Lawrence Panasci

Are frailty markers useful for predicting treatment toxicity and mortality in older newly diagnosed cancer patients? Results from a prospective pilot study

INTRODUCTIONnThe concept of frailty may be useful to characterize vulnerability. The aim of this pilot study was to explore the association between frailty/functional status and treatment toxicity at 3 months and mortality at 6 months.nnnMETHODSnPatients aged ≥65 years referred to the Jewish General Hospital, Montreal, with a new cancer diagnosis. Seven frailty markers and 4 functional status measures were examined. Logistic regression was used to examine the association between frailty/functional status and toxicity, and Cox models for time to death.nnnRESULTSn112 participated, median age 74.1, 31 had toxicity and 15 died. At baseline, 88% had ≥1 frailty marker. Low grip strength predicted toxicity (OR 8.47, 95%CI: 1.3-53.6), ECOG performance status and ADL disability predicted time to death.nnnCONCLUSIONnThe majority had ≥1 frailty marker. Low grip strength predicted toxicity, none of the functional measures did. Further researcher investigating the usefulness of frailty markers is needed.

Dasatinib sensitizes primary chronic lymphocytic leukaemia lymphocytes to chlorambucil and fludarabine in vitro

The dual c‐abl/Src kinase inhibitor, dasatinib, utilized to treat chronic myeloid leukaemia (CML) when used at clinically attainable sublethal concentrations, synergistically sensitized primary chronic lymphocytic leukaemia (CLL) lymphocytes to chlorambucil and fludarabine. In contrast, dasatinib alone demonstrated toxicity to CLL lymphocytes at concentrations that are generally not clinically attainable. Dasatinib resistance and poorer dasatinib‐mediated sensitization to chlorambucil and fludarabine was associated with higher expression of c‐abl protein levels. In contrast, chlorambucil and fludarabine resistance correlated with basal p53 protein levels. Moreover, Western blot analysis after in vitro treatment of primary CLL lymphocytes with dasatinib, chlorambucil and/or fludarabine, showed that dasatinib: (i) inhibited c‐abl function (e.g. downregulation of c‐abl protein levels and decreased the phosphorylation of a c‐abl downstream target, Dok2), (ii) decreased chlorambucil/fludarabine induced accumulation of p53 protein levels, (iii) altered the response to chlorambucil/fludarabine induced DNA‐damage as evidenced by an increase in chlorambucil/fludarabine‐induced H2AX phosphorylation, and (iv) accentuated the c‐abl downregulation induced by chlorambucil/fludarabine. Our results suggest that dasatinib in combination with chlorambucil or fludarabine may improve the therapy of CLL.

DNA repair protein levels vis-a' -vis anticancer drug resistance in the human tumor cell lines of the National Cancer Institute drug screening program

Nucleotide excision repair (NER) is a multi-enzyme DNA repair pathway in eukaryotes. Several NER genes in this pathway including XPB, XPD, XPA and ERCC-1 have been implicated in anticancer drug resistance in human tumor cells. In this study, we assessed the levels of the above-mentioned proteins in the NCI panel of 60 human tumor cell lines in relation to the cytotoxicity patterns of 170 compounds that constitute the standard agent (SA) database. The database consists of drugs used in the clinic for which a mechanism of action has been at least partially defined. The ERCC-1, XPD and XPB protein expression patterns yielded significant negative Pearson correlations with 13, 32 and 17 out of the 170 compounds, respectively (using p <0.05). XPA produced a random assortment of negative and positive correlations, and did not appear to confer an overall resistance or sensitivity to these drugs. Protein expression was also compared with a pre-defined categorization of the standard agents into six mechanism-of-action groups resulting in an inverse association between XPD and alkylating agent sensitivity. Our present data demonstrate that XPD protein levels correlate with resistance to alkylating agents in human tumor cell lines suggesting that XPD is implicated in the development of this resistance. NER activity, using the in vitro cell-free system repair assay, revealed no correlation between NER activity and the level of XPD protein in four cell lines with widely varying XPD protein levels. This lack of correlation may be due to the contribution of XPD to other functions including interactions with the Rad51 repair pathway.

The role of DNA repair in nitrogen mustard drug resistance.

The nitrogen mustards are an important class of DNA cross-linking agents, which are utilized in the treatment of many types of cancer. Unfortunately, resistance often develops in the treatment of patients and the tumor either never responds to or becomes refractory to these agents. Resistance to the nitrogen mustards in murine and human tumor cells has been reported to be secondary to alterations in (i) the transport of these agents, (ii) their reactivity, (iii) apoptosis and (iv) altered DNA repair activity. In the present review, we will discuss the role of DNA repair in nitrogen mustard resistance in cancer. The nitrogen mustards lethality is based on the induction of DNA interstrand cross-links (ICLs). Two DNA repair pathways are known to be involved in removal of ICLs: non-homologous DNA end-joining (NHEJ) and Rad51-related homologous recombinational repair (HRR). The reports discussed here lead us to hypothesize that low NHEJ activity defines a hypersensitive state, while high NHEJ activity, along with increased HRR activity, contributes to the resistant state in chronic lymphocytic leukemia. Studies on human epithelial tumor cell lines suggest that HRR rather than NHEJ plays a role in nitrogen mustard sensitivity.

