Vassilis L. Souliotis

Extent of Damage and Repair in the p53 Tumor-Suppressor Gene After Treatment of Myeloma Patients With High-Dose Melphalan and Autologous Blood Stem-Cell Transplantation Is Individualized and May Predict Clinical Outcome

PURPOSE To quantitate the individual levels of melphalan-induced DNA damage formation and repair in vivo and to search for possible correlations with clinical outcome in patients with multiple myeloma (MM). PATIENTS AND METHODS The formation and subsequent repair of DNA damage (monoadducts and interstrand cross-links) in the p53 tumor-suppressor gene, the proto-oncogene N-ras, and the housekeeping gene beta-actin during the first 24 hours after treatment with high-dose melphalan (HDM; 200 mg/m2) supported by autologous blood stem-cell transplantation (ABSCT) was measured in blood leukocytes of 26 patients with MM. The peak DNA adduct levels, the total amount of adducts over time, and the rate of adducts repair in each gene were correlated with response and time to progression after HDM. RESULTS The levels of gene-specific DNA damage formation and the individual repairing capacity varied up to 16-fold among patients, indicating that the melphalan-induced biologic effect in vivo is highly individualized. A significantly greater DNA damage and a slower rate of repair in p53 for all end points under study were found in patients who achieved tumor reduction compared with nonresponding patients. Furthermore, longer progression-free survival correlated with increased peak monoadduct levels in the p53 gene (P = .032). CONCLUSION Increased DNA damage and slower repairing capacity in the p53 gene from blood leukocytes after HDM correlate with improved outcome of patients with MM who undergo ABSCT. These results suggest that quantitation of such biologic end points may identify patients who are more likely to benefit from this procedure.

Adduct levels from benzo[a]pyrenediol epoxide: Relative formation to histidine in serum albumin and to deoxyguanosine in DNA in vitro and in vivo in mice measured by LC/MS-MS methods.

Stable and specific biomacromolecular adducts can be used to measure in vivo doses of reactive compounds. An LC/MS-MS method to measure adducts from the benzo[a]pyrene (BP) metabolite (±)-anti-BP-7,8-diol-9,10-epoxide ((±)-anti-BPDE) to His(146) in serum albumin (SA), earlier evaluated on in vitro alkylated human SA, was tested for its applicability to mouse. It was shown that (+)-anti-BPDE form BPDE-His adducts to mouse SA. The method was applied to samples from BP-exposed mice (100mg/kg of body weight for 1, 3, 7 and 28 days). BPDE-His in SA was close to the limit of quantification and showed the highest level (13fmol/mg) 3 days after exposure. The level was 400 times lower (calculated per gram macromolecule) than earlier measured level of BPDE-adduct to deoxyguanosine (dG) in DNA in the livers. The relative rate of formation of adducts from BPDE with His in SA and with dG in DNA was investigated. Quantification by LC/MS-MS of the adducts in human blood alkylated in vitro with (±)-anti-BPDE showed a 1850 times higher level of BPDE-dG compared to BPDE-His. The specific and stable BPDE-adducts to His in SA are potential biomarkers of in vivo dose of BPDE, though this requires a considerable improved analytical sensitivity of the LC/MS-MS method.

Aberrant DNA Damage Response Pathways May Predict the Outcome of Platinum Chemotherapy in Ovarian Cancer

Ovarian carcinoma (OC) is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR) pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/C30) to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs) from OC patients, sensitive (n = 7) or resistant (n = 4) to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9) were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05). Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03). Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05). We conclude that perturbations of DNA repair pathways as measured in PBMCs from OC patients correlate with the drug sensitivity of these cells and reflect the individualized response to platinum-based chemotherapy.

Benzo[a]pyrene-induced cell cycle arrest in HepG2 cells is associated with delayed induction of mitotic instability.

The environmental carcinogen benzo[a]pyrene (B[a]P) after being metabolised by cytochrome P450 enzymes forms DNA adducts. This abnormal situation induces changes in the cell cycle, DNA damage, chromosomal and mitotic aberrations, all of which may be related to carcinogenesis. In order to further investigate the mechanistic basis of these effects, HepG2 cells were treated with 3μM B[a]P for various time periods, followed by further incubation in the absence of B[a]P for up to 192h. B[a]P treatment led initially to S-phase arrest followed by recovery and subsequent induction of G2/M arrest, indicating activation of the corresponding DNA damage checkpoints. Immunofluorescence-based studies revealed accumulation of B[a]P-induced DNA adducts and chromosomal damage which persisted beyond mitosis and entry into a new cycle, thus giving rise to a new round of activation of the S-phase checkpoint. Prolonged further cultivation of the cells in the absence of B[a]P resulted in high frequencies of various abnormal mitotic events. Abrogation of the B[a]P-induced S-phase arrest by the Chk1 inhibitor UCN-01 triggered a strong apoptotic response but also dramatically decreased the frequency of mitotic abnormalities in the surviving cells, suggesting that events occurring during S-phase arrest contribute to the formation of delayed mitotic damage. Overall, our data demonstrate that, although S-phase arrest serves as a mechanism by which the cells reduce their load of genetic damage, its prolonged activation may also have a negative impact on the balance between cell death and heritable genetic damage.

