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Udgivelsesdato: 2010-Feb-12 Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy with a variable response to therapy. We have previously shown that DLBCL cell lines differ in their susceptibility to CD40-mediated cell death, and that resistance to CD40-targeted antibodies correlated with increased expression of markers of immature B-cell and absence of Vav-1 mRNA. We used gene expression profiling to investigate the mechanism of CD40 resistance in these cell lines, and found that resistance correlated with lack of Vav-1 and inability to activate NFkappaB upon CD40 ligation. Analysis of tissue microarrays of 213 DLBCL cases revealed that Vav-1 expression correlated with a higher proliferative index and the presence of the post-germinal centre marker Irf-4. Our results suggest that Vav-1 expression may be associated with activated B-cell DLBCL origin and higher proliferative activity, and indicate Vav-1 as a potential marker to identify tumours likely to respond to CD40-targeted therapies. Copyright (c) 2010 John Wiley & Sons, Ltd.

We report that 38% of primary large B-cell lymphoma (DLBCL) tested expressed active Src family kinases, which are targeted by dasatinib. The expression of active Src family of kinases (SFK) in primary DLBCL tumors correlated with unfavorable prognostic markers such as Ki67 and Mum1. Using four DLBCL cell lines we found that: (1) sensitivity to dasatinib (but not imatinib) varied 400-fold; (2) dasatinib resistance was associated with distinct signaling profiles downstream of BCR activation. In particular, although Src family kinase phosphorylation was inhibited by 100-150 nM dasatinib in all cell lines, this failed to inhibit BCR-mediated Blnk phosphorylation, calcium signaling and proliferation in a dasatinib resistant cell line.

Summary 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.

Checkpoint kinase 2 (CHEK2) is a protein involved in arresting cell cycle in response to DNA damage. To investigate whether it plays an important role in the development of prostate cancer (PRCA) in the Ashkenazi Jewish (AJ) population, we sequenced CHEK2 in 75 AJ individuals with prostate, breast, or no cancer (n=25 each). We identified seven coding SNPs (five are novel) that changed the amino-acid sequence, resulting in R3W, E394F, Y424H, S428F, D438Y, P509S, and P509L. We determined the frequency of each variant in 76 AJ families collected by members of the International Consortium for Prostate Cancer Genetics (ICPCG) where >or=2 men were affected by PRCA. Only one variant, Y424H in exon 11, was identified in more than two families. Exon 11 was then screened in nine additional AJ ICPCG families (a total of 85 families). The Y424H variant occurred in nine affected cases from four different families; however, it did not completely segregate with the disease. We performed bioinformatics analysis, which showed that Y424H is a non-conservative missense substitution that falls at a position that is invariant in vertebrate CHEK2 orthologs. Both SIFT and Align-GVGD predict that Y424H is a loss of function mutation. However, the frequency of Y424H was not significantly different between unselected AJ cases from Montreal/Memorial Sloan Kettering Cancer Centre (MSKCC) and AJ controls from Israel/MSKCC (OR 1.18, 95%CI: 0.34-4.61, p=.99). Moreover, functional assays using Saccharomyces cerevisiae revealed that the Y424H substitution did not alter function of CHEK2 protein. Although we cannot rule out a subtle influence of the CHEK2 variants on PRCA risk, these results suggest that germline CHEK2 mutations have a minor role in, if any, PRCA susceptibility in AJ men.

In vertebrate cells, Xrcc3 initiates the repair of exogenous induced-DNA breaks during S and G(2)/M phases of the cell cycle by homologous recombination. However, much less is known of the role of Xrcc3 in the response to spontaneous DNA breaks. Using a siRNA approach, we show that depletion of XRCC3 inhibits the proliferation of MCF7 breast cancer cells. This inhibition of replication coincides with the accumulation of DNA breaks, as shown by the comet assay. Cell cycle specific analysis of gammaH2AX expression shows that S and G2/M phase cells express the highest fraction of gammaH2AX positive cells. This is consistent with replication-dependent accumulation of DNA breaks and deficient homologous recombination. While the induction of gammaH2AX is followed by cell death in parental cells, a p53 knockdown derivative becomes more resistant to XRCC3 depletion-induced death without changes in the levels of gammaH2AX. These results show that XRCC3 is required for the proliferation of MCF7 cells, and that decrease in its expression leads to the accumulation of DNA breaks and the induction of p53-dependent cell death.