The phosphatidylinositol-3 kinase I inhibitor BKM120 induces cell death in B-chronic lymphocytic leukemia cells in vitro

BKM120, a pan class I PI3K inhibitor, was cytotoxic in the majority of primary B‐chronic lymphocytic leukemia (CLL) lymphocytes, including samples from patients who have a high‐risk for poor response to treatment (patient with del11 and del17) at clinically obtainable concentrations. The PI3Kδ inhibitor Cal‐101 is cytotoxic in B‐CLL lymphocytes in vitro and is active in the treatment of CLL in vivo. Interestingly, we demonstrated that BKM120 is 3.6 fold more toxic than Cal‐101 in malignant B‐CLL lymphocytes in vitro. BKM120 cytotoxicity correlated with the basal expression of proteins involved in the PI3K/Akt pathway. A protein signature of PI3K pathway proteins predicts the response to BKM120 treatment. In the primary B‐CLL lymphocytes tested in vitro, BKM120 decreased the phosphorylation status of molecular biomarkers used as indicators of PI3K pathway inhibition in vivo. Also, BKM120 induced apoptosis in primary B‐CLL cells culture in the presence and absence of stromal cell support. Our findings suggest that BKM120 should be tested clinically in CLL.

Enhanced host cell reactivation capacity and expression of DNA repair genes in human breast cancer cells resistant to bi-functional alkylating agents

Human breast carcinoma (MCF7-MLNr) cells resistant to the bifunctional drugs L-phenylalanine mustard (L-PAM, 5-fold resistance), mechlorethamine (9-fold), cisplatin (3-fold), and BCNU (3-fold) were used to investigate the role of DNA repair in the development of resistance to alkylating agents. We have previously shown that neither L-PAM transport and metabolism nor glutathione-associated enzymes were altered in MCF7-MLNr cells, compared to the sensitive cells MCF7-WT. This study shows that treatment of pRSV-CAT plasmid with L-PAM at concentrations up to 1 microM proportionally inhibit the expression of chloramphenicol acetyl transferase (CAT) activity, while higher concentrations abolished CAT activity. pRSV-CAT reactivation was significantly increased when plasmid was transfected into MCF7-MLNr cells, compared to MCF7-WT cells. This indicates that resistant cells have more efficient capacity to recognize and repair L-PAM induced DNA damage. The mRNA expression of DNA nucleotide excision repair genes ERCC1, XPD (ERCC2), XPB (ERCC3), and polymerase beta was found to be similar in both the MCF7-WT and MCF7-MLNr cells. Western blot analysis also reveals no difference in the expression of ERCC1, AP endonuclease, poly (ADP-ribose) polymerase, and alkyl-N-purine-DNA glycosylase proteins. The lack of correlation between enhanced host cell reactivation capacity in resistant cells, and the expression of these specific DNA repair genes suggests that proteins encoded by these genes are not rate limiting steps for resistance to bi-functional alkylating drugs in human breast cancer cells.

The PARP inhibitor ABT-888 synergizes irinotecan treatment of colon cancer cell lines.

SummaryPoly [ADP-ribose] polymerase-1 (PARP-1) localizes rapidly to sites of DNA damage and has been associated with various repair mechanisms including base excision repair (BER) and homologous recombination/non-homologous end joining (HRR/NHEJ). PARP-1 acts by adding poly-ADP ribose side chains to target proteins (PARylation) altering molecular interactions and functions. Recently small molecule inhibitors of PARP-1 have been shown to have significant clinical potential and third generation PARP inhibitors are currently being investigated in clinical trials. These drugs alone or in combination with radio/chemotherapy have resulted in meaningful patient responses and an increase in survival in metastatic breast cancer cases bearing BRCA-deficient or triple negative tumors and BRCA-deficient ovarian cancer patients. ABT-888, a potent PARP-1 inhibitor, sensitizes many cancer cells in-vitro and in-vivo to temozolomide. As such, we hypothesized that colon cancers would be sensitized to the DNA damaging chemotherapeutic agents, oxaliplatin and irinotecan, by ABT-888. Using colon cancer cell lines significant synergy was observed between ABT-888 and irinotecan at concentrations of ABT-888 as low as 0.125xa0μM. The level of synergy observed correlated with the degree of PARP1 inhibition as measured biochemically in cell lysates. ABT-888 at concentrations of 0.5–4xa0μM resulted in synergy with oxaliplatin. Furthermore, 24xa0h post treatment combinations of ABT-888/irinotecan generally resulted in increased G2/M cell cycle arrest and increased levels of DNA damage, followed by increased levels of apoptosis 48xa0h post treatment. In conclusion this study suggests that ABT-888 may be a clinically effective adjuvant to current colon cancer therapies that include the use of irinotecan and/or oxaliplatin.

