Inhibitor Decitabine Research Articles

CRISPR-Mediated Reactivation of DKK3 Expression Attenuates TGF-β Signaling in Prostate Cancer.

The DKK3 gene encodes a secreted protein, Dkk-3, that inhibits prostate tumor growth and metastasis. DKK3 is downregulated by promoter methylation in many types of cancer, including prostate cancer. Gene silencing studies have shown that Dkk-3 maintains normal prostate epithelial cell homeostasis by limiting TGF-β/Smad signaling. While ectopic expression of Dkk-3 leads to prostate cancer cell apoptosis, it is unclear if Dkk-3 has a physiological role in cancer cells. Here, we show that treatment of PC3 prostate cancer cells with the DNA methyltransferase (DNMT) inhibitor decitabine demethylates the DKK3 promoter, induces DKK3 expression, and inhibits TGF-β/Smad-dependent transcriptional activity. Direct induction of DKK3 expression using CRISPR-dCas9-VPR also inhibited TGF-β/Smad-dependent transcription and attenuated PC3 cell migration and proliferation. These effects were not observed in C4-2B cells, which do not respond to TGF-β. TGF-β signals can regulate gene expression directly via SMAD proteins and indirectly by increasing DNMT expression, leading to promoter methylation. Analysis of genes downregulated by promoter methylation and predicted to be regulated by TGF-β found that DKK3 induction increased expression of PTGS2, which encodes cyclooxygenase-2. Together, these observations provide support for using CRISPR-mediated induction of DKK3 as a potential therapeutic approach for prostate cancer and highlight complexities in Dkk-3 regulation of TGF-β signaling.

Methylation-associated silencing of BASP1 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

The AML1-ETO fusion protein (A/E), which results from the t(8;21) translocation, is considered to be a leukemia-initiating event. Identifying the mechanisms underlying the oncogenic activity of A/E remains a major challenge. In this study, we identified a specific down-regulation of brain acid-soluble protein 1 (BASP1) in t(8;21) acute myeloid leukemia (AML). A/E recognized AML1-binding sites and recruited DNA methyltransferase 3a (DNMT3a) to the BASP1 promoter sequence, which triggered DNA methylation-mediated silencing of BASP1. Ectopic expression of BASP1 inhibited proliferation and the colony-forming ability of A/E-positive AML cell lines and led to apoptosis and cell cycle arrest. The DNMT inhibitor decitabine up-regulated the expression of BASP1 in A/E-positive AML cell lines. In conclusion, our data suggest that BASP1 silencing via promoter methylation may be involved in A/E-mediated leukemogenesis and that BASP1 targeting may be an actionable therapeutic strategy in t(8;21) AML.

Decitabine augments cytotoxicity of cisplatin and doxorubicin to bladder cancer cells by activating hippo pathway through RASSF1A.

Genetic abnormalities and epigenetic alterations both play vital role in initiation as well as progression of cancer. Whereas genetic mutations cannot be reversed, epigenetic alterations such as DNA methylation can be reversed by the application of DNA methyltransferase inhibitor decitabine. Epigenetic silencing of RASSF1A and involvement of hippo pathway both have been shown to involve in chemo-resistance. Purpose of this study was to observe the effect of combination treatment of decitabine with cisplatin or doxorubicin on bladder cancer cells involving hippo pathway through RASSF1A. Bladder cancer cells (HT1376 & T24) were treated with decitabine and its effect on RASSF1A expression, hippo pathway molecules (MST & YAP), and its downstream targets (CTGF, CYR61 & CTGF) was observed. Effect of decitabine pretreatment on sensitivity of bladder cancer cells towards chemotherapeutic drugs was also studied. Decitabine treatment leads to restoration of RASSF1A, activation of hippo pathway followed by decreased expression of its oncogenic downstream targets (CTGF & CYR61). Further pretreatment of decitabine enhanced cytotoxicity of cisplatin and doxorubicin to bladder cancer cells.

