-Aza-2′deoxycytidine (5-AzadC), a DNMT1 inhibitor, induces re-expression of tumor suppressor genes by removing/erasing methylation marks from their promoters. Our studies highlighted another mechanism wherein 5-AzadC induced DNA damage, which then resulted in enhanced occupancy of NF-ĸB, P-TEFb, and serine 2 phosphorylated RNA Polymerase II on the HEXIM1 gene. As a consequence, 5-AzadC induced HEXIM1 expression in prostate cancer cell lines and triple negative breast cancers. 5-AzadC-induced DNA damage enhanced P-TEFb occupancy via a mechanism that involved activation of ATR and ATM and induction of NF-ĸB recruitment to the HEXIM1 promoter. Downregulation of NF-ĸB attenuated 5-AzadC-induced HEXIM1 expression in prostate and breast cancer cells. The functional relevance of 5-AzadC-induced HEXIM1 expression is revealed by studies showing the HEXIM1 is required for the induction of apoptosis. Collectively, our findings support a non-epigenetic mechanism for 5-AzadC-induced re-expression of HEXIM1 protein, and may contribute to the clinical efficacy of 5-AzadC.

Epigenetic silencing of miR-342-3p in B cell lymphoma and its impact on autophagy

BackgroundmiR-342-3p, localized to 14q32, is a tumor suppressor miRNA implicated in carcinogenesis. Given the presence of a promotor-associated CpG island for its host gene, EVL, we hypothesized that intronic miR-342-3p is a tumor suppressor co-regulated with host gene by promoter DNA methylation in B cell lymphoma.ResultsBy bisulfite pyrosequencing-verified methylation-specific PCR (MSP), EVL/MIR342 methylation was detected in five (50%) lymphoma cell lines but not normal peripheral blood and tonsils. EVL/MIR342 methylation correlated with repression of both miR-342-3p and EVL in cell lines. In completely methylated SU-DHL-16 cells, 5-AzadC treatment resulted in promoter demethylation and re-expression of miR-342-3p and EVL. In 132 primary lymphoma samples, EVL/MIR342 was preferentially methylated in B cell lymphomas (N = 68; 68.7%) than T cell lymphoma (N = 8; 24.2%) by MSP (P < 0.0001). Moreover, EVL/MIR342 methylation was associated with lower miR-342-3p expression in 79 primary NHL (P = 0.0443). In SU-DHL-16 cells, the tumor suppressor function of miR-342-3p was demonstrated by the inhibition of cellular proliferation and increase of cell death upon over-expression of miR-342-3p. Mechanistically, overexpression of miR-342-3p resulted in a decrease of LC3-II, a biomarker of autophagy, which was pro-survival for SU-DHL-16. Pre-treatment with 3-methyladenine, an autophagy inhibitor, abrogated tumor suppression associated with miR-342-3p overexpression. By luciferase assay, MAP1LC3B, a precursor of LC3-II, was confirmed as a direct target of miR-342-3p. Finally, in SU-DHL-16 cells, overexpression of miR-342-3p downregulated the known target DNMT1, with promoter demethylation and re-expression of tumor suppressor E-cadherin.ConclusionsIntronic miR-342-3p is co-regulated with its host gene EVL by tumor-specific promoter DNA methylation in B cell lymphoma. The tumor suppressor function of miR-342-3p was mediated via inhibition of pro-survival autophagy by targeting MAP1LC3B and downregulation of DNMT1 with demethylation and re-expression of tumor suppressor genes.

DNA Methylation as a Therapeutic Target for Bladder Cancer.

