Luca A. Petruccelli

Metformin Reduces Endogenous Reactive Oxygen Species and Associated DNA Damage

Pharmacoepidemiologic studies provide evidence that use of metformin, a drug commonly prescribed for type II diabetes, is associated with a substantial reduction in cancer risk. Experimental models show that metformin inhibits the growth of certain neoplasms by cell autonomous mechanisms such as activation of AMP kinase with secondary inhibition of protein synthesis or by an indirect mechanism involving reduction in gluconeogenesis leading to a decline in insulin levels and reduced proliferation of insulin-responsive cancers. Here, we show that metformin attenuates paraquat-induced elevations in reactive oxygen species (ROS), and related DNA damage and mutations, but has no effect on similar changes induced by H202, indicating a reduction in endogenous ROS production. Importantly, metformin also inhibited Ras-induced ROS production and DNA damage. Our results reveal previously unrecognized inhibitory effects of metformin on ROS production and somatic cell mutation, providing a novel mechanism for the reduction in cancer risk reported to be associated with exposure to this drug. Cancer Prev Res; 5(4); 536–43. ©2012 AACR.

Phosphorylation of eIF4E promotes EMT and metastasis via translational control of SNAIL and MMP-3

The progression of cancers from primary tumors to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Understanding the molecular events which promote metastasis is thus critical in the clinic. Translational control is emerging as an important factor in tumorigenesis. The messenger RNA (mRNA) cap-binding protein eIF4E is an oncoprotein that has an important role in cancer initiation and progression. eIF4E must be phosphorylated to promote tumor development. However, the role of eIF4E phosphorylation in metastasis is not known. Here, we show that mice in which eukaryotic translation initiation factor 4E (eIF4E) cannot be phosphorylated are resistant to lung metastases in a mammary tumor model, and that cells isolated from these mice exhibit impaired invasion. We also demonstrate that transforming growth factor-beta (TGFβ) induces eIF4E phosphorylation to promote the translation of Snail and Mmp-3 mRNAs, and the induction of epithelial-to-mesenchymal transition (EMT). Furthermore, we describe a new model wherein EMT induced by TGFβ requires translational activation via the non-canonical TGFβ signaling branch acting through eIF4E phosphorylation.

Vorinostat Induces Reactive Oxygen Species and DNA Damage in Acute Myeloid Leukemia Cells

Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.

Expression of Leukemia-Associated Fusion Proteins Increases Sensitivity to Histone Deacetylase Inhibitor–Induced DNA Damage and Apoptosis

Histone deacetylase inhibitors (HDI) show activity in a broad range of hematologic and solid malignancies, yet the percentage of patients in any given malignancy who experience a meaningful clinical response remains small. In this study, we sought to investigate HDI efficacy in acute myeloid leukemia (AML) cells expressing leukemia-associated fusion proteins (LAFP). HDIs have been shown to induce apoptosis, in part, through accumulation of DNA damage and inhibition of DNA repair. LAFPs have been correlated with a DNA repair–deficient phenotype, which may make them more sensitive to HDI-induced DNA damage. We found that expression of the LAFPs PLZF-RARα, PML-RARα, and RUNX1-ETO (AML1-ETO) increased sensitivity to DNA damage and apoptosis induced by the HDI vorinostat. The increase in apoptosis correlated with an enhanced downregulation of the prosurvival protein BCL2. Vorinostat also induced expression of the cell-cycle regulators p19INK4D and p21WAF1 and triggered a G2–M cell cycle arrest to a greater extent in LAFP-expressing cells. The combination of LAFP and vorinostat further led to a greater downregulation of several base excision repair (BER) enzymes. These BER genes represent biomarker candidates for response to HDI-induced DNA damage. Notably, repair of vorinostat-induced DNA double-strand breaks was found to be impaired in PLZF-RARα–expressing cells, suggesting a mechanism by which LAFP expression and HDI treatment cooperate to cause an accumulation of damaged DNA. These data support the continued study of HDI-based treatment regimens in LAFP-positive AMLs. Mol Cancer Ther; 12(8); 1591–604. ©2013 AACR.

