Grazia Graziani

Inhibition of poly(ADP-ribose) polymerase prevents irinotecan-induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma

Poly(ADP‐ribose) polymerase (PARP) inhibitors enhance the antitumor activity of the topoisom‐erase I inhibitor irinotecan (CPT‐11), which is used to treat advanced colorectal carcinoma. Since PARP inhibitors sensitize tumor cells also to the methylating agent temozolomide (TMZ) and clinical trials are evaluating CPT‐11 in combination with TMZ, we tested whether the PARP inhibitor GPI 15427 (10‐(4‐methyl‐piperazin‐1‐ylm‐ethyl)‐2H‐7‐oxa‐1,2‐diaza‐benzo[de]anthracen‐3‐one) increases the efficacy of CPT‐11 + TMZ against colon cancer. Moreover, due to the ability of PARP inhibitors to avoid cell death consequent to PARP‐1 overactivation, we evaluated whether oral administration of GPI 15427 provides protection from the dose‐limiting intestinal toxicity of CPT‐11. The results of colony formation assay indicated that GPI 15427 increased the antiproliferative effects (combination index <1) of TMZ + SN‐38 (the active metabolite of CPT‐11) against colon cancer cells. Accordingly, GPI 15427 (40 mg/kg/day×5 days per os) in combination with TMZ (10 mg/kg/day×5 days) + CPT‐11 (4 mg/kg/day×5 days) significantly reduced the growth of tumor xenografts. Oral administration of GPI 15427 (40 mg/kg/q2 × 3 days) prevented intestinal injury and diarrhea induced by CPT‐11 (30 mg/kg/day × 3 days) reducing inflammation and PARP‐1 overactivation, as evidenced by immunohistochemical staining of intestinal tissue with antipoly(ADP‐ribose) antibody(Ab). Inconclusion, the PARP inhibitor represents a novel strategy to enhance the antitumor efficacy and reduce toxicity of chemotherapy in colon cancer.—Tentori, L., Leonetti, C., Scarsella, M., Muzi, A., Mazzon, E., Vergati, M., Forini, O., Lapidus, R., Xu, W., Dorio, A. S., Zhang, J., Cuzzo‐crea, S., Graziani, G. Inhibition of poly(ADP‐ribose) polymerase prevents irinotecan‐induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma. FASEB J. 20, E1024–E1036 (2006)

Ipilimumab: A novel immunostimulatory monoclonal antibody for the treatment of cancer

Ipilimumab (Yervoy, developed by Medarex and Bristol-Myers Squibb) is a fully human monoclonal IgG1κ antibody against the cytotoxic T-lymphocyte antigen-4 (CTLA-4), an immune-inhibitory molecule expressed in activated T cells and in suppressor T regulatory cells. Interaction of the monoclonal antibody with CTLA-4 blocks inhibitory signals generated through this receptor and enhances T cell activation, leading to increased antitumor responses. Ipilimumab has been approved by FDA in March 2011 as monotherapy (3mg/kg every 3 weeks for 4 doses) for the treatment of advanced (unresectable or metastatic) melanoma both in pre-treated or chemotherapy naïve patients. Four months later, ipilimumab has received a rapid approval by the European Commission, after a positive opinion from the Committee for Medicinal Products for Human Use. However, the indication in the EU is limited to previously-treated patients with advanced melanoma. Ipilimumab is the first agent that has demonstrated to improve overall survival in patients with metastatic melanoma, which has a very poor prognosis, in randomized phase III clinical trials. The patterns of tumour response to ipilimumab differ from those observed with cytotoxic chemotherapeutic agents, since patients may have a delayed yet durable response and obtain long-term survival benefit despite an initial tumour growth. The major draw-back of ipilimumab is the induction of immune-related adverse effects; the latter can be life-threatening, unless promptly managed with immunosuppressive agents (most frequently corticosteroids) according to specific guidelines. Further development of ipilimumab includes its use in the neoadjuvant or adjuvant high-risk melanoma setting and for the treatment of other refractory and advanced solid tumours, either as single agent or in combination with additional immunostimulating agents or molecularly targeted therapies.

PARP1 is activated at telomeres upon G4 stabilization: Possible target for telomere-based therapy

New anti-telomere strategies represent important goals for the development of selective cancer therapies. In this study, we reported that uncapped telomeres, resulting from pharmacological stabilization of quadruplex DNA by RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate), trigger specific recruitment and activation of poly-adenosine diphosphate (ADP) ribose polymerase I (PARP1) at the telomeres, forming several ADP-ribose polymers that co-localize with the telomeric repeat binding factor 1 protein and are inhibited by selective PARP(s) inhibitors or PARP1-specific small interfering RNAs. The knockdown of PARP1 prevents repairing of RHPS4-induced telomere DNA breaks, leading to increases in chromosome abnormalities and eventually to the inhibition of tumor cell growth both in vitro and in xenografts. More interestingly, the integration of a TOPO1 inhibitor on the combination treatment proved to have a high therapeutic efficacy ensuing a complete regression of the tumor as well as a significant increase in overall survival and cure of mice even when treatments started at a very late stage of tumor growth. Overall, this work reveals the unexplored link between the PARP1 and G-quadruplex ligands and demonstrates the excellent efficacy of a multi-component strategy based on the use of PARP inhibitors in telomere-based therapy.

