p14ARF Gene Research Articles

A Genomic Approach for High-Grade B Cell Lymphoma with MYC and BCL2 and / or BCL6 Rearrangements: Two Clusters Stood out on Next Generation Sequencing Results

Introduction High-grade B cell lymphomas with double or triple hit rearrangement (HGBL DH/TH), which appeared on the 4th revised edition WHO classification of lymphoid neoplasms, are aggressive mature B-cell lymphomas with high-grade cytology (Burkitt-like, blastoid or DLBCL-like) that have a MYC (8q24) rearrangement in combination with a BCL2 (18q21) and / or a BCL6 (3q27) rearrangement. There are currently no treatment guidelines for this subtype, and the usual immunochemotherapy leads to few complete remissions. While the NGS technology has been used in diffuse large B cell lymphoma, there has been no specific study on additional genetic events which would predict the clinical outcome in HGBL DH/TH. Here, we report our preliminary results using a targeted DNA capture panel approach (NGS) to identify subgroups with different clinical outcomes among HGBL DH/TH. Methods We conducted a retrospective study on 44 patients diagnosed with DH or TH HGBL and treated at Centre Hospitalier Lyon Sud or Centre Léon Bérard starting in 2013. We collected the medical information regarding the treatment, time to response to treatment or relapse, progression free survival, and overall survival. They all benefited from a DNA-targeted NGS analysis covering 54 genes. Results The cohort included 20 females and 24 males, with a median age of 65 (31-87). Among them, 22 patients were MYC/ BCL2 DH (50%), 12 were MYC/ BCL6 DH (27,3%) and 10 were TH (22,7%). Eleven patients had B symptoms (25%). The Ann Arbor stage was high (III-IV) in 97.5%. The WHO Performance Status was low (0-1) for most of the patients (70%). The majority of them presented with an IPI above to 2 (82,5%) and an elevated LDH level (80%). The Ki67 index was above 70% (33/42) in 78,5% of the cases and above 90% (19/42) in nearly half of them. The major finding of the NGS analysis was the identification of 2 groups based on the TP53/ CDKN2A_p14ARF mutational status. The CDKN2A locus encodes for 2 alternative open reading frames leading to 2 unrelated proteins p14ARF and p16INK4A. P14ARF is involved in the regulation of the TP53 pathway whereas p16INK4A is not. We therefor defined 2 groups : the first group included patients with either TP53 or CDKN2A_p14ARF-only alterations (group A, n=10) while the second group had no alteration in these two coding sequences (group B, n=34) although 12 patients had a CDKN2A_p16INK4A loss or mutation affecting both CDKN2A transcripts. Most of TP53 mutations were missense mutations mostly in TP53 DNA-binding domain and 2 cases appeared to be single hit mutations with an allele frequency below 50%. The patient with a p14ARF only mutation had a start codon mutation p.(M1*) with an allele frequency of 96% without CDKN2A copy number loss. The median overall survival (OS) (Figure 1) was 6,2 months for group A, and 55,5 months for group B (p = 0,0076). The median follow-up time was 14 months (7,5 months in the group A, 18 in group B). Only 2 patients in group A survived and are still in complete remission, one was DH BCL6 and the other DH BCL2 Conclusion Our preliminary results indicate a strong negative prognostic impact of either CDKN2A_p14ARF or TP53 gene alterations on the OS of HGBL DH/TH patients. The biological basis for the difference between groups would be in favour of the alteration of the p14ARF-TP53 tumor suppressive pathway as major contributor of chemoresistance. A more detailed analysis is on-going to further identify mutation profiles of double hit and triple hit lymphomas and compare our finding with existing data. Our results confirm the more favourable outcome of DH BCL6 patients (data not shown), which is consistent with the 5 th revised OMS classification that removed that specific entity. Our results also indicate that routine NGS analysis of HGBL DH/TH patients offers new perspectives for patient stratification. Regarding their survival, patients with TP53 pathway alterations should probably not be treated with the standard RCHOP chemo-immunotherapy. Instead, a more intensive immunochemotherapy regimen or novel strategies such as first-line bispecific antibodies or CARTCells might be considered.

Expanding Opportunities in Viral Oncolysis.

Expanding Opportunities in Viral Oncolysis.

Induction of Immune-Stimulating Factors and Oncolysis Upon p14ARF Gene Transfer in Melanoma Cell Lines.