Abstract A30: Preclinical study of a combination of mocetinostat (HDAC inhibitor) and 5-AZA-dC (decitabine) in chondrosarcoma

Abstract Chondrosarcomas are the second most frequently occurring type of bone malignancy, and account for approximately 25% of all bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. They are largely considered to be resistant to conventional chemotherapy and radiotherapy. Previous reports have shown that the interaction of DNA methylation and histone modification regulates gene expression, and targeting these mechanisms could be a potentially novel approach to treat cancer. In the present study, we hypothesized that concurrent inhibition of histone acetylation and DNA methylation could result in decreased viability of chondrosarcoma cells. To test this, we used a panel of chondrosarcoma cell lines including IDH wild type (CH2879), IDH1 mutant (JJ012), and IDH2 mutant (SW1353) cell line. Results from our in vitro proliferation assay showed that combination of sub-IC50 concentrations of the DNA methyltransferase (DNMT) inhibitor decitabine (5-AZA-dC) and a spectrum-selective class I and IV histone deacetylase (HDAC) inhibitor mocetinostat (MGCD0103) resulted in decreased cell viability, which appears to be at least additive, in all the three chondrosarcoma cell lines when compared to either of the two drugs alone. Western blot analysis showed induction of cleaved Poly-ADP Ribose Polymerase (PARP) as well as cleaved caspase 3, known markers of apoptosis. Consistent with augmented DNA damage, combination of decitabine and MG103 markedly increased the levels of phospho-H2AX, a DNA damage marker, and proapoptotic BH3-only proteins such as Bim. Combination treatment also resulted in increased induction of histone acetylation (AcH3 and AcH4) and increased expression of E-cadherin, an important tumor suppressor gene. Cell cycle analysis showed induction of sub-G1 population phase in combination treatment. Previous reports have shown that CpG methylation is a critical event in transcriptional repression of hypermethylated genes in cancer. To this end, we show that combination treatment with mocetinostat and 5-AZA-dC results in downregulation of (H3K9) methylation. Taken together, our data strongly suggest that combination treatment with mocetinostat and decitabine is a novel treatment approach and merits evaluation in the treatment of chondrosarcoma. Citation Format: Tahir N. Sheikh, Parag Patwardhan, Gary K. Schwartz. Preclinical study of a combination of mocetinostat (HDAC inhibitor) and 5-AZA-dC (decitabine) in chondrosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A30.

MAGE-A11 is activated through TFCP2/ZEB1 binding sites de-methylation as well as histone modification and facilitates ESCC tumor growth.

Recently, we have reported that the product of Melanoma Antigens Genes (MAGE) family member MAGE-A11 is an independent poor prognostic marker for esophageal squamous cell carcinoma (ESCC). However, the reason how MAGE-A11 is activated in ESCC progression still remains unclear. In the current study, we demonstrated that DNA methylation and the subsequent histone posttranslational modifications play crucial roles in the regulation of MAGE-A11 in ESCC progression. We found that the methylation rate of TFCP2/ZEB1 binding site on MAGE-A11 promoter in ESCC tissues and cells is higher than the normal esophageal epithelial tissues and cells. Transcription factors TFCP2 and ZEB1 directly bind MAGE-A11 promoter and regulate the endogenous MAGE-A11 expression in a methylation-dependent manner in ESCC cells. Following MAGE-A11 promoter methylation, the methyl-CpG-binding protein MeCP2 was found to bind the methylated MAGE-A11 promoter to mediate histone deactylation by recruiting HDAC1 and HDAC2. Simultaneously, histone inactivation marks including H3K27me3 as well as H3K9me3 were increased, whereas histone activation mark H3K4me3 was decreased. HDAC inhibitor Trichostatin A (TSA) increased DNA methylase inhibitor Decitabine (DAC)-induced MAGE-A11 expression. siRNA-mediated knockdown of histone methltransferase EZH2 or DZNep (a EZH2 inhibitor) treatment increased DAC-induced MAGE-A11 expression. Our results indicate that MAGE-A11 is activated through DNA demethylation, histone acetylation and histone methylation in ESCC, and its activation promotes ESCC tumor growth.