Recent Approaches to Improve the Antitumor Efficacy of Temozolomide

Temozolomide (TMZ) is an oral anticancer agent approved for the treatment of newly diagnosed glioblastoma in combination with radiotherapy. Moreover, TMZ has shown comparable efficacy with respect to dacarbazine, the reference drug for metastatic melanoma. Due to its favorable toxicity and pharmacokinetic profile, TMZ is under clinical investigation for brain metastasis from solid tumors and refractory leukemias. TMZ interacts with DNA generating a wide spectrum of methyl adducts mainly represented by N-methylpurines. However, its antitumor activity has been mainly attributed to O(6)-methylguanine, since tumor cell sensitivity inversely correlates with the levels of O(6)-alkylguanine DNA alkyltransferase and requires an intact mismatch repair system. Therefore, an increasing number of studies have been performed in order to identify patients who will benefit from TMZ treatment on the basis of their molecular/genetic profile. Unfortunately, resistance to the methylating agent occurs relatively often and strongly affects the rate and durability of the clinical response in cancer patients. Thus, different approaches have been developed to abrogate resistance or to increase the efficacy of TMZ and for many of them investigation is still underway. Herein, we provide an overview on the recent findings of preclinical and clinical studies on TMZ in combination with inhibitors of DNA repair, chemotherapeutic drugs with different mechanisms of action or radiotherapy, anti-angiogenic agents and other biological modulators.

Poly(ADP-ribose) polymerase inhibitor increases growth inhibition and reduces G2/M cell accumulation induced by temozolomide in malignant glioma cells

Temozolomide (TZM) is a novel methylating agent currently under investigation for treatment of recurrent high‐grade gliomas. Although TZM generates a wide spectrum of methyl adducts, its cytotoxicity has been attributed to mismatch repair (MR)‐mediated processing of O6‐methylguanine:T mispairs. N3‐methyladenine and N7‐methylguanine adducts are promptly repaired by the base excision repair system, unless a poly(ADP‐ribose) polymerase (PARP) inhibitor is combined to TZM. In this case, the repair process of N‐methylpurines cannot be completed and the deriving DNA strand breaks contribute to cytotoxicity. In this study, we investigated the influence on cell growth and cell cycle of treatment with TZM + PARP inhibitor in glioma cells characterized by different susceptibility to TZM. The results indicated that PARP inhibitor increases growth inhibition induced by TZM in either p53–wild‐type or p53‐mutant glioblastoma cells, as early as 24 h after drug exposure. The enhancing effect exerted by PARP inhibitor was particularly evident in glioma cells characterized by a defective expression of MR, since these cells are tolerant to O6‐methylguanine damage and show low sensitivity to TZM. In O6‐alkylguanine‐DNA alkyltransferase (OGAT)‐deficient and MR‐proficient tumor cells bearing wild‐type p53, the drug combination markedly reduced cell accumulation in the G2/M phase of cell cycle and induction of the G2 checkpoint regulator Chk1 kinase. In short‐term cultures of glioma cells derived from surgical specimens, PARP inhibitor enhanced chemosensitivity to TZM and this effect was especially evident in OGAT‐proficient tumors. Thus, a pharmacological strategy based on the interruption of N‐methylpurine repair might represent a novel strategy to restore or increase glioma sensitivity to TZM. GLIA 40:44–54, 2002.

Challenging resistance mechanisms to therapies for metastatic melanoma

Melanoma is the most aggressive form of skin cancer and, if spread outside the epidermis, has a dismal prognosis. Before the approval of the anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) monoclonal antibody ipilimumab and the BRAF inhibitors vemurafenib and dabrafenib, no other agents had demonstrated better results in terms of overall survival than the DNA-methylating compound dacarbazine (or its oral analog temozolomide). However, most patients with metastatic melanoma do not obtain long-lasting clinical benefit from ipilimumab and responses to BRAF inhibitors are short lived. Thus, combination therapies with inhibitors of DNA repair (e.g., poly(ADP-ribose) polymerase [PARP] inhibitors), novel immunomodulators (monoclonal antibodies against programmed death-1 [PD-1] or its ligand PD-L1), targeted therapies (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase [MEK] or phosphatidylinositol 3-kinase [PI3K]/AKT/mammalian target of rapamycin [mTOR] inhibitors) or antiangiogenic agents are currently being investigated to improve the efficacy of antimelanoma therapies. This review discusses the implications of simultaneously targeting key regulators of melanoma cell proliferation/survival and immune responses to counteract resistance.