Bladder cancer (BC) is the tenth most frequent cancer worldwide and is associated with high mortality when diagnosed in its most aggressive form, which is not reverted by the current treatment options. Thus, the development of new therapeutic strategies, either alternative or complementary to the current ones, is of major importance. The disruption of normal epigenetic mechanisms, namely, DNA methylation, is a known early event in cancer development. Consequently, DNA methyltransferase (DNMT) inhibitors constitute a promising therapeutic target for the treatment of BC. Although these inhibitors, mainly nucleoside analogues such as 5-azacytidine (5-aza) and decitabine (DAC), cause re-expression of tumor suppressor genes, inhibition of tumor cell growth, and increased apoptosis in BC experimental models and clinical trials, they also show important drawbacks that prevent their use as a valuable option for the treatment of BC. However, their combination with chemotherapy and/or immune-checkpoint inhibitors could aid in their implementation in the clinical practice. Here, we provide a comprehensive review of the studies exploring the effects of DNA methylation inhibition using DNMTs inhibitors in BC, from in vitro and in vivo studies to clinical trials.

Novel prodrugs of decitabine with greater metabolic stability and less toxicity

BackgroundDNA demethylation therapy is now used in practice for hematological tumors and is being developed for solid tumors. Nevertheless, it is difficult to achieve stable pharmacokinetics with the current DNA-demethylating agents, azacitidine (AZA) and decitabine (DAC), because of their rapid deamination by cytidine deaminase in vivo and spontaneous hydrolytic cleavage. Here, we aimed to develop metabolically stable prodrugs of AZA and DAC as novel DNA-demethylating agents.ResultsThirty-five 5′-O-trialkylsilylated AZAs/DACs were synthesized with potential resistance to deamination. Out of these, 11 compounds exhibited demethylating activity similar to that of DAC and guadecitabine, and a suitable aqueous solubility. Pharmacokinetic analysis in mice showed that OR-2003 displayed the highest serum concentration and the area under the curve in an intraperitoneal experiment, whereas OR-2100 exhibited high stability to cytidine deaminase. Treatment of cells with OR-2003 and OR-2100 depleted DNA methyltransferase 1 completely and induced both gene-specific and genome-wide demethylation. The treatment suppressed the growth of multiple types of cancer cells and induced re-expression of tumor suppressor genes. The anti-tumor effect and DNA demethylation effect of OR-2003 and OR-2100 were comparable to that of DAC with fewer adverse effects in vivo.ConclusionsWe developed two novel prodrugs of DAC that exhibited greater stability, comparable DNA demethylation activity, and less toxicity. These compounds are expected to overcome the difficulty in achieving stable pharmacokinetics in patients, leading to maximum DNA demethylation activity with minimum adverse effects.

Synergistic Efficacy of the Demethylation Agent Decitabine in Combination With the Protease Inhibitor Bortezomib for Treating Multiple Myeloma Through the Wnt/β-Catenin Pathway.

Multiple myeloma (MM) is a hematopoietic malignancy characterized by the clonal proliferation of antibody-secreting plasma cells. Bortezomib (BZM), the first FDA-approved proteasome inhibitor, has significant antimyeloma activity and prolongs the median survival of MM patients. However, MM remains incurable predominantly due to acquired drug resistance and disease relapse. β-Catenin, a key effector protein in the canonical Wnt signaling pathway, has been implicated in regulating myeloma cell sensitivity to BZM. Decitabine (DAC) is an epigenetic modulating agent that induces tumor suppressor gene reexpression based on its gene-specific DNA hypomethylation. DAC has been implicated in modulating Wnt/β-catenin signaling by promoting the demethylation of the Wnt/β-catenin antagonists sFRP and DKK. In this study, we report the effects of single reagent DAC therapy and DAC combined with BZM on β-catenin accumulation, myeloma cell survival, apoptosis, and treatment sensitivity. Our study proved that DAC demethylated and induced the reexpression of the Wnt antagonists sFRP3 and DKK1. DAC also reduced GSK3β (Ser9) phosphorylation and decreased β-catenin accumulation in the nucleus, which were induced by BZM. Thus, the transcription of cyclin D1, c-Myc, and LEF/TCF was reduced, which synergistically inhibited cell proliferation, enhanced BZM-induced apoptosis, and promoted BZM-induced cell cycle arrest in myeloma cells. In summary, these results indicated that DAC could synergistically enhance myeloma cell sensitivity to BZM at least partly by regulating Wnt/β-catenin signaling. Our results can be used to optimize therapeutic regimens for MM.