MTHFR gene variants and non-MALT lymphoma development in primary Sjogren’s syndrome

Primary Sjogren’s syndrome (pSS) confers increased risk for non-Hodgkin lymphoma (NHL) development. Two common polymorphisms, the c. 677C > T and c. 1298A > C, of the methylene-tetrahydrofolate reductase (MTHFR) gene, an enzyme essential in DNA synthesis and methylation, have been associated with susceptibility to NHL. Herein, we tested the hypothesis that MTHFR variants contribute to pSS-related lymphomagenesis. 356 pSS patients, of whom 75 had MALT and 19 non-MALT NHL and 600 healthy controls were genotyped for the detection of MTHFR polymorphisms. DNA methylation levels were assessed by pyrosequencing of the LINE-1 retroelement promoter in DNA from 55 salivary gland tissues from pSS patients. DNA double-strand breaks were determined in peripheral blood mononuclear cells from 13 pSS patients, using comet assay. Αnalysis according to lymphoma subtype revealed increased frequency of c. 677C > T TT genotype and T allele, as well as reduced prevalence of the c. 1298A > C C allele in the pSS non-MALT group compared to controls and patients without NHL. MTHFR c. 677C > T TT genotype was associated with reduced DNA methylation levels, while MTHFR c. 1298A > C AC genotype with reduced DNA double-strand breaks levels. MTHFR variants may be involved in SS non-MALT NHL development, through contribution to defective DNA methylation and genomic instability.

Deficient double strand breaks repair of bone marrow plasma cells correlates with better clinical outcome of multiple myeloma patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA interstrand crosslinks (ICLs) are complex DNA lesions generated by bi-functional alkylating agents, a class of compounds extensively used in cancer chemotherapy. During ICL repair, replication forks stall at the ICL, inducing the formation of a lethal form of DNA damage termed DNA double-strand breaks (DSBs), which are repaired mainly by homologous recombination (HR) and non-homologous end joining (NHEJ). In this study we applied the properties of the ICL-inducing agent melphalan as a model, to elucidate the underlying mechanisms in processing and repair of ICLs. We studied two human multiple myeloma (HMCLs) cell lines (melphalan-sensitive RPMI-8226 and -resistant LR5) and CD138+ bone marrow plasma cells (BMPCs) from 15 newly diagnosed MM patients (8M/7F; median age 61 years) before they receive high-dose melphalan therapy supported by ASCT. HMCLs and BMPCs were treated with melphalan alone or in combination with RI-1 (selective inhibitor of HR) or SCR7 (selective inhibitor of NHEJ) and the extent of the N-ras-specific ICLs using a quantitative PCR assay and DSBs (intermediates of ICL repair) using γH2Ax foci measurements by confocal microscopy were evaluated. The induction of the melphalan-induced apoptosis using a photometric enzyme-assay was also evaluated. The ICLs repair efficiency correlated with response to treatment and could identify 2 groups of patients: non-responders (<PR, n=6), with higher ICLs repair efficiency (t1/2 23h) and responders (≥PR, n=9) with lower efficiency (t1/2 48h). Moreover, using γ-H2AX foci formation/removal measurement, we found that the repair efficiency of DSBs in BMPCs was significantly higher in non-responders (t1/2 9h) than in responders (t1/2 12h) (all p<0.001). Also, the melphalan-induced apoptosis in BMPCs inversely correlated with the repair efficiencies of ICLs and DSBs, with the toxicity being higher in responders than in non-responders. Furthermore, LR5 cells showed higher repair efficiency of both ICLs and DSBs and lower toxicity than RPMI-8226 cells. Interestingly, in all cellular populations analysed, significant correlation between ICL and DSBs levels was observed. To further elucidate the mechanism of drug-induced DSBs repair, HMCLs and BMPCs were treated with melphalan in combination with nontoxic doses of RI-1 or SCR7. We found that the combined treatment of melphalan with RI-1 or SCR7 significantly increased the induction of the melphalan-only phosphorylation of H2AX, delayed the repair of ICLs and strongly enhanced the cytotoxic activity of melphalan (all p<0.01). Collectively, our study demonstrates that significant changes in the repair efficiency of DSBs occur in MM. These changes affect the repair of ICLs, modify drug sensitivity of malignant BMPCs, and correlate with clinical outcome. Specific inhibition of HR and/or NHEJ might be an effective strategy to enhance sensitivity of cancer cells in MM. Citation Format: Maria Gkotzamanidou, Masood Shammas, Evangelos Terpos, Sathees C. Raghavan, Kenneth C. Anderson, Nikhil C. Munshi, Meletios - Athanasios Dimopoulos, Vassilis L. Souliotis. Deficient double strand breaks repair of bone marrow plasma cells correlates with better clinical outcome of multiple myeloma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4762. doi:10.1158/1538-7445.AM2014-4762