Together with an anti-tumor immune response, oncolysis using a recombinant viral vector promises to eliminate cancer cells by both gene transfer and host-mediated functions. In this study we explore oncolysis induced by nonreplicating adenoviral vectors used for p14ARF and interferon-β (hIFNβ) gene transfer in human melanoma cell lines, revealing an unexpected role for p14ARF in promoting cellular responses predictive of immune stimulation. Oncolysis was confirmed when UACC-62 (p53 wild-type) cells succumbed upon p14ARF gene transfer in vitro, whereas SK-Mel-29 (p53-mutant) benefitted from its combination with hIFNβ. In the case of UACC-62, in situ gene therapy in nude mice yielded reduced tumor progression in response to the p14ARF and hIFNβ combination. Potential for immune stimulation was revealed where p14ARF gene transfer in vitro was sufficient to induce emission of immunogenic cell death factors in UACC-62 and upregulate pro-immune genes, including IRF1, IRF7, IRF9, ISG15, TAP-1, and B2M. In SK-Mel-29, p14ARF gene transfer induced a subset of these factors. hIFNβ was, as expected, sufficient to induce these immune-stimulating genes in both cell lines. This work is a significant advancement for our melanoma gene therapy strategy because we revealed not only the induction of oncolysis, but also the potential contribution of p14ARF to immune stimulation.

P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7.

Background: It is well known that P16 INK4A , P14 ARF , P15 INK4B mRNAs, and ANRIL lncRNA are transcribed from the CDKN2A/2B locus. LncRNA P14AS is a lncRNA transcribed from antisense strand of P14 ARF promoter to intron-1. Our previous study showed that P14AS could upregulate the expression level of ANRIL and P16 INK4A and promote the proliferation of cancer cells. Because polycomb group protein CBX7 could repress P16 INK4A expression and bind ANRIL, we wonder whether the P14AS-upregulated ANRIL and P16 INK4A expression is mediated with CBX7. Results: In this study, we found that the upregulation of P16 INK4A , P14 ARF , P15 INK4B and ANRIL expression was induced by P14AS overexpression only in HEK293T and HCT116 cells with active endogenous CBX7 expression, but not in MGC803 and HepG2 cells with weak CBX7 expression. Further studies showed that the stable shRNA-knockdown of CBX7 expression abolished the P14AS-induced upregulation of these P14AS target genes in HEK293T and HCT116 cells whereas enforced CBX7 overexpression enabled P14AS to upregulate expression of these target genes in MGC803 and HepG2 cells. Moreover, a significant association between the expression levels of P14AS and its target genes were observed only in human colon cancer tissue samples with high level of CBX7 expression (n = 38, p < 0.05), but not in samples (n = 37) with low level of CBX7 expression, nor in paired surgical margin tissues. In addition, the results of RNA immunoprecipitation (RIP)- and chromatin immunoprecipitation (ChIP)-PCR analyses revealed that lncRNA P14AS could competitively bind to CBX7 protein which prevented the bindings of CBX7 to both lncRNA ANRIL and the promoters of P16 INK4A , P14 ARF and P15 INK4B genes. The amounts of repressive histone modification H3K9m3 was also significantly decreased at the promoters of these genes by P14AS in CBX7 actively expressing cells. Conclusions: CBX7 expression is essential for P14AS to upregulate the expression of P16 INK4A , P14 ARF , P15 INK4B and ANRIL genes in the CDKN2A/2Blocus. P14AS may upregulate these genes’ expression through competitively blocking CBX7-binding to ANRIL lncRNA and target gene promoters.

Aberrant hydroxymethylation in promoter CpG regions of genes related to the cell cycle and apoptosis characterizes advanced chronic myeloid leukemia disease, poor imatinib respondents and poor survival

BackgroundThere is strong evidence that disease progression, drug response and overall clinical outcomes of CML disease are not only decided by BCR/ABL1 oncoprotein but depend on accumulation of additional genetic and epigenetic aberrations. DNA hydroxymethylation is implicated in the development of variety of diseases. DNA hydroxymethylation in gene promoters plays important roles in disease progression, drug response and clinical outcome of various diseases. Therefore in this study, we aimed to explore the role of aberrant hydroxymethylation in promoter regions of different tumor suppressor genes in relation to CML disease progression, response to imatinib therapy and clinical outcome.MethodsWe recruited 150 CML patients at different clinical stages of the disease. Patients were followed up for 48 months and haematological/molecular responses were analysed. Haematological response was analysed by peripheral blood smear. BCR/ABL1 specific TaqMan probe based qRT-PCR was used for assessing the molecular response of CML patients on imatinib therapy. Promoter hydroxymethylation of the genes was characterized using MS-PCR.ResultsWe observed that promoter hydroxymethylation of DAPK1, RIZ1, P16INK4A, RASSF1A and p14ARFARF genes characterize advanced CML disease and poor imatinib respondents. Although, cytokine signalling (SOCS1) gene was hypermethylated in advanced stages of CML and accumulated in patients with poor imatinib response, but the differences were not statistically significant. Moreover, we found hypermethylation of p14ARF, RASSF1 and p16INK4A genes and cytokine signalling gene (SOCS1) significantly associated with poor overall survival of CML patients on imatinib therapy. The results of this study are in agreement of the role of aberrant DNA methylation of different tumor suppressor genes as potential biomarkers of CML disease progression, poor imatinib response and overall clinical outcome.ConclusionIn this study, we report that promoter hydroxymethylation of DAPK1, RIZ1, P16INK4A, RASSF1A and p14ARFARF genes is a characteristic feature of CML disease progressions, defines poor imatinib respondents and poor overall survival of CML patients to imatinib therapy.