Chlorambucil (CLB) treatment is used in chronic lymphocytic leukemia (CLL) but resistance to CLB develops in association with accelerated repair of CLB-induced DNA damage. Phosphorylated histone H2AX (gammaH2AX) is located at DNA double-strand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways. A key component of NHEJ is the DNA-dependent protein kinase (DNA-PK) complex. Increased DNA-PK activity is associated with resistance to CLB in CLL. We used the specific DNA-PK inhibitor 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) to sensitize CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and in primary CLL-lymphocytes, chlorambucil plus NU7026 has synergistic cytotoxic activity at nontoxic doses of NU7026. CLB treatment results in G(2)/M phase arrest, and NU7026 increases this CLB-induced G(2)/M arrest. Moreover, a kinetic time course demonstrates that CLB-induced DNA-PK activity was inhibited by NU7026, providing direct evidence of the ability of NU7026 to inhibit DNA-PK function. DSBs, visualized as gammaH2AX, were enhanced 24 to 48 h after CLB and further increased by CLB plus NU7026, suggesting that the synergy of the combination is mediated by NU7026 inhibition of DNA-PK with subsequent inhibition of DSB repair.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Enhanced DNA damage repair is one mechanism behind colon cancer drug resistance. Thus, targeting molecular components of repair pathways with specific small molecule inhibitors may improve the efficacy of chemotherapy. ABT-888 and VE-821, inhibitors of poly-ADP-ribose-polymerase (PARP) and the serine/threonine-kinase Ataxia telangiectasia related (ATR), respectively, were used to treat colon cancer cell lines in combination with the topoisomerase-I inhibitor irinotecan (SN38). Our findings show that each of these molecules at nontoxic single agent concentrations synergized with SN38 to produce a 2.2 to 3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 (Table 1). When combined, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5 to 27 fold reduction in the IC50 of SN38. Furthermore, the combination of all three agents was associated with maximal G2-M arrest and enhanced DNA-damage (γH2AX). The mechanism of this enhanced synergy was associated with maximal suppression of SN38 induced PARP activity in the presence of both inhibitors. Furthermore, VE-821 enhancement of SN38 induced DNA-PK phosphorylation was abrogated by ABT-888, resulting in more unrepaired DNA damage. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help circumvent resistance to this drug in colon cancer. | Cell Line | VE-821 (μM) | ABT-888 (μM) | SN38 (IC50) ± SE (nM) | I value | P value | |:--------- | ----------- | ------------ | --------------------- | ------- | ------- | | LoVo | | | 13.5±1.2 | | | | | 1 | | 4.5±1.9 | 0.3 | 0.02 | | | 0.5 | | 5.8±2.0 | 0.4 | 0.03 | | | | 0.5 | 9.4±1.0 | 0.7 | 0.06 | | | 1 | 0.5 | 3.0±1.2 | 0.2 | 0.01 | | | 0.5 | 0.5 | 4.7±1.9 | 0.4 | 0.02 | | HCT-116 | | | 8.1±0.7 | | | | | 1 | | 3.5±0.5 | 0.5 | 0.01 | | | 0.5 | | 5.2±0.6 | 0.7 | 0.03 | | | | 0.5 | 3.9±1.1 | 0.5 | 0.03 | | | 1 | 0.5 | 0.3±0.03 | 0.1 | 0.01 | | | 0.5 | 0.5 | 2.1±0.4 | 0.3 | 0.01 | | HT-29 | | | 20.5±1.8 | | | | | 1 | | 9.3±0.6 | 0.6 | 0.01 | | | 0.5 | | 12.2±1.3 | 0.7 | 0.02 | | | | 0.5 | 12.9±0.7 | 0.6 | 0.02 | | | 1 | 0.5 | 3.9±0.5 | 0.3 | 0.01 | | | 0.5 | 0.5 | 6.1±0.2 | 0.4 | 0.01 | Table 1 Colon cancer cell lines LoVo, HCT-116 and HT-29 were treated with SN38 alone or in combination with various concentrations of the ATR inhibitor VE-821 and/or the PARP inhibitor ABT-888. The IC50 values of SN38 were significantly reduced in the presence of VE-821 and/or ABT-888 for all cell lines. Drug synergy (I) values were determined, according to Berenbaum (1978), by calculating the ratio: (IC50 SN38 with inhibitors/IC50 SN38 alone) + (inhibitor concentration used/IC50 inhibitor alone); I less than 1 = synergism, I equal to 1 = additive effect and I greater than 1 = antogonism. Citation Format: David Davidson, Atlal Abu-Sanad, Yunzhe Wang, Fatemeh Hasheminasab, Justin Panasci, Raquel Aloyz, Lawrence Panasci. Simultaneous inhibition of ATR and PARP greatly sensitizes colon cancer cell lines to irinotecan. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5327. doi:10.1158/1538-7445.AM2015-5327