The Transcriptional Regulator Sin3A Contributes to the Oncogenic Potential of STAT3.

Epigenetic silencing of promoter and enhancer regions is a common phenomenon in malignant cells. The transcription factor STAT3 is aberrantly activated in several tumors, where its constitutive acetylation accounts for the transcriptional repression of a number of tumor suppressor genes (TSG) via molecular mechanisms that remain to be understood. Using nucleophosmin-anaplastic lymphoma kinase-positive (NPM-ALK+) anaplastic large-cell lymphoma (ALCL) as model system, we found in cells and patient-derived tumor xenografts that STAT3 is constitutively acetylated as a result of ALK activity. STAT3 acetylation relied on intact ALK-induced PI3K- and mTORC1-dependent signaling and was sensitive to resveratrol. Resveratrol lowered STAT3 acetylation, rescued TSG expression, and induced ALCL apoptotic cell death. STAT3 constitutively bound the Sin3A transcriptional repressor complex, and both STAT3 and Sin3A bound the promoter region of silenced TSG via a resveratrol-sensitive mechanism. Silencing SIN3A caused reexpression of TSG, induced ALCL apoptotic cell death in vitro, and hindered ALCL tumorigenic potential in vivo. A constitutive STAT3-Sin3A interaction was also found in breast adenocarcinoma cells and proved critical for TSG silencing and cell survival. Collectively, these results suggest that oncogene-driven STAT3 acetylation and its constitutive association with Sin3A represent novel and concomitant events contributing to STAT3 oncogenic potential. SIGNIFICANCE: This study delineates the transcriptional regulatory complex Sin3A as a mediator of STAT3 transcriptional repressor activity and identifies the STAT3/Sin3A axis as a druggable target to antagonize STAT3-addicted tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/12/3076/F1.large.jpg.See related commentary by Monteleone and Poli, p. 3031.

Trial in progress abstract phase I trial of 5-aza-4’-thio-2’-deoxycytidine (Aza-TdC) in patients with advanced solid tumors.

TPS3148 Background: The nucleoside analog 5-aza-4’-thio-2’-deoxycytidine (Aza-TdC) inhibits DNA methyltransferase 1 (DNMT1), a methyltransferase involved in methylation-mediated silencing of tumor suppressor genes. Attenuation of DNA methylation via DNMT1 inhibitors results in reactivation of silenced tumor suppressor genes and can lead to tumor growth arrest and apoptosis. The DNMT1 inhibitors decitabine and 5-azacytdine are currently FDA-approved for use in myelodysplastic syndromes and are also used in patients with acute myeloid leukemia. Relative to these compounds, Aza-TdC exhibits enhanced stability and incorporation into DNA and has shown improved preclinical antitumor activity in both leukemia and solid tumor xenograft models. This study seeks to evaluate the safety and maximum tolerated dose (MTD) of oral Aza-TdC in patients with advanced solid tumors. Secondary study objectives include assessing objective response by RECIST 1.1, pharmacokinetic (PK) analysis, and examining re-expression of tumor suppressor genes inhibited by methylation in circulating tumor cells (CTCs). Methods: Patients are treated with Aza-TdC on days 1-5 and 8-12 of each 21-day cycle. The study follows Simon accelerated titration design 3, with 100% dose increments and 1 patient per dose level. Accelerated titration will continue until 1 patient experiences a dose-limiting toxicity (DLT) or 2 patients experience drug-related grade 2 toxicity at any dose level, after which, a 3 + 3 dose escalation design will be used. Blood samples are collected for PK and CTC analyses. An MTD expansion cohort is planned, in which tumor biopsies will be collected for further pharmacodynamic assessments. Patients included in this study must be ≥18 years old and have histologically documented solid tumors that have progressed on standard therapy and for which there is no other standard therapy available. Dose level 3 has been completed without any DLTs; enrollment to dose level 4 began in February 2019. Funded by NCI Contract No. HHSN261200800001E. Clinical trial information: NCT03366116.