Abstract IA15: Multiple targetable pathways for epigenetic therapy

Abstract Epigenetic reprogramming erases the malignant potential of some transformed cells and the aim of epigenetic therapy is to achieve some degree of reprogramming in-vivo by targeting epigenetic writers/readers. The resultant gene expression response includes reactivation of tumor-suppressor genes and differentiation pathways as well as activation of an immune response program, all of which contribute to eventual clinical activity. The DNA methyltransferase inhibitors azacitidine, decitabine and guadecitabine induce clinically meaningful remissions or improvements in 30-60% of patients with myeloid leukemias and prolong survival compared to standard approaches including chemotherapy. This therapy is accompanied by (i) global and gene specific demethylation, (ii) reactivation of silenced gene expression, (iii) delayed responses that correlate with early epigenetic reactivation and result in clonal elimination and (iv) eventual relapses and resistance the mechanisms of which appear to involve genetic evolution in some cases. Multiple other classes of epigenetic modifiers have now been targeted therapeutically including histone acetylases and deacetylases, histone methylases and demethylases, and readers of the epigenetic marks such as the BET family of proteins. Clinical trials of these drugs are ongoing and have already shown impressive clinical activity in selected cases. Using a parallel approach, we have shown that gene expression directed unbiased phenotypic screens can also uncover new and repurposed drugs for epigenetic therapy (e.g. cardiac glycosides, arsenic trioxide, natural products), and unbiased genetic screens have suggested novel targets as well (SWI/SNF proteins, CHD4 etc.). An even greater promise of epigenetic therapy lies in sensitization to other treatment modalities such as chemotherapy and immunotherapy. Ultimately, epigenetic therapy will be integrated into comprehensive treatment plans aimed long term disease control in many human malignancies. Citation Format: Jean-Pierre Issa. Multiple targetable pathways for epigenetic therapy [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA15.

Untargeted DNA-Demethylation Therapy Neither Prevents Nor Attenuates Ischemia-Reperfusion-Induced Renal Fibrosis

Background: Current treatment options for chronic kidney disease (CKD) are limited and their focus is on slowing its progression by addressing comorbidities. Fibrosis, the common histopathological process in CKD, is a major therapeutic research target. In CKD, fibroblasts are terminally activated due to alterations in their DNA-methylation pattern, particularly hypermethylation. Preventing the copying of pathological DNA-methylation patterns in proliferating fibroblasts could be a new effective therapeutic strategy for treating CKD. Methods: To evaluate the therapeutic effect of short-term treatment with the DNA-methyltransferase (DNMT)-inhibitor decitabine on fibrosis (either developing or already established), male C57Bl/6 mice underwent warm unilateral ischemia-reperfusion injury. Respectively 3 days, 3 and 6 weeks after surgery, decitabine treatment (0.25 mg/kg) was initiated for 10 days after which animals were followed up to 12 weeks after ischemia. The efficacy of therapy on fibrosis was evaluated by collagen I and tgfβ gene expression and histological quantification of collagen I staining. In addition, the effect of decitabine treatment on tubular injury (Kim-1, Ngal), inflammation (TNFa, IL6), DNA-methyltransferases (Dnmt1, 3a, and 3b), and global methylation status was determined. Results: Following ischemia there was a significant increase in fibrotic, injury, and inflammatory markers as well as an increase of the various dnmts. Although decitabine treatment transiently increased renal injury and had a moderately decreasing effect on dnmt expression and on global DNA-methylation upon immediate treatment, none of the treatment regimens succeeded in preventing, attenuating, or diminishing fibrosis in the long run. Conclusion: Administration of untargeted nucleoside analogues seems unsuitable as a first-line treatment option in developing or established CKD.