Irinotecan and DNA-PKcs inhibitors synergize in killing of colon cancer cells

SummaryThis study sought to measure the degree of synergy induced by specific small molecule inhibitors of DNA-PK [NU7026 and IC486241 (ICC)], a major component of the non-homologous end-joining (NHEJ) pathway, with SN38 or oxaliplatin. Synergy between the DNA damaging drugs and the DNA-PK inhibitors was assessed using the sulforhodamine-B assay (SRB). Effects of drug combinations on cell cycle and DNA-PK activity were determined using flow cytometry and western blot analysis. DNA damage was assessed via comet assay and quantification of γH2AX. The role of homologous recombination repair (HRR) was determined by nuclear Rad51 protein levels and a GFP reporter recombination assay. Significant reductions in the IC50 values of SN38 were observed at 5 and 10xa0μM of DNA-PK inhibitors. Moreover, at 1–2xa0μM (attainable concentrations with ICC in mice) these DNA-PKcs inhibitors demonstrated synergistic reductions in the IC50 of SN38. Flow cytometric data indicated that SN38 and SN38 in combination with DNA-PKcs inhibitors showed dramatic G2/M arrest at 24xa0h. Furthermore, reduced phosphorylation of DNA-PKcs and increased DNA damage were observed at this time point with SN38 in combination with DNA-PKcs inhibitors as compared to cells treated with SN38 alone. SN38 alone and in the presence of ICC increased nuclear Rad51 protein levels. Furthermore, inhibition of DNA-PKcs increased HRR suggesting that NHEJ is a negative regulator of HRR. These data indicate that small molecule inhibitors of DNA-PKcs dramatically enhance the efficacy of SN38 in colon cancer cell lines.

Effects of the novel DNA dependent protein kinase inhibitor, IC486241, on the DNA damage response to doxorubicin and cisplatin in breast cancer cells

SummaryThe purpose of this study was to determine the degree to which the novel DNA-PKcs inhibitor, IC486241 (ICC), synergizes the cytotoxicity of DNA damaging agents in 3 genetically diverse breast cancer cell lines. The sulforhodamine B (SRB) assay was employed as a primary screening method to determine the in-vitro cytotoxicity and the degree of synergy of ICC in combination with the topoisomerase II inhibitor, doxorubicin, or the DNA cross linking agent, cisplatin. Molecular mechanisms underlying drug toxicity were probed using immunostaining and flow cytometry, as well as, the alkaline comet assay to detect DNA damage. In this study, improved cytotoxicity and significant synergy were observed with both anticancer agents in the presence of nontoxic concentrations of ICC. Moreover, ICC decreased doxorubicin-induced DNA-PKcs autophosphorylation on Ser2056 and increased doxorubicin-induced DNA fragmentation. In conclusion, the novel DNA-PKcs inhibitor, ICC, synergistically sensitized 3 breast cancer cell lines to doxorubicin and cisplatin. Enhanced efficacy of doxorubicin was achieved by inhibiting non-homologous end joining resulting in increased accumulation of DNA damage.

DNA repair in cancer therapy

Clinical Resistance to Alkylators: Status and Perspective Brian Leyland-Jones Role of Nonhomologous End-Joining and Recombinational DNA Repair in Resistance to Nitrogen Mustard and DNA Crosslinking Agents Lawrence C. Panasci, Zhi-Yuan Xu, and Raquel Aloyz Repair of DNA Interstrand Crosslinks Produced by Cancer Chemotherapeutic Drugs Peter J. McHugh and John A. Hartley Chemosensitization to Platinum-Based Anticancer Drugs: Current Trends and Future Prospects Bertrand J. Jean-Claude Regulation of DNA Repair and Apoptosis by p53 and Its Impact on Alkylating Drug Resistance of Tumor Cells Istvan Boldogh, Kishor K. Bhakat, Dora Bocangel, Gokul C. Das, and Sankar Mitra Stress-Activated Signal Transduction Pathways in DNA Damage Response: Implications for Repair, Arrest, and Therapeutic Interventions Moulay A. Alaoui-Jamali, P. James Scrivens, and Martin Loignon Overcoming Resistance to Alkylating Agents by Inhibitors of O6-Alkylguanine-DNA Alkyltransferase Anthony E. Pegg and M. Eileen Dolan Cellular Protection Against the Antitumor Drug Bleomycin Dindial Ramotar, Huijie Wang, and Chaunhua He Potential Role of PARP Inhibitors in Cancer Treatment and Cell Death Michele Rouleau and Guy G. Poirier Relationship Among DNA Repair Genes, Cellular Radiosensitivity, and the Response of Tumors and Normal Tissues to Radiotherapy David Murray and Adrian C. Begg Strand-Break Repair and Radiation Resistance George Shenouda V(D)J Recombination and DNA Double-Strand-Break Repair: From Immune Deficiency to Tumorigenesis Despina Moshous and Jean-Pierre de Villartay Inherited Disorders of Genomic Instability and Cancer Susceptibility Gary M. Kupfer Role of BRCA1 and BRCA2 in DNA Repair and Potential Impact on Therapeutics Razqallah Hakem Genetic Variations in DNARepair: Their Implications in Human Cancer Risk, Prevention, and Therapy Jennifer J. Hu Index