Treatment with temozolomide and poly(ADP-ribose) polymerase inhibitors induces early apoptosis and increases base excision repair gene transcripts in leukemic cells resistant to triazene compounds.

Methylating triazenes have shown marked antileukemic effects, possibly through generation of a variety of DNA adducts. Cells tolerant to O6-methylguanine due to a defect in the mismatch repair system (MRS), might become sensitive to other methyl adducts, by inhibiting the N-methylpurine repair, which requires base excision repair (BER) and poly(ADP-ribose) polymerase (PADPRP). Therefore, MRS-deficient Jurkat leukemic cells resistant to methylating triazenes, have been treated with temozolomide (TZM) and PADPRP inhibitors. Expression of PADPRP or molecules involved in the BER system [3-methylpurine-DNA glycosylase (MPG) and X-ray repair cross-complementing 1 (XRCC1)], have been explored. Cytotoxic effects of TZM associated with PADPRP inhibitors are evident shortly after treatment, suggesting that completion of cell division is not required for the lethal effect of the drug combination. Increase of PADPRP or MPG transcripts was found after treatment with TZM alone or combined with PADPRP inhibitor. XRCC1 transcript was positively modulated only in the case of drug combination. This could suggest that in the presence of PADPRP inhibitor, persistence of DNA damage triggers XRCC1 transcription. Our results suggest that association of TZM and PADPRP inhibitors might be of benefit for MRS-deficient malignancies unresponsive to the methylating agent.

Pharmacological Strategies to Increase the Antitumor Activity of Methylating Agents

Among methylating agents of clinical interest, temozolomide is a novel antitumor compound that has raised particular interest due to its acceptable safety profile and activity against tumors poorly responsive to conventional chemotherapy, such as malignant glioma and metastatic melanoma. Moreover, the drug has recently shown promising antitumor activity in a patient affected by primary brain lymphoma and is currently under phase II clinical trials for leptomeningeal metastases from leukemia and lymphoma or for brain metastases from lung and breast cancers. The antitumor activity of TMZ, that generates different types of methyl adducts (70% N7-methylguanine, 10% N3-methyladenine and 9% O6-methylguanine), has been mainly attributed to the formation of O6-methylguanine adducts. Indeed, tumor cell susceptibility to TMZ is strongly affected by the functional status of DNA repair systems, involved either in the removal of methyl adducts from O6G or in the apoptotic signaling triggered by O6-methylG:T mispairs. This review will focus on the different pharmacological strategies aimed at overcoming tumor resistance to TMZ such as new formulations of the drug or dosing schedules, and combined treatments with other chemotherapeutic agents, modulators of DNA repair systems, or gene therapy. The potential use of N3-methyladenine selective agents in the case of tumors tolerant to O6-methylguanine will be also discussed.

Evidence that corticotropin-releasing hormone inhibits cell growth of human breast cancer cells via the activation of CRH-R1 receptor subtype

It has been previously shown that corticotropin-releasing hormone (CRH) exerts antiproliferative activity on an estrogen-dependent tumor cell line, i.e. human endometrial adenocarcinoma Ishikawa (IK) cells. Here we have investigated the effects of CRH on another estrogen-dependent tumor cell line, human breast cancer MCF7 cells. In this paradigm, CRH given at a fixed concentration of 100 nM significantly inhibited cell growth induced by 100 nM estradiol (E2) after 48 and 72 h of incubation. This effect was not associated with the induction of apoptosis. CRH inhibition of cell proliferation was counteracted in a concentration-dependent manner by the non-selective CRH receptor antagonist, astressin, as well as by a CRH-R1 selective receptor antagonist, antalarmin. RNase protection assays carried out on MCF7 under basal conditions showed that these cells express in a constitutive manner the CRH-R1 receptor subtype. We have also investigated the putative source of CRH acting on breast cancer cells; we found that MCF7 cells express CRH mRNA under basal conditions and secrete sizable amounts of immunoreactive CRH, which leads to postulate the existence of paracrine-autocrine inhibitory mechanism operated by CRH in breast cancer cells.

Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T-cell leukemia line tolerant to methylating agents.

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6‐alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6‐methylguanine (O6meG) induces cell death in OGAT‐deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T‐cell leukemic Jurkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSα, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15–21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA→TGA). In addition, a (C)8→(C)7 frameshift mutation in codon 1085–1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSα. Genes Chromosomes Cancer 23:159–166, 1998.