Abstract 5066: Combination treatment with SAHA and 5-Azacytidine (Decitabine) induces apoptosis and suppresses tumor growth in preclinical models of chondrosarcoma

Abstract Chondrosarcomas are the second most frequently occurring type of bone malignancy, and account for approximately 25% of all bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. They are largely considered to be resistant to conventional chemotherapy and radiotherapy. Several studies have reported that targeting epigenetic mechanisms including DNA methylation or histone acetylation are novel approaches for the treatment of some human cancers. Previous reports have shown that the interaction of DNA methylation and histone modification regulates gene expression. In the present study, we hypothesized that concurrent inhibition of histone acetylation and DNA methylation could result in decreased viability of chondrosarcoma cells both in vitro and in vivo. To test this, we used a panel of chondrosarcoma cell lines including IDH wild type (CH2879), IDH1 mutant (JJ012) and IDH2 mutant (CS1) cell line. Results from our in vitro proliferation assay showed that combination of sub-IC50 concentrations of the DNA methyltransferase (DNMT) inhibitor Decitabine (5-AZA-dC) and histone deacetylase (HDAC) inhibitor (SAHA) resulted in decreased cell viability of all the three chondrosarcoma cell lines tested when compared to either drug alone. Western blot analysis showed induction of cleaved Poly-ADP Ribose Polymerase (PARP), a known marker of apoptosis. Consistent with augmented DNA damage, combination of Decitabine and SAHA markedly increased the levels of phospho-H2AX, a DNA damage marker, and pro-apoptotic BH3-only proteins such as Bim. Combination treatment also resulted in increased induction of histone acetylation (AcH3) and increased expression of E-Cadherin. Previous reports have shown that E-cadherin is critical for apoptosis and depletion and inhibition of E-Cadherin impairs apoptosis induction via DR4 and DR5 death receptors. To this end, we showed that induction of cleaved PARP by Apo2L/TRAIL stimulation decreased over time when E-Cadherin was knocked down by siRNA. Xenograft studies using IDH2 mutant (CS1) tumor model showed a significant suppression of tumor volume when animals were treated in combination with SAHA and Decitabine compared to single agent treatments. Taken together, our data strongly suggests that combination treatment with SAHA and Decitabine is a novel treatment approach and merits evaluation in the treatment of chondrosarcoma. Citation Format: Tahir Sheikh, Parag Patwardhan, Gary K. Schwartz. Combination treatment with SAHA and 5-Azacytidine (Decitabine) induces apoptosis and suppresses tumor growth in preclinical models of chondrosarcoma [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 5066. doi:10.1158/1538-7445.AM2017-5066

Abstract 1422: Enhancing the therapeutic effects of PARP inhibitors in combination DNA methyl transferase inhibitors, using low doses of ionizing radiation in non small cell lung cancers