Combined analysis of KRAS mutation and p16INK4a and p14ARF methylation status in locally advanced rectal carcinoma treated with preoperative chemoradiotherapy

Current management of locally advanced rectal carcinoma (LARC) involves preoperative chemoradiotherapy (preCRT) before surgery. Despite improved local control rate, the response to preCRT of individual patients is variable and may reflect heterogeneous biological properties among tumors of the same clinical stage. Identifying novel molecular parameters with predictive and/or prognostic value is of great clinical importance for a personalized therapeutic approach. In this study, KRAS mutation status was analyzed by direct sequencing, while methylation-specific polymerase chain reaction (MSP) was used to examine p16INK4a and p14ARF gene methylation status in pretreatment tumor biopsies of 60 patients with LARC. The examined molecular changes of KRAS, p16INK4a and p14ARF genes were mutually independent (p16INK4a/KRAS, P=0.272; p14ARF/KRAS, P=0.923; p16INK4a/p14ARF, P=0.715). However, the simultaneous presence of p14ARF methylation and KRAS mutation was associated with a more frequent appearance of local recurrences and distant metastasis (P=0.027). Moreover, patients with the simultaneous presence of p16INK4a and p14ARF methylation and KRAS mutation had significantly shorter overall survival (P=0.011). The obtained results strongly suggest that combined analyses of examined genetic and epigenetic molecular alterations could contribute to the identification of LARC patient subgroups with more aggressive tumor behavior and worse disease outcome.

Abstract 12513: Post-GWAS Functional Analysis of the CDKN2A/B Locus Identifies CUX1 as a Regulator of Endothelial Senescence by Modulating the CAD-Associated p16 INK4A Expression

Introduction: Genome-wide association studies (GWAS) have validated a strong association of the CDKN2A/B locus with more than 10 different age-related diseases including atherosclerosis and its complications. Hypothesis: The CDKN2A/B locus is one of the most complicated genetic loci that harbors p16INK4a, one of the markers for cellular senescence. As a single locus associates with multiple different age-related diseases, this region may contribute to the progression of these diseases by promoting aging itself, perhaps through modulating cellular senescence. Methods: By coupling regulatory element-sequencing (Reel-seq) with SNP-specific DNA competition pulldown-mass spectrometry (SDCP-MS), two techniques recently developed in our lab, the disease-associated functional SNPs (fSNP) on the CDKN2A/B locus and the fSNP binding proteins were identified. The mechanism underlying the contribution of an atherosclerosis-associated fSNP rs1537371 to the pathogenesis of, and the susceptibility to atherosclerosis was revealed by contemporary techniques. Results: We demonstrate that the homeodomain transcription factor CUX1 specifically binds to the atherosclerosis-associated fSNP rs1537371 within the CDKN2A/B locus. Although it binds at a considerable distance (&gt; 100 kb), CUX1 is shown to regulate the CDKN2A/B encoded genes p14 ARF , p16 INK4A , p15 INK4B and ANRIL in endothelial cells (ECs). In-depth analysis demonstrates that endothelial CUX1 expression correlates with telomeric length and is induced by both DNA-damaging agents and oxidative stress. Moreover, induction of CUX1 expression triggers both replicative and stress-induced premature senescence via activating p16 INK4A expression. Consistent with these findings, we also detect elevated expression of CUX1 and p16 INK4A in the plaques of patients with carotid artery disease. Conclusions: CUX1 is a transcriptional regulator of p16 INK4A expression. It regulates p16 INK4A -dependent endothelial senescence via binding to the atherosclerosis-associated fSNP rs1537371.