Treatments for hematological malignancies have improved considerably over the past decade, but the growing therapeutic arsenal has not benefited adult T-cell leukemia (ATL) patients. Oncolytic viruses such as vesicular stomatitis virus (VSV) have recently emerged as a potential treatment of solid tumors and leukemias in vitro and in vivo. In the current study, we investigated the ability of VSV to lyse primary human T-lymphotropic virus type 1 (HTLV-1)-infected T-lymphocytes from patients with ATL. Ex vivo primary ATL cells were permissive for VSV and underwent rapid oncolysis in a time-dependent manner. Importantly, VSV infection showed neither viral replication nor oncolysis in HTLV-1-infected, nonleukemic cells from patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and in naive CD4(+) T-lymphocytes from normal individuals or in ex vivo cell samples from patients with chronic lymphocytic leukemia (CLL). Interestingly, activation of primary CD4(+) T-lymphocytes with anti-CD3/CD28 monoclonal antibody, and specifically with anti-CD3, was sufficient to induce limited viral replication and oncolysis. However, at a similar level of T-cell activation, VSV replication was increased fourfold in ATL cells compared to activated CD4(+) T-lymphocytes, emphasizing the concept that VSV targets genetic defects unique to tumor cells to facilitate its replication. In conclusion, our findings provide the first essential information for the development of a VSV-based treatment for ATL.

Eukaryotic cells respond to DNA damage by activation of DNA repair, cell cycle arrest, and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signaling other cellular responses. The Rad51-guided homologous recombination repair system plays an important role in the recognition and repair of DNA interstrand crosslinks (ICLs), and cells deficient in this repair pathway become hypersensitive to ICL-inducing agents such as cisplatin and melphalan. We investigated the possible role of the Rad51-paralog protein Xrcc3 in drug resistance. Xrcc3 overexpression in MCF-7 cells resulted in 1) a 2- to 6-fold resistance to cisplatin/melphalan, 2) a 2-fold increase in drug-induced Rad51 foci, 3) an increased cisplatin-induced S-phase arrest, 4) decreased cisplatin-induced apoptosis, and 5) increased cisplatin-induced DNA synthesis arrest. Interestingly, Xrcc3 overexpression did not alter the doubling time or cell cycle progression in the absence of DNA damage. Furthermore, Xrcc3 overexpression is associated with increased Rad51C protein levels consistent with the known interaction of these two proteins. Our results demonstrate that Xrcc3 is an important factor in DNA cross-linking drug resistance in human tumor cells and suggest that the response of the homologous recombinational repair machinery and cell cycle checkpoints to DNA cross-linking agents is intertwined.

We have recently completed screening of the National Cancer Institute human tumor cell line panel and demonstrated that among four nucleotide excision repair proteins (XPA, XPB, XPD, and ERCC1), only the TFIIH subunit XPD endogenous protein levels correlate with alkylating agent drug resistance. In the present study, we extended this work by investigating the biological consequences of XPD overexpression in the human glioma cell line SK-MG-4. Our results indicate that XPD overexpression in SK-MG-4 cells leads to cisplatin resistance without affecting the nucleotide excision repair activity or UV light sensitivity of the cell. In contrast, in SK-MG-4 cells treated with cisplatin, XPD overexpression leads to increased Rad51-related homologous recombinational repair, increased sister chromatid exchanges, and accelerated interstrand cross-link removal. Moreover, we present biochemical evidence of an XPD-Rad51 protein interaction, which is modulated by DNA damage. To our knowledge, this is the first description of functional cross-talk between XPD and Rad51, which leads to bifunctional alkylating agent drug resistance and accelerated removal of interstrand cross-links.