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in the US. Treatment most commonly relies on ionizing radiation (IR) and platinum-based DNA damaging agents, but long-term survival is poor and patients tend to suffer chronic side-effects due to the high radiation dose to the surrounding normal tissues. Therefore, new treatments are needed that can be used in combination lower radiation doses. We have recently reported that low, non-cytotoxic doses Poly ADP ribose polymerase inhibitors (PARPi) Talazoparib in combination with DNA methyltransferase inhibitors (DNMTi) Decitabine (DAC) or azacytidine (AZA) significantly increase cytotoxicity in acute myeloid leukemia and breast cancer models in vitro and anti-tumor effects in vivo. Simultaneous administration of both inhibitors result in increased PARP binding in DNA, leading to higher levels of DNA double strand breaks (DSBs), yielding increased cytotoxicity, compared with each agent treatment alone. We first studied the efficacy of Talazoparib and AZA combination therapy in multiple NSCLC cell lines (A549, H358 and H838) in vitro through colony forming assays. Results showed, compared to single agent treatments, combination drug treatment significantly decreased colony formation. Cell viability was also significantly decreased with the drug combination in MTS assays (P<0.05). These results also showed synergistic activity between the two drugs, with a combination index of less than 1 for all tested NSCLC cell lines. Importantly, the drug combination increased PARP trapping in chromatin and DSB formation, as measured by immunofluorescent staining for γH2AX. We next determined whether AZA/Talazoparib in combination with a single dose of radiation (2Gy) had an increased anti-cancer effect compared to each modality alone in in vivo mouse xenografts of NSCLC A549. While the combination of PARPi+DNMTi with radiation treatment decreased tumor growth, compared to PARPi/DNMTi alone or RT alone, no significant enhancement with radiation was observed. We next determined whether low doses of fractionated IR (2Gy 3 fractions) would improve the efficacy of DNTMi and PARPi combination treatment. In vitro studies with colony forming assays of NSCLC cell line A549 show that IR doses in combination with PARPi and DNMTi decrease clonogenicity, compared with non-IR controls. Furthermore, A549 xenografts were treated with the drug combination and then irradiated 1 week later with 2Gy daily for 3 consecutive days. Mice treated with the drug combination followed by IR had significant decreases in tumor volume and survival (P<0.05). This suggests that low doses of PARPi and DNMTi therapy in combination with low dose IR can potentially target NSCLC tumors. This represents a novel treatment approach for NSCLC patients that may reduce chronic side-effects of high dose IR. Citation Format: Christopher Biondi, Daniel Fontaine, Lora Stojanovic, Pratik Nagaria, Rena Lapidus, Eun Yong Choi, Javed Mahmood, Stephen Baylin, Feyruz V. Rassool. Enhancing the therapeutic effects of PARP inhibitors in combination DNA methyl transferase inhibitors, using low doses of ionizing radiation in non small cell lung cancers [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 1422. doi:10.1158/1538-7445.AM2017-1422

Synergistic inhibitory effects of deferasirox in combination with decitabine on leukemia cell lines SKM-1, THP-1, and K-562

A multi-center study from the French Myelodysplastic Syndrome (MDS) Group confirmed that iron chelation therapy is an independent prognostic factor that can increase the survival rate of patients who are suffering from transfusion-dependent low-risk MDS. In this study, we aimed to explore this clinical phenomena in vitro, by exploring the synergistic effect of the iron chelator Deferasirox (DFX) and the DNA methyl transferase inhibitor Decitabine (DAC) in the leukemia cell lines SKM-1, THP-1, and K-562. Treatment with both DFX or DAC promoted apoptosis, induced cell cycle arrest, and inhibited proliferation in all three of these cell lines. The combination of DFX and DAC was much greater than the effect of using either drug alone. DFX showed a synergistic effect with DAC on cell apoptosis in all three cell lines and on cell cycle arrest at the G0/G1 phase in K-562 cells. DFX decreased the ROS levels to varying degrees. In contrast, DAC increased ROS levels and an increase in ROS was also noted when the two drugs were used in combination. Treatment of cells with DAC induced re-expression of ABAT, APAF-1, FADD, HJV, and SMPD3, presumably through demethylation. However the combination of DAC and DFX just had strong synergistic effect on the re-expression of HJV.

The combination of dimethoxycurcumin with DNA methylation inhibitor enhances gene re-expression of promoter-methylated genes and antagonizes their cytotoxic effect