Time dependent of epigenetic effect of disulfiram on tumor suppressor gene of RASSF1A in Hela cancer cell line

Introduction: Cervical cancer is the third most common tumor among women. Surgery, radiotherapy, and chemotherapy are common treatments, however high stage tumors have frequently poor prognosis. Nowadays, the epigenetic reversion introduced as an efficient strategy of treatment of cervical cancer. In the process, inhibitors of DNA methyltransferase (DNMT) induce re-expression of tumor suppressor genes. Among these inhibitors, disulfiram (DSF) has been suggested as non-nucleoside analogous. In this research, we evaluated the epigenetic effect of DSF on demethylation of the tumor suppressor gene, RASSF1A, in Hela cell line. Materials and methods: Hela cells were cultured and treated with different doses from 2.5 to 37.5µM during 24, 48 and 72 hours. MTT assay was carried out to find half maximal inhibitory concentration (IC50). The methylation specific PCR (MSP) assay was applied to evaluate methylation pattern. Results: The IC50 of DSF was determined at the 2.5, 12.5, and 15µM after72 hours. The MSP results showed partial demethylation at mentioned concentrations after 72h but unmethylated band was not observed after 24h. Conclusion: Our findings indicated that, IC50 of DSF exerted a biphasic effect in Hela cell line and at least 72 hours treatment is needed for the epigenetic reversion of DSF on RASSF1Ain Hela cell line.

Re-Expression of Bone Marrow Proteoglycan-2 by 5-Azacytidine is associated with STAT3 Inactivation and Sensitivity Response to Imatinib in Resistant CML Cells

Background:Epigenetic silencing of tumor suppressor genes (TSG) is involved in development and progression of cancers. Re-expression of TSG is inversely proportionate with STAT3 signaling pathways. Demethylation of DNA by 5-Azacytidine (5-Aza) results in re-expression of silenced TSG. Forced expression of PRG2 by 5-Aza induced apoptosis in cancer cells. Imatinib is a tyrosine kinase inhibitor that potently inhibits BCR/ABL tyrosine kinase resulting in hematological remission in CML patients. However, majority of CML patients treated with imatinib would develop resistance under prolonged therapy.Methods:CML cells resistant to imatinib were treated with 5-Aza and cytotoxicity of imatinib and apoptosis were determined by MTS and annexin-V, respectively. Gene expression analysis was detected by real time-PCR, STATs activity examined using Western blot and methylation status of PRG2 was determined by pyrosequencing analysis.Result:Expression of PRG2 was significantly higher in K562-R+5-Aza cells compared to K562 and K562-R (p=0.001). Methylation of PRG2 gene was significantly decreased in K562-R+5-Aza cells compared to other cells (p=0.021). STAT3 was inactivated in K562-R+5-Aza cells which showed higher sensitivity to imatinib.Conclusion:PRG2 gene is a TSG and its overexpression might induce sensitivity to imatinib. However, further studies are required to evaluate the negative regulations of PRG2 on STAT3 signaling.

Epigenetic targeting of clear cell renal cell carcinoma (ccRCC) with ascorbic acid (AA) via upregulation of Ten-Eleven Translocation (TET) methylcytosine dioxygenase activity.