ONCOLYSIS MEDIATED BY INFB AND ARF GENE TRANSFER ACTIVATES KEY COMPONENTS OF THE IMMUNE SYSTEM

<h3>Background</h3> The importance of the immune system for controlling neoplasms was highlighted with the success of checkpoint blockade therapy. New cancer therapeutics seek to break the immune tolerance inherent in tumor progression. To this end, some virus-mediate gene therapy approaches have been shown to induce oncolysis accompanied by emission of immunogenic cell death (ICD) factors, thus attracting and activating cells of the immune system. <h3>Objective</h3> To use combined adenovirus-mediated gene transfer of p14ARF and human interferon-β (IFNβ) to human melanoma cells to induce oncolysis, ICD and subsequent activation of immune cells. <h3>Methods</h3> Cell death was measured by PI and/or AnnexinV staining. Detection of calreticulin was performed by cytometry, while ATP and IFNβ were detected by ELISA. DC and lymphocytes from healthy donors were cultivated ex vivo. <h3>Results</h3> IFNβ alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. However, detection of critical markers of ICD) was stronger when cells were treated with combined p14ARF and IFNβ gene transfer. In situ gene therapy for s.c. SK-MEL-147 tumors in Nod-Scid mice controlled tumor growth and increased animal survival in response to IFNβ alone or in combination with p14ARF. However, the immunocompromised animal model cannot demonstrate the expected immune activation. Thus, we co-cultured SK-MEL-147 transduced with our combination of adenoviruses with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors. These Mo-DCs showed increased levels of activation markers HLA-DR, CD80, and CD86. Next, we used these activated Mo-DCs to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. T cells primed in this way were co-cultured with fresh non-transduced SK-MEL-147, resulting in tumor cell killing. Currently we are expanding these studies to include three-dimensional cultures of melanoma cell lines and also the use of human melanoma samples in patient derived organoid (PDO) models. We expect to reveal the influence of oncolysis on tumor infiltrating cells, including DCs, macrophages, NK and T cells. <h3>Conclusion</h3> Collectively, our results indicate that p14ARF and IFNβ delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.

Methylation status of p16INK4a and p14ARF gene in saliva of smokeless tobacco users: A pilot descriptive study

ObjectivesThe tumor suppressor genes p16INK4a and p14ARF are frequently silenced through hypermethylation in oral potentially malignant disorders (OPMDs) and oral cancers. This study aimed to evaluate the methylation status of p16INK4a and p14ARF in saliva of smokeless tobacco users as an early diagnostic marker of dysplasia. MethodsUnstimulated whole saliva with exfoliated buccal cells were collected from 30 non-tobacco users and 30 smokeless tobacco users without any clinically evident tobacco associated oral lesions from South Indian population. DNA extraction, bisulfite modification and methylation specific polymerase chain reaction were done to identify methylation with respect to p16INK4a and p14ARF. The amplified sequences were visualized in 1% agarose gel electrophoresis. ResultsOf the 60 samples collected, 26 samples were rejected owing to the poor quality and insufficient quantity of DNA. A total of 34 DNA samples were assessed for methylation status of three sets of primers for p16INK4a and one set of primer for p14ARF. Methylation was not detected in saliva from non-tobacco users. In case of smokeless tobacco users, partial methylation was detected in 33.3 % samples corresponding to set II primer of p16INK4a. ConclusionDetection of methylation in p16INK4a in oral mucosa of smokeless tobacco users without any associated oral lesions, indicates its role in early dysplastic changes.

Regulation of p14ARF by Siva1 in H. pylori-infected gastric epithelial cells.

243 Background: Helicobacter pylori ( H. pylori) is the strongest known risk factor for gastric cancer. Bacterial degradation of tumor suppressor proteins affect the host microbe’s interactions and host cellular response, which contribute to tumorigenesis. p14ARF, a crucial tumor suppressor protein that activates p53 protein under oncogenic stress plays a major role in oncogenic stress response (OSR) regulation. However, little is known about the mechanism of ARF and OSR regulation in H. pylori-infected gastric epithelial cells. Methods: The expression of p14ARF and cytotoxin-associated gene A (CagA) were analyzed in gastric cells co-cultured with H. pylori strains isolated from high-gastric risk and low-gastric risk areas by immunoblotting. To investigate the potential role of CagA in regulation of p14ARF, we employed isogenic cagA− and cagE− H. pylori mutants in gastric epithelial cells, and C57BL/6 mice (n = 10). We also analyzed the expression of Siva1 in human individual infected with cagA-positive (n = 13) and cagA-negative (n = 13) bacteria as well as uninfected human subjects (n = 6). siRNA was used to inhibit activity of Siva1 protein. Results: In this study, H. pylori strains expressing high levels of CagA virulence factor and associated with a higher gastric cancer risk more strongly suppress p14ARF compared with low-risk strains in vivo and in vitro. We found that degradation of p14ARF induced by CagA is mediated by E3 ubiquitin ligase Siva1, which works in concert with another E3 ubiquitin ligase TRIP12. Decreased expression of Siva1 protein and consequent up-regulation of p14ARF was also found in gastric mucosa of H. pylori-infected mice and human individuals. Tumorigenic strain 7.13 was more potent in upregulation of Siva1 and downregulation of p14ARF than non-tumorigenic strain B128. Inhibition of p14ARF protein by H. pylori causes inhibition of autophagy in infected cells. Conclusions: Our results provide first evidence that carcinogenic H. pylori strains significantly alter the host tumor suppressor protein p14ARF, leading to suppression of host OSR and autophagy, which may affect host-bacteria interactions and tumorigenic alteration in the stomach.