The novel arsenical Darinaparsin circumvents BRG1-dependent, HO-1-mediated cytoprotection in leukemic cells

Darinaparsin (Dar) is a more potent cytotoxic arsenical than arsenic trioxide (ATO). We hypothesized that the increased cytotoxicity of Dar may be because of a decreased cytoprotective response. We observed that, unlike ATO, Dar does not induce heme oxygenase-1 (HO-1), even though it induces expression of other nuclear factor (erythroid-derived 2)-like 2 (NRF2)-dependent detoxifying enzymes to a greater extent than ATO, in both cancer cell lines and patient-derived leukemic cells. This strengthens the emerging evidence, showing that response to reactive oxygen species (ROS) is stimuli specific. Dar treatment prevents recruitment of the transcriptional coregulator Brahma-related gene 1 (BRG1) to the HMOX1 promoter, which is required for HMOX1 expression. The inability of Dar to induce HO-1 correlates with arrest in G2/M cell cycle phase and BRG1 phosphorylation. Inhibition of HO-1 increases the toxicity of ATO, but has no effect on Dar-induced apoptosis. Accordingly, the lack of HO-1 induction is involved in Dar’s enhanced antileukemic properties. Our data highlight cytoprotective responses mediated by HO-1 and BRG1 as a novel target for enhancing the therapeutic range of arsenicals.

Abstract 288: TGF-beta induced epithelial-to-mesenchymal transition is attenuated when the MNK/eIF4E pathway is functionally impaired.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The epithelial-mesenchymal-like transition (EMT) is a process enabling epithelial cells to gain the motile characteristics of mesenchymal cells, in a manner resembling metastasis. TGF-beta, via well-defined transcriptional mechanisms, is considered a master regulator of EMT. However, the idea that TGF-beta can regulate the translational machinery to drive EMT remains largely unexplored. The eukaryotic translation initiation factor eIF4E is known to be overexpressed in breast cancer, has been linked to increased invasiveness, and is a promising target for the treatment of breast cancer. Our hypothesis is that phosphorylation of eIF4E stimulated by TGF-beta is required for inducing EMT and metastasis in breast cancer. Our novel preliminary data show that TGF-beta can stimulate eIF4E phosphorylation as normal mammary epithelial cells become mesenchymal. Silencing of eIF4E attenuates molecular and behavioral changes associated with EMT. Moreover, decreasing eIF4E levels can impair TGF-beta induced migration and invasion of ErbB2-expressing breast cancer cells. In keeping with a role of phosphorylated eIF4E in driving the metastatic phenotype, we show that chemically and genetically inhibiting the eIF4E kinase MNK1 attenuates TGF-beta-stimulated EMT. We hypothesized that the expression of master regulators of EMT could be restricted when the eIF4E/MNK pathway is functionally impaired, which prompted us to look at the expression of Twist and Snail in TGF-beta treated eIF4E- and MNK- silenced cells. Our data shows that TGF-beta-induced Snail protein expression, but not mRNA level, is repressed when the MNK/eIF4E pathway is functionally impaired. Our results indicate that MNK and eIF4E are essential for EMT and suggest that therapeutic inhibition of MNK/eIF4E pathway may be a useful strategy for the control of tumor invasion and metastasis. Citation Format: Sonia V. del Rincon, Bonnie Huor, Elaine Ngan, Luca Petruccelli, Peter Siegel, Wilson H. Miller, Jr. TGF-beta induced epithelial-to-mesenchymal transition is attenuated when the MNK/eIF4E pathway is functionally impaired. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 288. doi:10.1158/1538-7445.AM2013-288