ABSTRACTCurcumin and its analogs exhibited antileukemic activity either as single agent or in combination therapy. Dimethoxycurcumin (DMC) is a more metabolically stable curcumin analog that was shown to induce the expression of promoter-methylated genes without reversing DNA methylation. Accordingly, co-treatment with DMC and DNA methyltransferase (DNMT) inhibitors could hypothetically enhance the re-expression of promoter-methylated tumor suppressor genes. In this study, we investigated the cytotoxic effects and epigenetic changes associated with the combination of DMC and the DNMT inhibitor decitabine (DAC) in primary leukemia samples and cell lines. The combination demonstrated antagonistic cytotoxic effects and was minimally cytotoxic to primary leukemia cells. The combination did not affect the metabolic stability of DMC. Although the combination enhanced the downregulation of nuclear DNMT proteins, the hypomethylating activity of the combination was not increased significantly compared to DAC alone. On the other hand, the combination significantly increased H3K27 acetylation (H3K27Ac) compared to the single agents near the promoter region of promoter-methylated genes. Furthermore, sequential chromatin immunoprecipitation (ChIP) and DNA pyrosequencing of the chromatin-enriched H3K27Ac did not show any significant decrease in DNA methylation compared to other regions. Consequently, the enhanced induction of promoter-methylated genes by the combination compared to DAC alone is mediated by a mechanism that involves increased histone acetylation and not through potentiation of the DNA hypomethylating activity of DAC. Collectively, our results provide the mechanistic basis for further characterization of this combination in leukemia animal models and early phase clinical trials.

An Epigenomic Approach to Improving Response to Neoadjuvant Cisplatin Chemotherapy in Bladder Cancer.

Bladder cancer is among the five most common cancers diagnosed in the Western world and causes significant mortality and morbidity rates in affected patients. Therapeutic options to treat the disease in advanced muscle-invasive bladder cancer (MIBC) include cystectomy and chemotherapy. Neoadjuvant cisplatin-based combination chemotherapy is effective in MIBC; however, it has not been widely adopted by the community. One reason is that many patients do not respond to neoadjuvant chemotherapy, and no biomarker currently exists to identify these patients. It is also not clear whether a strategy to sensitize chemoresistant patients may exist. We sought to identify cisplatin-resistance patterns in preclinical models of bladder cancer, and test whether treatment with the epigenetic modifier decitabine is able to sensitize cisplatin-resistant bladder cancer cell lines. Using a screening approach in cisplatin-resistant bladder cancer cell lines, we identified dysregulated genes by RNA sequencing (RNAseq) and DNA methylation assays. DNA methylation analysis of tumors from 18 patients receiving cisplatin-based chemotherapy was used to confirm in vitro results. Cisplatin-resistant bladder cancer cells were treated with decitabine to investigate epigenetic sensitization of resistant cell lines. Our results show that HOXA9 promoter methylation status is associated with response to cisplatin-based chemotherapy in bladder cancer cell lines and in metastatic bladder cancer. Bladder cancer cells resistant to cisplatin chemotherapy can be sensitized to cisplatin by the DNA methylation inhibitor decitabine. Our data suggest that HOXA9 promoter methylation could serve as potential predictive biomarker and decitabine might sensitize resistant tumors in patients receiving cisplatin-based chemotherapy.

Erratum: Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity.

Scientific Reports 6: Article number: 31022; published online: 12 August 2016; updated: 21 September 2016 This Article contains typographical errors. In the Results section under subheading ‘DNA demethylating agents protect neurons from mutant Htt-induced cytotoxicity.’ “Following validation assays of possible screen “hits” using the MTS assay, we identified the cytosine nucleoside-analog DNA methyltransferase (DNMT) inhibitor decitabine, as the most effective drug in our mutant Htt neuroprotection screen (Fig.

Fulvestrant inhibits growth of triple negative breast cancer and synergizes with tamoxifen in ERα positive breast cancer by up-regulation of ERβ.