Effect of 5-fluoro-2′-deoxycytidine (FdCyd) on p16INK4a, p14ARF, p15INK4b, and DNA methyltransferase 1, 3a, and 3b Genes Expression, Apoptosis Induction, and Cell Growth Inhibition in Pancreatic Cancer AsPC-1 and Hepatocellular Carcinoma LCL-PI 11 Cell Li

Background: Aberrant DNA methylation of the promoter region is one of the most epigenetic changes in numerous cancers. DNA methyltransferase inhibitors (DNMTIs) can revert DNA hypermethylation in tumor suppressor genes (TSGs). The present study was designed to investigate the effect of 5-fluoro-2′-deoxycytidine (FdCyd) on p16INK4a, p14ARF, p15INK4b, and DNA methyltransferase 1, 3a, and 3b genes expression, apoptosis induction, cell growth inhibition in pancreatic cancer AsPC-1 and hepatocellular carcinoma LCL-PI 11 cell lines. Materials and Methods: The cells were treated with FdCyd at different periods. Then, the MTT assay, cell apoptosis assay, and qRT-PCR were done to determine cell viability, cell apoptosis, and the relative gene expression level respectively. Results: The FdCyd decreased DNA methyltransferase 1, 3a, and 3b and increased p16INK4a, p14ARF, and p15INK4b genes expression significantly (P&lt;0.001). Besides, LCL-PI 11 cell was more sensitive to FdCyd in comparison to AsPC-1 cell. FdCyd induced significant cell growth inhibition with a dose- and time-dependent manner (P&lt;0.001). The IC50 value of FdCyd was obtained with approximately 1μM. Further, FdCyd induced cell apoptosis significantly as a time-dependent manner. The number of apoptotic cells was significantly increased in all groups. The percentage of apoptotic cells after 24 and 48 h were 13.86 and 29.6 % in AsPC-1 and 21.04 and 41.52 % in LCL-PI 11 cell line respectively (P&lt;0.001). Conclusion: The FdCyd can reactivate the p16INK4a, p14ARF, and p15INK4b through inhibition of DNA methyltransferase 1, 3a, and 3b gene expression.

Combined p14ARF and Interferon-β Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation.

Immune evasion is an important cancer hallmark and the understanding of its mechanisms has generated successful therapeutic approaches. Induction of immunogenic cell death (ICD) is expected to attract immune cell populations that promote innate and adaptive immune responses. Here, we present a critical advance for our adenovirus-mediated gene therapy approach, where the combined p14ARF and human interferon-β (IFNβ) gene transfer to human melanoma cells led to oncolysis, ICD and subsequent activation of immune cells. Our results indicate that IFNβ alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. In situ gene therapy of s.c. SK-MEL-147 tumors in Nod-Scid mice revealed inhibition of tumor growth and increased survival in response to IFNβ alone or in combination with p14ARF. Emission of critical markers of ICD (exposition of calreticulin, secretion of ATP and IFNβ) was stronger when cells were treated with combined p14ARF and IFNβ gene transfer. Co-culture of previously transduced SK-MEL-147 cells with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors resulted in increased levels of activation markers HLA-DR, CD80, and CD86. Activated Mo-DCs were able to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. Preliminary data showed that T cells primed by Mo-DCs activated with p14ARF+IFNβ-transduced SK-MEL-147 cells were able to induce the loss of viability of fresh non-transduced SK-MEL-147 cells, suggesting the induction of a specific cytotoxic population that recognized and killed SK-MEL-147 cells. Collectively, our results indicate that p14ARF and IFNβ delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.