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.

Developmental sympathetic neuron death is determined by functional interactions between the TrkA/NGF receptor and the p75 neurotrophin receptor (p75NTR). A key question is whether p75NTR promotes apoptosis by directly inhibiting or modulating TrkA activity, or by stimulating cell death independently of TrkA. Here we provide evidence for the latter model. Specifically, experiments presented here demonstrate that the presence or absence of p75NTR does not alter Trk activity or NGF- and NT-3–mediated downstream survival signaling in primary neurons. Crosses of p75NTR−/− and TrkA−/− mice indicate that the coincident absence of p75NTR substantially rescues TrkA−/− sympathetic neurons from developmental death in vivo. Thus, p75NTR induces death regardless of the presence or absence of TrkA expression. These data therefore support a model where developing sympathetic neurons are “destined to die” by an ongoing p75NTR-mediated apoptotic signal, and one of the major ways that TrkA promotes neuronal survival is by silencing this ongoing death signal.

BACKGROUND The generation of DNA interstrand cross-links is thought to be important in the cytotoxicity of nitrogen mustard alkylating agents, such as melphalan, which have antitumor activity. Cell lines with mutations in recombinational repair pathways are hypersensitive to nitrogen mustards. Thus, resistance to melphalan may require accelerated DNA repair by either recombinational repair mechanisms involving Rad51-related proteins (including x-ray repair cross-complementing proteins Xrcc2, Xrcc3, and Rad52) or by nonhomologous endjoining involving DNA-dependent protein kinase (DNA-PK) and Ku proteins. We investigated the role of DNA repair in melphalan resistance in epithelial tumor cell lines. METHODS Melphalan cytotoxicity was determined in 14 epithelial tumor cell lines by use of the sulforhodamine assay. Homologous recombinational repair involving Rad51-related proteins was investigated by determining the levels of Rad51, Rad52, and Xrcc3 proteins and the density of nuclear melphalan-induced Rad51 foci, which represent sites of homologous recombinational repair. Nonhomologous endjoining was investigated by determining the levels of Ku70 and Ku86 proteins and DNA-PK activity. Linear regression analysis was used to analyze correlations between the various protein levels, DNA-PK activity, or Rad51 foci formation and melphalan cytotoxicity. All statistical tests were two-sided. RESULTS Melphalan resistance was correlated with Xrcc3 levels (r =.587; P =.027) and the density of melphalan-induced Rad51 foci (r =.848; P =.008). We found no correlation between melphalan resistance and Rad51, Rad52, or Ku protein levels or DNA-PK activity. CONCLUSION Correlations of melphalan resistance in epithelial tumor cell lines with Xrcc3 protein levels and melphalan-induced Rad51 foci density suggest that homologous recombinational repair is involved in resistance to this nitrogen mustard.

The retinoblastoma tumor suppressor protein (pRb) family is essential for cortical progenitors to exit the cell cycle and survive. In this report, we test the hypothesis that pRb collaborates with basic helix-loop-helix (bHLH) transcription factors to regulate cortical neurogenesis, taking advantage of the naturally occurring dominant-inhibitory HLH protein Id2. Overexpression of Id2 in cortical progenitors completely inhibited the induction of neuron-specific genes and led to apoptosis, presumably as a consequence of conflicting differentiation signals. Both of these phenotypes were rescued by coexpression of a constitutively activated pRb mutant. In contrast, Id2 overexpression in postmitotic cortical neurons affected neither neuronal gene expression nor survival. Thus, pRb collaborates with HLHs to ensure the coordinate induction of terminal mitosis and neuronal gene expression as cortical progenitors become neurons.