479 Background: We and others have previously shown that the ccRCC epigenome is characterized by widespread DNA hypermethylation (CCR 2014, Nature 2014). Various important tumor suppressor genes (TSGs), such as SMAD7 (inhibitor of oncogenic TGF-β signaling), are under-expressed due to aberrantly methylated promoters or enhancers. We hypothesized that the hypermethylation in ccRCC could be due to low activity of the TET enzymes, which convert methylcytosine (5-mc) to hydroxymethylcytosine (5-hmc). Loss of function of TET enzymes can occur with an inactivating mutation (TET-2 is mutated in about 6% of ccRCC (Science Signaling 2013) or hypoactivity of normal TET enzymes, through inhibition by metabolic intermediates. AA is an essential co-factor for TET enzymes (JACS 2015). We hypothesized that high dose AA treatment of ccRCC could potentially increase the functional activity of TET enzymes leading to demethylation of the RCC genome, and enhance expression of TSGs. Methods: In vitro TET activity was performed on ccRCC cell line 769P with increasing doses of AA (L-AA). Genome wide quantitative 5-mc and 5-hmc was evaluated using mass spectrometry. SMAD7 expression was determined using qRT-PCR. Proliferation assay (MTT) was performed with AA in combination with pazopanib. Cell cycle and apoptosis assays were performed. Results: AA, at doses achieved only by the intravenous route (1-10mM), increased TET activity in ccRCC cell line 769P. Genome wide 5-mc was significantly reduced and 5-hmc was increased, correlating with increase in TET activity. SMAD7 expression was increased with AA treatment. High dose AA led to proliferation inhibition with a cell cycle arrest in the G1 phase, and had a synergistic effect with pazopanib. Since AA leads to the generation of H2O2 in-vitro, catalase was used as control. This did not reverse the effect of AA on epigenetic changes; and proliferation inhibition was seen despite catalase control. Conclusions: High dose AA causes TET mediated DNA demethylation of the hypermethylated RCC genome, resulting in the re-expression of TSGs, and proliferation inhibition. Sequencing and xenograft studies are underway.

Combination epigenetic therapy in metastatic colorectal cancer (mCRC) with subcutaneous 5-azacitidine and entinostat: a phase 2 consortium/stand Up 2 cancer study

PurposeTherapy with demethylating agent 5-azacitidine and histone deacetylase inhibitor entinostat shows synergistic re-expression of tumor-suppressor genes and growth inhibition in colorectal (CRC) cell lines and in vivo studies.Experimental DesignWe conducted a phase II, multi-institutional study of the combination in metastatic CRC patients. Subcutaneous azacitidine was administered at 40 mg/m2 days 1-5 and 8-10 and entinostat was given 7 mg orally on days 3 and 10. An interim analysis indicated toxicity crossed the pre-specified safety boundary but was secondary to disease. A 2nd cohort with added eligibility restrictions was accrued: prior therapies were limited to no more than 2 or 3 (KRAS-mutated and KRAS-wildtype cancers, respectively) and <30% of liver involvement. The primary endpoint was RECIST response. Serial biopsies were performed at baseline and after 2 cycles of therapy.ResultsForty-seven patients were enrolled (24:Cohort 1, 23:Cohort 2). Patients were heavily pre-treated (median prior therapies 4: Cohort 1 and 2.5: cohort 2). No responses were observed. Median progression-free survival was 1.9 months; overall survival was 5.6 and 8.3 months in Cohorts 1 and 2, respectively. Toxicity was tolerable and as expected. Unsupervised cluster analysis of serial tumor biopsies suggested greater DNA demethylation in patients with PFS above the median.ConclusionIn this first trial of CRC patients with combination epigenetic therapy, we show tolerable therapy without significant clinical activity as determined by RECIST responses. Reversal of hypermethylation was seen in a subset of patients and correlated with improved PFS.

SiRNA and epigenetic aberrations in ovarian cancer.

Ovarian cancer has the most noteworthy lethal rate around gynecologic malignancies, and it is also considered as the fourth most frequent cancer in the woman in world. Two most critical barriers to treatment of ovarian malignancy are absence of early diagnostic markers and advancement of drug resistance after therapy, especially in advanced stages. Various epigenetic changes have been recognized in ovarian cancer. Recent progresses in our understanding of molecular pathogenesis of ovarian malignancy have dramatically provided potential new targets for molecularly targeted therapies. In very recent years, small interfering RNA (siRNA)-mediated gene silencing has been emerging as a novel treatment modality in preclinical studies in the light of its strong gene-specific silencing. Gene suppression mediated by RNA interference (RNAi) significantly suppressed gene expression at the messenger RNA (mRNA) and protein levels. SiRNAs have therapeutic potential for ovarian cancer through various mechanisms. In this review, we not only provide an overview of siRNA designing for epigenetic silencing of genes aberrantly expressed in ovarian cancer but also we will highlight that the epigenetically silenced genes offer new targets for therapeutic approaches based on re-expression of tumor suppressor genes via demethylating and deacetylating drugs.