Abstract 5548: Indicators of immune activation upon p14ARF and interferon-beta gene transfer in a human melanoma cell line

Abstract Immunoevasion adopted by tumor cells is an important cancer hallmark and understanding of its mechanisms has generated promising therapeutic approaches. In this context, triggering immunogenic cell death (ICD) is a promising idea since it alarms the immune system by attracting a myriad of cell populations that promote innate and adaptive immune responses. Since melanomas preserve wild-type p53 in over 80% of cases, our group developed a gene therapy strategy that utilizes an adenoviral vector with a p53-responsive promoter (PG). Here we present a critical advance for our approach, examining the immune activating potential of combined p14ARF and IFN-beta gene transfer in human melanoma cells. Our results indicate that IFN-beta alone or in combination with p14ARF was able to induce massive cell death accompanied by caspase 3/7 activation in the human melanoma cell line SKMEL147. In our animal model of in situ gene therapy, s.c. SKMEL147 tumors were established in Nod-scid mice before intratumoral virus injection, revealing tumor inhibition and improved survival in response to IFN-beta alone or in combination with p14ARF. However, this model cannot reveal immune activation and so far the benefit of p14ARF has not been revealed. Strikingly, three markers of ICD (exposition of calrecticulin, secretion of ATP and IFN-beta) were induced only when cells were treated with combined p14ARF and IFN-beta gene transfer. We then used PBMCs derived from normal donors as a source of DCs and lymphocytes. Coculture of DCs with previously-transduced SKMEL147 exhibit a typical phenotype of activation. Finally, coculture of these exposed DCs with lymphocytes results in high-level secretion of proinflammatory cytokines, followed by the increase of IL-10 in the samples treated with combination of p14ARF and IFN-beta. Collectively, our results indicate that the advantage of p14ARF and INF-beta delivered by our adenoviral system is the induction of cell death in human melanoma cells by an immune-activating mechanism. Future studies will include examination of this phenomenon in tumors from melanoma patients and using their own PBMCS. In parallel, the use of PDX model for in situ treatment will also be explored. Citation Format: Otto Luiz Cerqueira, Maria Alejandra Salomon, Elaine Cristina Cardoso, Tharcisio Citrangulo Tortelli, Bryan Eric Strauss. Indicators of immune activation upon p14ARF and interferon-beta gene transfer in a human melanoma cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5548.

Effect of Zebularine on p16INK4a, p14ARF, p15INK4b, and DNA Methyltransferase 1 Gene Expression, Cell Growth Inhibition, and Apoptosis Induction in Human Hepatocellular Carcinoma PLC/PRF5 and Pancreatic Cancer PA-TU-8902 Cell Lines.

Tumorigenesis must be understood as a summary of altered genetic and genomic changes resulting in the inactivation of tumor suppressor genes (TSGs). One of the characterizations of epigenetic alterations is DNA methylation. Epigenetic alteration of the p16INK4a, p14ARF, p15INK4b, and DNA methyltransferase 1 gene (DNMT1) expression occurs in hepatocellular carcinoma (HCC) and pancreatic cancer frequently. DNA methyltransferase inhibitors (DNMTIs), such as zebularine, play a significant effect on the demethylation and reactivation of TSGs. This study aimed to investigate the effect of zebularine on p16INK4a, p14ARF, p15INK4b, and DNA methyltransferase 1 gene expression, cell growth inhibition, and apoptosis induction in HCC PLC/PRF5 and pancreatic cancer PA-TU-8902 cell lines. Both cell lines were cultured and treated with zebularine at different times. The MTT assay, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and flow cytometry were used to determine cell viability, gene expression, and apoptotic cells, respectively. The result indicated that zebularine inhibited cell growth of both cell lines significantly as time- and dose-dependent manner (P < 0.007). The agent induced significant down-regulation of DNMT1 and up-regulation of p16INK4a, p14ARF, p15INK4b (P < 0.028). Besides, it had a significant apoptosis effect on both cell lines (P < 0.001). This compound had a strong significant effect on PLC/PRF5 in comparison to PA-TU-8902 cells. Concluding, zebularine inhibited PLC/PRF5 and PA-TU-8902 cell growth and induced apoptosis in these cell lines. The most likely mechanism underlying the zebularine played its role involves down-regulation of DNMT1 and up-regulation of p16INK4a, p14ARF, and p15INK4b genes.

The Expression of P53, MDM2, c-myc, and P14ARF Genes in Newly Diagnosed Acute Lymphoblastic Leukemia Patients.

The treatment response of acute lymphoblastic leukemia (ALL) depends on the percentage of lymphoblasts, cytogenetic aberrations, and altered gene expression. The analysis of the gene expression is applicable for determination of risk stratification and prognosis of cancers. c-MYC, P14ARF, MDM2, and P53 play a vital role in cell survival through a functional network. This study aimed to investigate the expression of these genes, also their correlation with immunophenotypic subtypes of ALL and the percentage of blasts. Real-time PCR was performed for the expression analysis of P53, MDM2, c-MYC, and P14ARF in the bone marrow or peripheral blood samples of 52 ALL patients and 13 normal samples as controls. The morphological analysis and flow cytometry were carried out to examine the phenotypes and percentage of lymphoblasts. The decreased expression levels of P53 and MDM2 were seen in ALL patients compared with control group. In T cell subgroup of ALL the expression of P14ARF gene was more decreased among other subgroups. The expression of MDM2 was decreased in ALL patients who were under the age of 16. Based on our study, the interaction between P53 and MDM2 might be more complex and different from reports published in previous studies. Our findings showed that MDM2 is not negatively correlated with P53, at least in our samples. It can be very effective on the current and future studies to use different techniques for analysis of genome, transcriptome, and proteome in definitive risk stratification and prognosis determination.