In this report, we examine how the Ras protein regulates neuronal survival, focusing on sympathetic neurons. Adenovirus-expressed constitutively activated Ras (RasV12) enhanced survival and the phosphorylation of Akt (protein kinase B) and MAP kinase (MAPK), two targets of Ras activity. Functional inhibition of endogenous Ras by adenovirus-expressed dominant-inhibitory Ras (N17Ras) decreased nerve growth factor (NGF)-dependent survival and both Akt and MAPK phosphorylation as well. To determine the signaling pathways through which Ras mediates survival, we used Ras effector mutants and pharmacological inhibitors that selectively suppress phosphatidylinositol 3-kinase (PI3-K)/Akt or MAP kinase kinase (MEK)/MAPK pathways. The Ras effector mutant RasV12Y40C, which selectively stimulates PI3-K and Akt, rescued survival in the absence of NGF, and the PI3-K inhibitor LY 294002 inhibited both Ras- and NGF-dependent survival. RasV12T35S, which activates MEK/MAPK but not PI3-K/Akt, was less effective at rescuing survival, whereas the MEK inhibitor PD 098059 also partially suppressed Ras-dependent survival. To investigate the mechanisms by which Ras suppresses neuronal death, we examined whether Ras functions by inhibiting the proapoptotic p53 pathway (Jun-N-terminal kinase/p53/BAX) that is necessary for neuronal death after NGF withdrawal and p75NTR activation. We found that RasV12 suppressed c-jun, BAX, and p53 levels, whereas inhibition of NGF-induced Ras-survival activity via N17Ras increased the levels of these proteins. Furthermore, the E1B55K protein, which suppresses p53 activity, blocked N17Ras-induced neuronal death. Together, these results indicate that Ras is, in part, both necessary and sufficient for survival of sympathetic neurons and that this effect is mediated by activation of both the PI3-K– and MEK-signaling cascades, which in turn suppress a proapoptotic p53 pathway.

We have asked whether p75NTRmay play a role in neuronal apoptosis by producing transgenic mice that express the p75NTRintracellular domain within peripheral and central neurons. These animals showed profound reductions in numbers of sympathetic and peripheral sensory neurons as well as cell loss in the neocortex, where there is normally little or no p75NTRexpression. Developmental loss of facial motor neurons was not observed, but induced expression of the p75NTRintracellular domain within adult animals led to increased motor neuron death after axotomy. Biochemical analyses suggest that these effects were not attributable to a p75NTR-dependent reduction in trk activation but instead indicate that the p75NTRintracellular domain may act as a constitutive activator of signaling cascades that regulate apoptosis in both peripheral and central neurons.

The mRNA encoding brain-derived neurotrophic factor (BDNF) is widely distributed in central nervous system neurons, including in hippocampus and cortex. However, little is known about the physiology of BDNF protein within neurons, including how it is processed or packaged and the mechanisms that control its release. In this study, we have used antibodies to monitor the subcellular distribution of BDNF in cortical extracts from adult rats treated with kainic acid. BDNF immunoreactivity is elevated in rat cortex 12 h after kainic acid treatment. The protein is enriched in a vesicular fraction isolated from lysed synaptosomes, its distribution being similar to that of synaptotagmin, which is associated with synaptic vesicles and large dense core vesicles at nerve terminals. The vesicular pool of BDNF is digested by proteinase K only in the presence of Triton X-100 suggesting localization of BDNF in membrane fractions. Immunocytochemistry detects diffuse and punctate BDNF staining within cell bodies and processes of cortical neurons from kainic acid-treated rats, as well as in mossy fiber terminals of rat hippocampus. Taken together, these data show that BDNF can accumulate axonally within a vesicular compartment of brain neurons. Results support the idea that endogenous BDNF may be transported anterogradely and released by regulated secretory mechanisms.

Reply to ‘Imatinib induces apoptosis in CLL lymphocytes with high expression of Par-4’ by Chow et al