Low concentrations of 5-aza-2'-deoxycytidine induce breast cancer stem cell differentiation by triggering tumor suppressor gene expression.

BackgroundBreast cancer stem cells (BCSCs) are considered the cause of tumor growth, multidrug resistance, metastasis, and recurrence. Therefore, differentiation therapy to reduce self-renewal of BCSCs is a promising approach. We have examined the effects of 5-aza-2′-deoxycytidine (DAC) on BCSC differentiation.Materials and methodsBCSCs were treated with a range of DAC concentrations from 0.625 to 100 µM. The differentiation status of DAC-treated BCSCs was graded by changes in cell proliferation, CD44+CD24− phenotype, expression of tumor suppressor genes, including BRCA1, BRCA2, p15, p16, p53, and PTEN, and antitumor drug resistance.ResultsDAC treatment caused significant BCSC differentiation. BCSCs showed a 15%–23% reduction in proliferation capacity, 3.0%–21.3% decrease in the expression of BCSC marker CD44+/CD24−, activation of p53 expression, and increased p15, p16, BRCA1, and BRCA2 expression. Concentrations of DAC ranging from 0.625 to 40 µM efficiently induce cell cycle arrest in S-phase. ABCG2, highly expressed in BCSCs, also decreased with DAC exposure. Of particular note, drug-sensitivity of BCSCs to doxorubicin, verapamil, and tamoxifen also increased 1.5-, 2.0-, and 3.7-fold, respectively, after pretreatment with DAC.ConclusionDAC reduced breast cancer cell survival and induced differentiation through reexpression of tumor suppressor genes. These results indicate the potential of DAC in targeting specific chemotherapy-resistant cells within a tumor.

Abstract LB-213: Combination of epigenetic modifiers achieves complete remission in xenograft models of pediatric acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is the second most-common form of leukemia in children. Although, the 5-year survival rate for children with AML is estimated at 64%, nearly half of the patients have refractory disease. Novel targeted therapeutics with minimal side effects are required to improve the outcome in kids with refractory disease. One class of molecular targeted therapeutics that shows specific anti-leukemic effects while sparing normal hematopoeitic progenitor cells includes inhibitors of epigenetic modifications such as DNA methylation and histone deacetylation. Although DNA methyltransferase (DNMT) inhibitors are currently on clinical trials for AML, their efficacy as single agents is limited and the mechanism of action is unknown. This limited efficacy could be due to co-regulatory effects of DNA and histone modifications on gene expression. Therefore, DNA hypomethylating agents when used in combination with other epigenetically active agents such as histone deacetylase (HDAC) inhibitors should achieve greater efficacy by optimal re-expression of tumor suppressor genes silenced in AML. Our data shows a synergistic induction of cytotoxicity upon treatment with azacytidine (DNMT inhibitor) and panobinostat (HDAC inhibitor) with combination indices ranging from 0.6 to 0.8 in a variety of pediatric AML cell lines bearing distinct chromosomal abnormalities. Furthermore, immune-deficient (NSG-B2m) mice transplanted with MV4;11 cells via the tail vein were treated i.p. with 20 doses of maximally tolerated dose of 2.5 mg/Kg azacytidine and/or 2.5 mg/Kg panobinostat over a period of 33 days. The mice treated with azacytidine or panobinostat increased median survival by 26 and 6 days respectively. However, mice treated with both drugs showed a drastic reduction in leukemic burden leading to complete remission till the end of their life (around 2 years). Reduced leukemic burden and prolonged survival was also observed in AML-193 xenografted mice treated with the drug combination. In an effort to understand the mechanism of synergism between azacytidine and panobinostat, an Infinium Human Methylation 450K Bead Chip array was performed to identify methylation status of 400,000 CpG islands spanning promoter regions and island shores. Azacytidine treatment reduced the number of hypermethylated CpG islands by 30%, while panobinostat had no effect. However, treatment with drug combination did not change the number of hypermethylated CpG islands indicating that the observed synergy both in vitro and in vivo might result from specific activation of a subset of gene targets. Identification and validation of targets that mediate these synergistic effects is in progress to improve the selection of patients most likely to benefit from combination therapy. Citation Format: Anilkumar Gopalakrishnapillai, E Anders Kolb, Sonali P. Barwe. Combination of epigenetic modifiers achieves complete remission in xenograft models of pediatric acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-213. doi:10.1158/1538-7445.AM2015-LB-213