High Positive Correlations between ANRIL and p16-CDKN2A/p15-CDKN2B/p14-ARF Gene Cluster Overexpression in Multi-Tumor Types Suggest Deregulated Activation of an ANRIL-ARF Bidirectional Promoter.

The CDKN2B-AS1 gene, also called ANRIL, is located at the human CDKN2A/B locus at 9p21.3 and transcribed by RNA polymerase II into a long non-coding RNA of 3834 bp. The CDKN2B-AS1 gene overlaps a critical region of 125 kb covering the CDKN2B gene. The CDKN2A/B locus encompasses three major tumor suppressors juxtaposed and joined into a p16-CDKN2A/p15-CDKN2B/p14-ARF gene cluster. CDKN2A encodes splice variants p16-CDKN2A and p14-ARF, and CDKN2B encodes p15-CDKN2B. ANRIL shares a bidirectional promoter with the p14-ARF gene and is transcribed from the opposite strand to the cluster. We performed an analysis of the expression level of ANRIL and tumor suppressor p16-CDKN2A, p15-CDKN2B, and p14-ARF genes using quantitative RT-PCR in a multitumor panel. We observed the overexpression of the four genes ANRIL, p16-CDKN2A, p15-CDKN2B, and p14-ARF in the great majority of the 17 different cancer types. ANRIL was upregulated in 13/17 tumors compared to normal tissues, ranging from 5% (prostate cancer) to 91% (cervix cancer), with variable expression of p16-CDKN2A, p15-CDKN2B, and p14-ARF genes. A high positive correlation was identified between levels of expression of ANRIL and the three tumor suppressors. The strongest positive association was observed with p14-ARF (p < 0.001) in all but one (lung squamous cell carcinoma) of the examined tumor types. This correlation suggests coordinated deregulated mechanisms in all cancer types through aberrant activation of a bidirectional p14-ARF/ANRIL promoter. Furthermore, significant positive correlation was unexpectedly established in prostatic carcinomas, in contradiction with previous data.

Abstract 831: Hypermethylation in promoter regions of CpG islands of multiple genes is associated with disease progression and molecular response in chronic myelogenous leukemia

Abstract Chronic myelogenous leukemia (CML) has a typical progressive course with transition from a less aggressive chronic phase to a terminal more aggressive blast crisis. The molecular mechanisms leading to CML disease progression remain to be elucidated. However, it has been proposed in many studies that alterations in the methylation status of various genes may contribute to the progression of different malignancies. Hence, in this study we tried to understand the role of aberrant methylation of six tumor suppressor genes in the disease progression and molecular response in CML. We investigated 100 CML patients and 100 age and sex matched healthy controls for the methylation status of six tumor suppressor genes. The methylation status of these genes was evaluated by MS-PCR. In this study, 100 CML samples and 100 controls were analyzed for the DAPK1, RASSF1A, RIZ-1, p16, p14ARF and SOCS-1 promoter methylation status. These genes, except p14ARF and SOCS-1 showed significant differences in methylation status among CML patients and healthy controls. Also DAPK1, RASSF1A, RIZ-1, p16 and p14ARF genes were observed to be hypermethylated during progression of CML disease to advanced phases. The methylation frequencies of these five genes were seen to increase progressively during CML disease progression. This increase in hypermethylation of these five genes from was statistically significant with a p-value of &amp;lt;0.0001, 0.001, 0.009,0.002 and 0.01 respectively. Although, there was a progressive increase in the promoter methylation of SOCS1 gene during the CML disease progression (CP= 4%, AP=8% and BC=16%) but this did not reached a statistical significance (p-value 0.1). We also assessed the molecular response of these 100 CML patients after imatinib therapy and the patients were categorized into two groups; major molecular response group and loss of molecular response group. Out of 100 CML patients on imatinib therapy 52 achieved major molecular response while 48 patients showed loss of molecular response. Out of the above studied genes RASSF1 and RIZ1 promoter hypermethylation was significantly associated with loss of molecular response in CML patients receiving imatininb therapy. Out of 48 CML patients who were in loss of molecular response, 23.0% and 16.77% showed promoter hypermethylation of RASSF1 and RIZ1 genes respectively while out of 52 patients that were in major molecular response only 9.6% and 1.92% of patients showed hypermethylation in RASSF1 and RIZ1 genes respectively. These results suggest that hypermethylation of tumor suppressor genes plays a significant role in the progression and response in CML. Note: This abstract was not presented at the meeting. Citation Format: Mamta P. Sumi, Sameer A. Guru, Imtiyaz A Najar, Mariyam Zuberi, N. Gupta, Alpana Saxena. Hypermethylation in promoter regions of CpG islands of multiple genes is associated with disease progression and molecular response in chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 831.