Abstract 2136: Expression of fusion proteins in acute myeloid leukemia cells increases sensitivity to histone deacetylase inhibitors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Acute myeloid leukemias (AMLs) are often characterized by chromosomal translocations resulting in the expression of fusion proteins (FP). Some FP have been demonstrated to recruit histone deacetylases (HDACs) and repress the expression of DNA repair genes. This may result in an increased sensitivity to histone deacetylase inhibitors (HDI) due to their ability to also down-regulate DNA repair gene expression and induce DNA damage. We have tested the sensitivity of PLZF-RARα, PML-RARα and AML1-ETO inducible cell lines to HDI in order to characterize changes in the mechanisms of cell death. Treatment of U937 cells with vorinostat results in DNA damage as measured by the COMET assay, followed by cell death. To test the effect of FP expression, U937 cells stably transfected with PLZF-RARα, PML-RARα or AML1-ETO cDNA under the control of a tetracycline-off or a zinc-inducible system were treated with vorinostat and assayed for cell death in the presence/absence of FP. FP expression resulted in increased cell death and caspase-3/7 activation. This effect was found to be largely caspase driven as pre-treatment with the pan-caspase inhibitor Z-VAD-FMK resulted in protection against vorinostat-induced cell death. In addition, FP expressing cells were also exposed to the DNA-targeting agents Doxorubicin, Etoposide, Cisplatin and ionizing radiation. Again, FP expression resulted in increased cell death. In addition, we investigated the effect of FP expression on vorinostat sensitivity using a PLZF-RARα murine hematopoietic model. A retroviral expression system was used to overexpress PLZF-RARα in lineage-depleted (Lin-) murine hematopoietic progenitors, followed by vorinostat treatment in the absence/presence of PLZF-RARα. The increased sensitivity of FP expressing cells to HDI and DNA-targeting agents suggests a mechanism where the combination of an FP expression and HDI results in an increased accumulation of DNA damage, leading to enhanced cell death. These findings are significant as they point to FP expressing AMLs as a target group that may respond better to HDI-based therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2136. doi:10.1158/1538-7445.AM2011-2136

Abstract 3449: Tungsten induces DNA damage and alters growth of developing B lymphocytes

Tungsten is used widely in modern life in everything from household goods to technologically advanced material. However, little is known about the consequences of tungsten exposure. The lack of information concerning the toxicities associated with tungsten has been highlighted recently when high tungsten levels were discovered at the sites of several pediatric leukemia clusters. In these clusters, the majority of the children developed acute lymphoblastoid leukemia of the preB lymphocyte subtype. Thus, we investigated whether tungsten exposure alters the growth of preB cells and defined signaling pathways induced by tungsten. We exposed a co-culture system of the BU-11 preB cell line grown on BMS2 stromal cells to tungsten at doses found at the sites of the leukemia clusters. Growth, cell cycle, and apoptosis were determined in both cell types. While no significant changes in any of these parameters were observed in the BMS2 stromal cells, tungsten treatment for 48 hours decreased the BU-11 cell number, which correlated with an accumulation of cells in G0/G1 and increased apoptosis. Unlike polycyclic aromatic hydrocarbons, tungsten induced apoptosis in BU-11 cells grown in rIL-7 and therefore, is independent of the stromal cell layer. These results were confirmed in primary murine bone marrow cultures and in human peripheral blood mononuclear cells. Tungsten can polymerize forming polytungstates in water and soil. Interestingly, these polytungstates are significantly more effective at inhibiting growth in BU-11 and BMS2 cells. In order to begin analysis of signaling pathways induced by tungsten, cDNA microarrays were performed in a leukemic cell line following 24 hours of tungsten treatment and integrated pathway analyses were performed. Genes involved in DNA damage and ER stress were highly upregulated following tungsten exposure and validated by qPCR. An increase in DNA damage was confirmed by COMET assay. These results suggest that the developing B lymphocyte population is sensitive to tungsten-induced toxicities and that tungsten exposure may contribute to leukemogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3449.