The estrogen receptor-alpha (ERα) is used as a predictive marker for anti-estrogen therapy in breast cancer patients. In addition to aromatase inhibitors, ERα can be targeted at the receptor level using the receptor modulator tamoxifen or by the pure anti-estrogen fulvestrant. The role of the second ER, ER-beta (ERβ), as a therapeutic target or prognostic marker in breast cancer is still elusive. Hitherto, it is not known if ERα+/ERβ+ breast cancers would benefit from a treatment strategy combining tamoxifen and fulvestrant or if fulvestrant exert any therapeutic effects in ERα-/ERβ+ breast cancer. Here, we report that fulvestrant up-regulated ERβ in ERα+/ERβ+ breast cancer and in triple negative ERβ+ breast cancers (ERα-/ERβ+). In ERα+/ERβ+ breast cancer, a combination therapy of tamoxifen and fulvestrant significantly reduced tumor growth compared to either treatment alone both in vivo and in vitro. In ERα-/ERβ+ breast cancer fulvestrant had potent effects on cancer growth, in vivo as well as in vitro, and this effect was dependent on intrinsically expressed levels of ERβ. The role of ERβ was further confirmed in cells where ERβ was knocked-in or knocked-down. Inhibition of DNA methyltransferase (DNMT) increased the levels of ERβ and fulvestrant exerted similar potency on DNMT activity as the DNMT inhibitor decitabine. We conclude that fulvestrant may have therapeutic potential in additional groups of breast cancer patients; i) in ERα+/ERβ+ breast cancer where fulvestrant synergizes with tamoxifen and ii) in triple negative/ERβ+ breast cancer patients, a subgroup of breast cancer patients with poor prognosis.

Abstract 3200: New epigenetic mechanism of disulfiram targeting chromatin remodeling in neuroblastoma

Abstract Pediatric neuroblastoma is one of the most common extra cranial cancers in children. Despite an improvement in survival with the currently available therapies, neuroblastoma with an amplification of the transcription factor MYCN have a very poor prognosis. New therapeutic approaches must be developed to increase the survival of patients. One such approach is epigenetic drug therapy. Neuroblastoma, like many other pediatric cancers, contains several epigenetic alterations at the level of DNA methylation and histone modifications. In a screening of FDA approved drugs, we discovered some molecules having characteristics of epigenetic drugs that were unknown until now. Our study seeks to demonstrate the efficacy of these molecules in the treatment of neuroblastoma cell lines. Following preliminary tests, one of the molecules approved by the FDA stood out: disulfiram, a medication approved for the treatment of chronic alcoholism. We treated neuroblastoma cell lines (MYCN amplified: IMR-32, N91 and SK-N-DZ; MYCN non-amplified: SK-N-AS and SK-N-SH) for 48 hours with disulfiram at clinically relevant concentrations (from 10 nM to 50 μM). Our results demonstrate a 50% growth inhibition (IC50) of 50nM for the cell lines tested. In addition, after analysis by flow cytometry, we found a cell cycle block in G2/M. We also observed a decrease in the transcription factor MYCN and a reduction in acetylation of several histone marks by Western blots analysis. Further studies are underway to determine the mechanism of action of disulfiram and we also plan on doing RNA sequencing and ChIP sequencing. This study will evaluate the efficacy of disulfiram alone as well as in combination with other epigenetic drugs, such as DNA methyltransferase inhibitor decitabine, for the treatment of neuroblastoma. Citation Format: Simon Jacques-Ricard, Noel Raynal, Gregory Armaos, Elodie Da Costa, Annie Beaudry. New epigenetic mechanism of disulfiram targeting chromatin remodeling in neuroblastoma. [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 3200.

MUC1-C induces DNA methyltransferase 1 and represses tumor suppressor genes in acute myeloid leukemia.

Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML); however, the regulation of DNA methyltransferase 1 (DNMT1), which is responsible for maintenance of DNA methylation patterns, has largely remained elusive. MUC1-C is a transmembrane oncoprotein that is aberrantly expressed in AML stem-like cells. The present studies demonstrate that targeting MUC1-C with silencing or a pharmacologic inhibitor GO-203 suppresses DNMT1 expression. In addition, MUC1 expression positively correlates with that of DNMT1 in primary AML cells, particularly the CD34+/CD38− population. The mechanistic basis for this relationship is supported by the demonstration that MUC1-C activates the NF-κB p65 pathway, promotes occupancy of the MUC1-C/NF-κB complex on the DNMT1 promoter and drives DNMT1 transcription. We also show that targeting MUC1-C substantially reduces gene promoter-specific DNA methylation, and derepresses expression of tumor suppressor genes, including CDH1, PTEN and BRCA1. In support of these results, we demonstrate that combining GO-203 with the DNMT1 inhibitor decitabine is highly effective in reducing DNMT1 levels and decreasing AML cell survival. These findings indicate that (i) MUC1-C is an attractive target for the epigentic reprogramming of AML cells, and (ii) targeting MUC1-C in combination with decitabine is a potentially effective clinical approach for the treatment of AML.

