Azacitidine and Lenalidomide Combination Therapy in Myelodysplastic Syndromes: Topography and Translational Relevance of Nuclear Phospholipase C β - dependent Signalling

Nuclear inositide signalling pathways, and particularly those regulated by PI-PLCβ1, are associated with cell proliferation and differentiation. Myelodysplastic syndromes (MDS) are a heterogeneous spectrum of chronic myeloid hemopathies with associated symptomatic cytopenias and substantial potential for evolution to acute myeloid leukemia (AML). MDS patients are currently treated with two main approaches, epigenetic (Azacitidine) and immunomodulatory (Lenalidomide: above all in cell clones bearing a deletion of the long arm of the chromosome 5 [del(5q)]). As Azacitidine and Lenalidomide alone can show adverse effects or patients can be refractory, an experimental current approach is the combination of the two drugs. Clinically, this combination therapy is promising, while its molecular effect has to be clarified. Stemming from these data, in this study the effect of an Azacitidine-Lenalidomide combination therapy was studied, in both MDS patients and hematopoietic cell lines. The specific aims of this study were to evaluate the effect of Azacitidine and Lenalidomide MDS therapy on: cell cycle regulation, hematopoietic differentiation, gene mutation and miR expression. Lenalidomide alone, via PI-PLCβ1/PKC pathway, was able to induce a selective G0/G1 arrest of the cell cycle in del(5q) cells, slowing down their rate proliferation and favouring erythropoiesis activation. In addition, although the mutation profile at baseline was not entirely capable of predicting the clinical effect of Azacitidine and Lenalidomide therapy, the presence of specific point mutations affecting three inositide genes (PI3KCD, AKT3, PLCG2) was correlated to and anticipated a negative clinical outcome. Moreover, the differential miR expression was detectable even from the 4th cycle of therapy in responder patients, as compared to non-responders. In MDS, this is the first evidence that the molecular mutation profiling of inositide genes or a specific mini-cluster of differentially expressed miRs, targeting inositide signaling molecules, can be associated with the clinical response, thus possibly predicting the effect of the therapy.

Role of gene methylation in antitumor immune response: Implication for tumor progression

Cancer immunosurveillance theory has emphasized the role of escape mechanisms in tumor growth. In this respect, a very important factor is the molecular characterization of the mechanisms by which tumor cells evade immune recognition and destruction. Among the many escape mechanisms identified, alterations in classical and non-classical HLA (Human Leucocyte Antigens) class I and class II expression by tumor cells are of particular interest. In addition to the importance of HLA molecules, tumor-associated antigens and accessory/co-stimulatory molecules are also involved in immune recognition. The loss of HLA class I antigen expression and of co-stimulatory molecules can occur at genetic, transcriptional and post-transcriptional levels. Epigenetic defects are involved in at least some mechanisms that preclude mounting a successful host-antitumor response involving the HLA system, tumor-associated antigens, and accessory/co-stimulatory molecules. This review summarizes our current understanding of the role of methylation in the regulation of molecules involved in the tumor immune response.

Impact of genomic methylation on radiation sensitivity of colorectal carcinoma.

PURPOSE: To investigate the influence of demethylation with 5-aza-cytidine (AZA) on radiation sensitivity and to define the intrinsic radiation sensitivity of methylation deficient colorectal carcinoma cells. METHODS AND MATERIALS: Radiation sensitizing effects of AZA were investigated in four colorectal carcinoma cell lines (HCT116, SW480, L174 T, Co115), defining influence of AZA on proliferation, clonogenic survival, and cell cycling with or without ionizing radiation. The methylation status for cancer or DNA damage response-related genes silenced by promoter methylation was determined. The effect of deletion of the potential target genes (DNMT1, DNMT3b, and double mutants) on radiation sensitivity was analyzed. RESULTS: AZA showed radiation sensitizing properties at >or=1 micromol/l, a concentration that does not interfere with the cell cycle by itself, in all four tested cell lines with a sensitivity-enhancing ratio (SER) of 1.6 to 2.1 (confidence interval [CI] 0.9-3.3). AZA successfully demethylated promoters of p16 and hMLH1, genes associated with ionizing radiation response. Prolonged exposure to low-dose AZA resulted in sustained radiosensitivity if associated with persistent genomic hypomethylation after recovery from AZA. Compared with maternal HCT116 cells, DNMT3b-defcient deficient cells were more sensitive to radiation with a SER of 2.0 (CI 0.9-2.1; p = 0.03), and DNMT3b/DNMT1-/- double-deficient cells showed a SER of 1.6 (CI 0.5-2.7; p = 0.09). CONCLUSIONS: AZA-induced genomic hypomethylation results in enhanced radiation sensitivity in colorectal carcinoma. The mediators leading to sensitization remain unknown. Defining the specific factors associated with radiation sensitization after genomic demethylation may open the way to better targeting for the purpose of radiation sensitization.