Abstract 5449: Histone deacetylase inhibitor treatment in B-cell non-Hodgkin's lymphoma - effect of ex vivo treatment on primary patient samples

The use of histone deacetylase inhibitors (HDACis) is approved in cutaneous T-cell lymphoma, but the efficacy in other lymphomas, particularly B-cell Non-Hodgkin9s lymphoma (B-NHL), remains to be established. To investigate the hypothesis that HDACis have anti-neoplastic effects in B-NHL, we have tested a series of primary B-NHL patient samples for response to the two HDACis Vorinostat (SAHA) and Panobinostat (LBH589). Lymph node biopsies from patients with B-NHL were obtained with patient consent. A single-cell suspension was prepared and mononuclear cells isolated by Ficoll gradient separation. Lymphocytes were treated with clinically achievable doses of SAHA (2.5 μM, 5 μM and 7 μM) and LBH589 (10 nM, 25 nM and 50 nM) ex vivo for 48 hours. Treatment effect was evaluated by assays of cell viability (trypan blue staining and Cell TiterGlo luminescence assay) and cell death (propidium iodide staining analyzed by flow cytometric quantification of the sub-G0 cell population). Expression of acetylated histone H4, Bcl-2 and BCL6 was evaluated by western blot. To date, 16 patient samples, covering a wide variety of B-NHL, have been analyzed. Diagnoses include: follicular lymphoma (n=7), diffuse large B-cell lymphoma (n=5), mantle cell lymphoma (n=1), nodal marginal zone lymphoma (n=1), hairy cell leukemia (n=1) and small lymphocytic lymphoma/chronic lymphocytic leukemia (n=1). Fifteen out of 16 patient samples analyzed showed moderate to high sensitivity to one or both HDACis tested at the given concentrations. Overall, the effects of SAHA and LBH589 were comparable. Western blots of lymphocytes treated with both types of HDACi showed increased acetylation of histone H4 compared to untreated cells. No change in Bcl-2 expression was seen (n=2) and no expression of BCL6 was detected in the samples tested for this (n=2). Based on the data outlined herein, B-NHL is sensitive to the growth inhibitory effects of HDACis at clinically achievable doses. Building upon this pilot study, we are developing cell line models of B-NHL in order to characterize the mechanism of action of HDACis in B-NHL, including combination therapy with currently used agents such as Rituximab. A phase 2 clinical trial of LBH589 in diffuse large B-cell lymphoma, including an arm evaluating combination therapy with Rituximab, is also planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5449.

Abstract LB-100: Optimal modulation of DNA repair in CLL therapy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Resistance to chlorambucil (CLB) in chronic lymphocytic leukemia (CLL) can occur as a consequence of increased DNA repair including c-abl stimulated Rad-51 related homologous recombinational repair (HRR) and DNA-PK related nonhomologous endjoining (NHEJ). Recent reports suggest that the nonreceptor tyrosine kinase c-abl plays an important role in CLL. In particular, we have previously demonstrated that imatinib inhibition of c-abl or NU7026 inhibition of DNA-PK in CLL lymphocytes results in sensitization to CLB in most samples. Here we report that nilotinib, a superpotent (20-30 fold greater than niltinib) inhibitor of c-abl is more efficacious than imatinib in sensitizing CLL lymphocytes to CLB in the majority of the CLL lymphocyte samples associated with a greater nilotinib related inhibition of c-abl autophosporylation, increased apoptosis and decreased repair of CLB-induced DNA damage (increased activated H2AX). Furthermore, in CLL samples in which c-abl was inhibited by either inhibitor, there was an increased activation of DNA-PK. Utilizing NU7026, a specific inhibitor of DNA-PK, with nilotinib or imatinib resulted in further sensitization to CLB but there was a greater sensitization to CLB with nilotinib than imatinib. These results suggest: (1) a more potent inhibition of c-abl is more efficacious in sensitizing CLL lymphocytes to CLB, (2) inhibition of c-abl results in a compensatory increase in DNA-PK and (3) inhibiting both DNA repair systems optimally sensitizes CLL lymphocytes to CLB, an effect which is most pronounced with the more potent c-abl inhibitor, nilotinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-100.