Disruption of STAT3-DNMT1 interaction by SH-I-14 induces re-expression of tumor suppressor genes and inhibits growth of triple-negative breast tumor.

Epigenetic regulation of gene expression is an emerging target to treat several human diseases including cancers. In cancers, expressions of many tumor suppressor genes are suppressed by hyper-methylation in their regulatory regions. Herein, we describe a novel carbazole SH-I-14 that decreased the level of the acetyl-STAT3 at the K685 residue. Mutation analysis revealed that SH-I-14 disrupted STAT3-DNMT1 interaction by removing acetyl group from K685 of STAT3. Finally, the inhibition of STAT3-DNMT1 interaction by SH-I-14 resulted in re-expression of tumor suppressor genes such as VHL and PDLIM4 through de-methylation of their promoter regions. In addition, SH-I-14 showed anti-proliferative effect in triple-negative breast cancer (TNBC) cell lines in vitro and anti-tumor effect in a mouse xenograft model of MDA-MB-231 tumor. Taken together, our results suggest that targeting acetyl-STAT3 (K685) provides potential therapeutic opportunity to treat a subset of human cancers.

Molecular Mechanism of Action of 5‐Azacytidine and its Resistance in MDA‐MB 435 Cells

DNA methyltransferase inhibitors such as 5‐Azacytidine (5‐Aza) have shown promise in cancer treatment because of their ability to allow re‐expression of tumor suppressor genes in cancer cells. Preliminary studies showed that high doses of 5‐Aza are clearly cytotoxic to MDA‐MB 435 tumor cells. However, we have begun treating the MDA‐MB 435 cells with a low dose so that the majority of the cells survive. A resistant cell line has been developed through continuous growth in the presence of 5‐Aza. Tests were done to determine if the resistance inhibited growth in a methylcellulose assay. The number of colonies decreased in the 5‐Aza resistant cell line compared to the control. Control 435 cells were found to have an average colony count of 969±332 but when 5‐Aza was added the colony count dropped to 464±31. In order to look at the mechanism of 5‐Aza and its resistance, quantitative reverse transcriptase PCR was done to measure the expression of various genes. Between the control cells and the control cells plus 5‐Aza there was a five‐fold decreased expression of Mammaglobin B, a member of the secretoglobin family associated with chemoresistance, while the other genes tested did not have any change (TP53, RB1, BCL‐2, Myc). When the same tests were done on the resistant cell line the colony counts showed little change, 287±129 and 357±46, respectively, nor was there a difference between the resistant and resistant plus 5‐Aza in terms of Mammaglobin B expression. However, the resistant and the resistant plus 5‐aza both showed a six‐fold decrease in Mammaglobin B expression compared to the control cells. Future experiments will investigate a phenotypic role for this gene in the resistance.

Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2'-deoxycytidine in human leukemic KG1 cells.

5-azacytidine and 5-aza-2'-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2'-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2'-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2'-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2'-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.