15-hydroxyprostaglandin dehydrogenase is down-regulated in gastric cancer.

PURPOSE: We have investigated the expression and regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in gastric cancer. EXPERIMENTAL DESIGN: Clinical gastric adenocarcinoma samples were analyzed by immunohistochemistry and quantitative real-time PCR for protein and mRNA expression of 15-PGDH and for methylation status of 15-PGDH promoter. The effects of interleukin-1beta (IL-1beta) and epigenetic mechanisms on 15-PGDH regulation were assessed in gastric cancer cell lines. RESULTS: In a gastric cancer cell line with a very low 15-PGDH expression (TMK-1), the 15-PGDH promoter was methylated and treatment with a demethylating agent 5-aza-2'-deoxycytidine restored 15-PGDH expression. In a cell line with a relatively high basal level of 15-PGDH (MKN-28), IL-1beta repressed expression of 15-PGDH mRNA and protein. This effect of IL-1beta was at least in part attributed to inhibition of 15-PGDH promoter activity. SiRNA-mediated knockdown of 15-PGDH resulted in strong increase of prostaglandin E(2) production in MKN-28 cells and increased cell growth of these cells by 31% in anchorage-independent conditions. In clinical gastric adenocarcinoma specimens, 15-PGDH mRNA levels were 5-fold lower in gastric cancer samples when compared with paired nonneoplastic tissues (n = 26) and 15-PGDH protein was lost in 65% of gastric adenocarcinomas (n = 210). CONCLUSIONS: 15-PGDH is down-regulated in gastric cancer, which could potentially lead to accelerated tumor progression. Importantly, our data indicate that a proinflammatory cytokine linked to gastric carcinogenesis, IL-1beta, suppresses 15-PGDH expression at least partially by inhibiting promoter activity of the 15-PGDH gene.

Potential target antigens for immunotherapy in human pancreatic cancer.

BACKGROUND: To be effective and selective, immunotherapy ideally targets specifically tumor cells and spares normal tissues. Identification of tumor specific antigens is a prerequisite to establish an effective immunotherapy. Still very little is known about the expression of tumor-related antigens in pancreatic neoplasms. Cancer Testis antigens (CT) are antigens shared by a variety of malignant tumors, but not by normal tissues with the exception of germ cells in testis. Restricted expression in neoplastic tissues and inherent immunogenic features make CT antigens ideal for use in immunotherapy. We analyzed the expression of a selected panel of nine CT antigens that have been proven to elicit an efficient immunogenic response in other malignancies. In addition we analyzed the expression of HERV-K-MEL, an immunogenic antigen of viral origin. METHODS: Pancreatic adenocarcinoma tumor samples (n=130) were obtained intraoperatively, control tissues (n=23) were collected from cadaveric donor and from patients with chronic pancreatitis. Tumor-associated antigen expression of MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, LAGE-1, NY-ESO-1, SCP-1, SSX-2, SSX-4 and HERV-K-MEL was assessed by PCR. Sequencing of PCR products were performed to assess the expression of SSX-4 in neoplastic and normal pancreatic tissues. RESULTS: Three of 10 tested antigens were expressed in over 10% of malignant pancreatic tissue samples. SSX-4 was found positive in 30% of cases, SCP-1 in 19% and HERV-K-MEL in 23% of cases. No expression of CT antigens was found in non-malignant pancreatic tissue with the exception of SSX-4 and and SSX-2. CONCLUSIONS: Fifty two percentage of the analyzed tissues expressed at least one CT antigen. The concomitant expression of SSX-4 in both malignant and non-malignant pancreatic tissue is a new finding which may raise concerns for immunotherapy. However, HERV-K-MEL is expressed with a relatively high prevalence and may be a candidate for specific immunotherapy in a large subgroup of pancreatic cancer patients. This study advocates the analysis of patients with regard to their immunogenic profile before the onset of antigen-specific immunotherapy.

Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.

Chromosome damage induced by 5-azacytidine under the influence of caffeine or cytosine arabinoside in CHO-K1 (wild-type) and XRS-5 (mutant) cell lines

5-azacytidine (5-azaC) treatment combined with cytosine arabinoside (ara-C) or caffeine were performed in vitro in Chinese hamster cells, CHO-K1 (wild-type) and xrs-5 (mutant) cell lines, in order to compare the cell response to the induction of chromosomal aberrations. Exponentially growing cells were treated with 5-azaC (4-16 uM) for 1 h, the cells were washed and incubated for 7 h, and 500 uM caffeine or 5 uM ara-C were added to the cultures for the last 2 h. In both cell lines, 5-azaC induced a significantly increase (P<0.01) in the frequencies of aberrations; in the combined treatments (5-azaC + Ara-C), a significant reduction (P<0.05) was observed for the aberrations which were randomly distributed. Caffeine had no influence at the same conditions. 5-azaC induced-DNA lesions were probably processed at S/G2 phase in a common pathway in both cell lines, but alternatively, 5-azaC may cause xrs-5 cells to revert to the wild-type.

Efeito das baixas concentrações de agentes antineoplásicos sobre linfócitos humanos de sangue periférico

Our previous studies have shown that low concentrations (noncytotoxics) of antineoplastic agents modulate positively the dendritic cells, favoring their in vitro maturation and improving their antigen presenting function. The effects on colorectal cancer cells (HCT-116) were also investigated and we have observed an increased immunogenicity and susceptibility to cytotoxic T cells. Thereby, this study aimed to investigate the effect of 5-fluorouracil (5-FU) an azacitidine (AZA), in minimum effective and noncytotoxic concentrations on lymphocytes of healthy donors. In this study we have analyzed the cytotoxic effect of drugs at these concentrations as well as the proliferative ability of lymphocytes. In vitro production of IL-10 and IFN-γ has been also evaluated. We have observed that low concentrations of those chemotherapeutic agents are not cytotoxic for lymphocytes. However, the minimum effective concentrations (5-FU: 0,410±0,088 e AZA: 0,757±0,233; p<0, 05) have reduced the cell number. Proliferative activity of allogeneic lymphocytes in a mixed reaction (MLR) was not affected by the treatment. The cytokine production was not affected by the treatments, either. In conclusion, low concentrations of 5-FU and AZA has no deleterious effects on human peripheral blood lymphocytes and seems to be safe for combinatory administration with DC vaccines

Effetti clinici e biologici della terapia epigenetica nelle sindromi mielodisplastiche a basso rischio