ASSESSMENT OF A PROGNOSTIC VALUE OF CDKN2A AND TIMP3 GENE METHYLATION IN BLADDER CANCER

Promoter hypermethylation of tumor suppressor genes is one of the mechanisms of epigenetic regulation disturbance of gene expression and is often observed in different cancer types. The profile of mutational and epigenetic changes characterizes a malignant potential of a tumor, as well as its ability to invade and metastasize.The aim of the study was to determine a prognostic value of p16, p14ARF and TIMP3 gene methylation in the group of 158 bladder cancer patients. Epigenetic changes in these genes were observed with a frequency of 11.4, 0 and 10.8 %, respectively, and did not depend on clinic-morphological characteristics.A statistically significant association of p16 and TIMP3 abnormal methylation with smoking was found, indicating a possible influence of tobacco smoke carcinogens on the occurrence of these epigenetic changes. In the multivariate Cox regression analysis, p16 promoter hypermethylation was an independent predictor for bladder cancer progression (HR 6.84; 95 % CI 1.6–29.9; р = 0.011).The use of the data on the p16 methylation status may improve the accuracy of prognosis of the bladder cancer clinical course and the selection of appropriate treatment strategy.

Promoter methylation analysis of CDH1 and p14ARF genes in patients with urothelial bladder cancer.

Background/aimUrothelial bladder cancer arises from the accumulation of multiple epigenetic and genetic changes. We aimed to investigate the specificity and sensitivity of gene-specific promoter methylation of CDH1 and p14ARF genes in the early diagnosis of bladder cancer and compare those with other diagnostic tests in our population.Patients and methodsIn the current study, 65 patients with urothelial bladder cancer and 35 controls without any history of cancer were recruited. Methylation profiles of CDH1 and p14ARF genes from tumor and urine samples were determined by methylation-specific polymerase chain reaction method.ResultsMethylation of CDH1 and p14ARF genes in tumor samples was 95.4% and 78.5%, respectively. The methylation frequencies were found to be 68.8% for CDH1 gene and 72.9% for p14ARF gene in urine samples. Sensitivities of CDH1, p14ARF and urine cytology were found to be 67.4%, 72.1% and 34.9%, respectively, while their specificities were 93.9%, 63.6% and 93.9%, respectively.ConclusionAberrant promoter methylation of CDH1 and p14ARF genes can be used to detect urothelial bladder cancer. In low-grade tumors, when compared with urine cytology, combined methylation analysis of CDH1 and p14ARF genes may not increase the sensitivity to identify malignant cells in urine samples.

The 9p21 locus as a potential therapeutic target and prognostic marker in colorectal cancer.

Colorectal cancer (CRC) is a major cause of cancer-related-death worldwide. Despite extensive efforts to identify valid biomarkers for the risk stratification of CRC patients, there are few of proven clinical utility. It is recognized that genetic factors play a major role in determining susceptibility to CRC. Recent genome-wide association studies have demonstrated common genetic variants in a region on chromosome 9p21 associated with an increased risk of CRC. Several genetic polymorphisms have been identified in this region that are associated with CRC. Three genes are located at this locus; CDKN2B(encoding-p15ink4b), CDKN2A (encoding-p16ink4a/p14ARF) and 3' end of CDKN2BAS (termed-antisense-noncoding-RNA in the INK4-locus [ANRIL]). ANRIL has a post-transcriptional modulatory activity, which has been shown to perturb the expression of nearby genes. It also plays an important role in coordinating tissue remodeling through regulation of cell proliferation, apoptosis, aging, extra-cellular matrix remodeling and inflammatory response. However, the role of ANRIL is not well understood in CRC. Hypermethylation of the p14ARF and p16INK4a genes is often found in some tumors, including CRC. However, further studies are necessary to explore the clinical utility of these putative markers in risk stratification, and in the assessment of prognosis. In this review, we have summarized the prognostic and therapeutic potential of the p14ARF and p16INK4a genes in patients with colorectal cancer.