DNA demethylating agent decitabine broadens the peripheral T cell receptor repertoire.

PurposeDecitabine, a promising epi-immunotherapeutic agent has shown clinical responses in solid tumor patients, while the anti-tumor mechanisms were unclear. We aimed to investigate the immunomodulatory effect of decitabine in peripheral T cells.Experimental designWe applied next-generation sequencing to investigate the complementarity-determining region 3 (CDR3) of the TCRβ gene, the diversity of which acts as the prerequisite for the host immune system to recognize the universal foreign antigens. We collected the peripheral blood mononuclear cells (PBMCs) from 4 patients, at baseline and after 2 cycles of low-dose decitabine therapy.ResultsAn increase of the unique productive sequences of the CDR3 of TCRβ was observed in all of the 4 patients after decitabine treatment, which was characterized by a lower abundance of expanded clones and increased TCR diversity compared with before decitabine treatment. Further analysis showed a tendency for CD4 T cells with an increased CD4/CD8 ratio in response to decitabine therapy. In addition, the genome-wide expression alterations confirmed the effects of decitabine on immune reconstitution, and the increase of TCR excision circles (TRECs) was validated.ConclusionsThe low-dose DNMT inhibitor decitabine broadens the peripheral T cell repertoire, providing a novel role for the epigenetic modifying agent in anti-tumor immune enhancement.

CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.

Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

Simultaneous quantitative determination of 5-aza-2′-deoxycytidine genomic incorporation and DNA demethylation by liquid chromatography tandem mass spectrometry as exposure-response measures of nucleoside analog DNA methyltransferase inhibitors

The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2'-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2'-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2'-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100mm×2.1mm, 5μm) and isocratic elution with a 10mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2'-deoxycytidine, 2'-deoxycytidine, and 5-methyl-2'-deoxycytidine. The assay range was 2-400ng/mL for 5-aza-2'-deoxycytidine, 50-10,000ng/mL for 2'-deoxycytidine, and was 5-1000ng/mL for 5-methyl-2'-deoxycytidine. The assay proved to be accurate (93.0-102.2%) and precise (CV≤6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at -70°C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine.

Effects of the combination of decitabine and homoharringtonine in SKM-1 and Kg-1a cells

The methylation inhibitor decitabine (DAC) has great therapeutic value for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, DAC monotherapy is associated with relatively low rates of overall response and complete remission. Previous studies have shown promising results for combination treatment regimens including DAC. Homoharringtonine (HHT), an alkaloid from Chinese natural plants and Cephalotaxus, has demonstrated potential for leukemia treatment. Our studies have suggested that the combination of DAC and HHT has synergistic effects for inhibiting the viability of SKM-1 and Kg-1a cells. This combination leads to enhanced inhibition of colony formation and apoptosis induction compared with DAC alone in SKM-1 but not Kg-1a cells. Only high-dose DAC and HHT significantly up-regulate caspase-3 and caspase-9 and inhibit BCL-XL in the SKM-1 cell line. The combined effects of DAC plus HHT on apoptosis may not only depend on regulation of the apoptosis-related genes we examined but others as well. HHT had no demethylation effects, and HHT in combination with DAC had no enhanced effects on hypomethylation and DNMT1, DNMT3A and DNMT3B mRNA expression in SKM-1 cells. Overall, these results suggest that DAC used in combination with HHT may have clinical potential for MDS treatment.