Background: Nucleoside 5-Azacitidine (5-Aza) in high risk MDS patients (pts) at a dose of 75mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates (hematologic improvement (HI) 50-60%, complete remission (CR) 10-30%) and prolongation of survival (at 2 years 50,8%). Aim: The role of 5-Aza in low-risk MDS patients is not well defined but its use in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial a low-dose schedule of 5-Aza (75 mg/mq daily for 5 consecutive days every 28 days) was given to low-risk MDS pts in order to evaluate its efficacy and tolerability and to identify biological markers to predict the response. Methods: From September 2008 to February 2010, 34 patients were enrolled into the study. Fifteen patients had refractory anemia (RA), 5 patients refractory anemia with ringed sideroblasts (RARS), 7 patients refractory cytopenia with multilineage dysplasia (RCMD) and 7 patients refractory anemia with excess blasts-1 (RAEB-1). All patients failed previously EPO therapy and were in chronic red blood cell (RBC) supportive care with a median transfusions requirement of 4 units/monthly. The response treatment criteria was according to IWG 2006. Results: At present time 31 out of 34 pts are evaluable: 12/31 pts (39%) completed the treatment plan (8 courses), 7/31 pts (22%) performed the first 4 courses, 8/31 (26%) made 1 to 3 courses and 4/31 (13%) died during the treatment period. Out of 12 pts who completed the 8 courses of therapy 10 (83%) obtained an HI, 2/12 (17%) maintained a stable disease. Out of 10 pts who obtained HI, 4 pts (40%) achieved a CR. Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%) but they were transitory and usually no delay of treatment was necessary. 2/4 pts died early after the 1th cycle for septic shock and gastrointestinal hemorrage respectively whereas 2/4 pts died in a condition of stable disease after the 4th cycle for pneumonia and respiratory distress. Samples for biologic studies have been collected from the pts before starting the therapy and at the end of 4th and 8th course. Preliminary data on the lipid signalling pathways suggested a direct correlation between PI-PLC-β1 gene expression and 5-Aza responsiveness. Conclusion: Interim analysis of our study based on the small number of cases who completed the treatment program, shows that 83% of pts obtain an HI and 40% obtain a CR. 4 patients died during the treatment and even if the causes were reported as no related to the therapy it has been considered that caution has to be reserved in given 5-Aza in these pts who are elderly and frail. Preliminary data of PI-PLC-β1 gene expression suggest that this and probably other biological markers could help us to know a priori who are the patients who have more chances to respond.

La terapia demetilante con 5-azacitidina nelle sindromi mielodisplastiche: esperienza clinica del Nostro Istituto e correlazione con i dati biologici

Sulla base delle evidenze della letteratura (Fenaux, 2009; Lyons, JCO 2009), a partire da Settembre 2004 nel Nostro Istituto sono stati trattati 57 pazienti affetti da Sindrome Mielodisplastica (MDS) mediante terapia demetilante con 5-Azacitidina. Sono stati utilizzati differenti regimi terapeutici a seconda della classe di rischio IPSS: i pazienti a rischio basso/intermedio-1 hanno ricevuto Azacitidina 75 mg/mq/die sottocute per 5 giorni/mese (schema 5) per 8 cicli; i pazienti a rischio alto/intermedio-2 hanno ricevuto Azacitidina 50 mg/mq/die sottocute per 10 giorni/mese (schema 5+2+5) o Azacitidina 75 mg/mq/die per 7 giorni/mese (schema 7) fino a perdita della risposta. Su una casistica totale di 57 pazienti (15 a rischio basso/int-1; 41 rischio alto/int-2), l’87.7% (50 pazienti) sono risultati valutabili. Tra questi le risposte osservate sono state del 68% (34 pazienti), di cui il 14% (7 pazienti) ha ottenuto una Remissione Completa (CR) ed il 54% (27 pazienti) ha ottenuto un Hematologic Improvement (HI). La valutazione della risposta è stata eseguita secondo i criteri dell’International Working Group 2006 (IWG, Cheeson 2006). Le principali tossicità osservate sono state rappresentate da reazioni cutanee locali nel sito d’iniezione, tossicità gastrointestinale (stipsi e/o diarrea), mielotossicità, neutropenia febbrile, sepsi (3 pazienti). Tra i pazienti trattati abbiamo osservato la presenza di risposta ematologica prolungata (≥ 20 mesi) in 10 pazienti (20% dei pazienti valutabili). Inoltre, grazie alla collaborazione con il Dipartimento di Anatomia Umana dell’Università di Bologna (Prof. L. Cocco, Dott.ssa M.Y. Follo), tutti i pazienti trattati sono stati valutati per i livelli di espressione genica e metilazione del gene della fosfolipasi PI-PLC-beta1. I dati biologici così ottenuti sono stati correlati con quelli clinici, evidenziando la presenza di una correlazione tra i livelli di espressione genica e mutilazione della PI-PLC-beta1 e la risposta alla terapia demetilante con 5-Azacitidina.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.