Eukaryotic translation initiation factor 4E (eIF4E) is a rate-limiting factor for cap-dependent protein synthesis regulated by the PI3K/AKT/mTOR signaling pathway as well as MNK1/2-mediated phosphorylation.1, 2 In addition to its cytoplasmic functions in translation, nuclear eIF4E aids in the cytoplasmic export of specific mRNAs.1, 3 eIF4E is overexpressed in many cancers and has been reported to have important roles in the development and progression of hematological malignancies in animal models.2 However, the role of eIF4E in drug resistance in primary human cancer cells is less well documented.4, 5, 6 In this study, we sought to assess the contribution of eIF4E to fludarabine (FLU) resistance in primary chronic lymphocytic leukemia (CLL) lymphocytes as this nucleoside analog is used as a first-line treatment for the disease.7 To this end, we used a panel of primary CLL samples from 26 affected patients (Supplementary Table). To interfere with eIF4E function, we used Ribavirin, a well-characterized antiviral drug that has also been shown to target eIF4E in a variety of systems, including patients with acute myeloid leukemia (AML).6, 8, 9 A clinically achievable concentration of Ribavirin (10 μM), which was not cytotoxic to primary CLL lymphocytes in culture, significantly sensitized 76% of the samples tested to FLU with the sensitization index (R) ranging from 1.25 to eightfold (Figures 1a, P

Introduction: The understanding of DNA repairs pathways and their interactions allowed for the development of several targeted therapies, which sensitize neoplastic cells to the effects of DNA damaging chemotherapy. Poly-ADP ribose polymerase (PARP) inhibitors can increase the cytotoxicity of several anticancer agents by inhibiting various DNA repair pathways including Rad51 related homologous recombinational repair (HRR). However, inhibition of PARP may result in compensatory activation of the ATR pathway partially limiting the sensitization of chemotherapeutic agents seen with PARP inhibitors. Recently, a specific ATR inhibitor, VE-821 has demonstrated excellent sensitization to various chemotherapeutic agents with preferential antitumor activity in tumor cells as compared to normal cells (Reaper PM et al Nature Chem Biol 7: 428-30, 2011) Colon cancer cell lines: HCT116/Lovo (p53 wild type) and HT29 (p53 mutated) were treated with SN38, the PARP inhibitor (ABT-888) and/or the ATR inhibitor (VE821) at different concentrations. The SRB cytotoxicity assay was used to determine the IC50 of each drug separately and in combination. DNA damage caused by these agents was quantified by phosphorylated-H2AX. Cell cycle alterations and protein levels (western analysis) were determined after drug treatment. Results: Nontoxic concentrations (0.5-1 μM) of ABT-888 or VE-821 demonstrated a two to three fold decrease in the IC50 of SN38 in the colon cancer cell lines. Significantly, the combination of both inhibitors at the same nontoxic concentrations resulted in a dramatic 4 to 25-fold decrease in the IC50 of SN38 [HCT116 (8.1 nM to 0.3 nM), HT29 (20.5 nM to 3.9 nM) and Lovo (13.5nM to 3 nM)]. Notably, the observed effect was dose-dependent. In the Lovo cell line, the combination of SN38/VE821/ABT888 increased G2/M cell cycle arrest compared to SN38/VE821, SN38/ABT888 and SN38 alone. Similarly, increased phosphorylated-H2AX and apoptosis were seen with SN38/VE821/ABT888 in the Lovo and HCT-116 cell lines. Preliminary data utilizing the annexin-5 apoptosis assay in the HCT116 cell line supports the increased cell killing by the combination of all three agents. In the HCT116 cell line, treated with the SN38/VE821/ABT888 combination as compared to cells treated with SN38 alone, increased expression of p21 along with decreased expression of phospho-Chk1 was observed. Conclusion: The SN38/ABT888/VE821 combination resulted in maximal synergy/chemosensitivity in all colon cancer cell lines, associated with increased DNA damage and apoptosis. Our data suggests that this combination may effectively overcome colon cancer resistance to irinotecan-based chemotherapy. Citation Format: Atlal Abu-Sanad, David Davidson, Yunzhe Wang, John Pollard, Raquel Aloyz, Lawrence Panasci. ATR and PARP inhibition enhances topoisomerase I-dependent DNA damage in colon cancer cell lines. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B13.

Introduction Personalized medicine in oncology relies on translational research efforts to identify biomarkers that will influence clinical management. This is a concerted effort requiring an organizational framework that is often underestimated. The Quebec Clinical Research Organization in Cancer (Q-CROC) consortium is a multi-disciplinary and multi-institutional group of scientists and clinicians devoted to integrating and enhancing translational and clinical research capacity in Quebec. We describe here the organizational framework driving a multicenter, prospective study to identify biomarkers of clinical resistance to first-line therapy in metastatic colorectal cancer (NCT00984048, Q-CROC-01). Results The Q-CROC consortium has put in place an organizational infrastructure to support the activities and operations of its translational projects. We identified and addressed several critical issues during the course of the Q-CROC-01 translational project that were also common to our subsequent biomarker-driven trial in lymphoma (Q-CROC-02, NCT01238692) and breast cancer (Q-CROC-03, NCT01276899). Examples of these issues include: (i) feasibility and burden of tissue collection at participating sites, (ii) limiting pre-analytical variability in blood and tissue specimens for functional downstream applications, (iii) verification of tumor content on biopsy specimens, (iv) tracking sample flow, (v) integration of clinical data with discovery platforms, and (vi) engaging participation throughout all steps of the project. A critical element in these projects was a scientific project management team to ensure that objectives were aligned and deliverables were met. This academic framework for translational research may be comparable to that of multicenter clinical trials undertaken by industry, but some challenges, including financial and time constraints, data sharing and IP agreements, and engagement of its members, may be more palpable in the academic setting. Conclusion Infrastructure science is underestimated and under-reported in translational cancer research and is crucial to the success of any large-scale biomarker discovery effort. Our experience with three multi-institutional biomarker-driven trials is that progress hinges upon the availability of an infrastructure that provides a concrete link between each component. The Q-CROC-01 project is funded by a Pfizer-FRSQ Innovation Fund award and by Sanofi-Aventis. Q-CROC-02 is funded by Novartis and Roche, and Q-CROC-03 is funded by a Genome Quebec grant.

Introduction: Personalized medicine (PM) is a concept that has raised high expectations amongst scientists, clinicians, and patients. An emerging approach is to examine tumor biopsy material for genomic changes that are known targets of currently available therapeutic agents, with the assumption that a clinical benefit will be observed if the target is inhibited. While striking anecdotal reports are predictable from this approach, the clinical impact of these agents is limited by the inevitable development of therapeutic resistance. Our focus is on the design of parallel research programs using both in vitro and in vivo strategies, in an effort to delay or inhibit resistance. We present here preliminary data for a signature of resistance to standard first-line treatment - fluorouracil, folinic acid, oxaliplatin and bevacizumab (FOLFOX/B) using cell line models of resistance to this regimen. In parallel, we are conducting a prospective study to identify biomarkers of clinical resistance to first-line therapy in patients with metastatic colorectal cancer (CRC) (NCT00984048). Methods: Ten established CRC cell lines were treated with FOLFOX/B and categorized as resistant or sensitive based on IC50 values. In parallel, patients who consented to an initial biopsy and one at disease progression following an initial response were identified as intrinsically resistant or as having acquired resistance during treatment. CRC cell lines that were initially sensitive were rendered resistant to mimic the acquired resistance in patients, by serial passages with gradual increases in concentration of the combination regimen. We compared microarray data from three sensitive and three resistant cell lines. Results: We found a different expression pattern from microarray data comparing sensitive and resistant cell lines, thereby indicating a potential signature of resistance to FOLFOX/B. Interestingly, we found that the Src family kinase Lyn was overexpressed in resistant cells lines. Treating cells with non-cytotoxic concentration of dasatinib, a dual Src family kinase and Abl inhibitor, sensitized both the parental sensitive cells and the cells with acquired resistance to FOLFOX/B, thereby suggesting that combination treatment with dasatinib may be effective in delaying or inhibiting resistance. We have thus far collected needle core biopsies from liver metastases from forty patients who agreed to partake in this multi-center trial. Eligible patients have confirmed metastatic CRC, measurable disease, and consent to three needle-core biopsies (NCBs) of a non-resectable liver metastasis before treatment and at resistance, as well as serial blood collection throughout the study. Using standard operating procedures developed for this trial, we were able to both preserve morphology and obtain high-quality genomic material from biopsy tissue. We will determine if the resistance signature and overexpression of Lyn observed in the resistant CRC cell lines are similarly demonstrated in patients that were intrinsically resistant to FOLFOX/B. Conclusions: We have designed parallel in vitro and in vivo experiments to study resistance to standard first-line treatment for mCRC. These studies provide insight on metastatic signatures of resistance and suggest combination therapies to delay or inhibit therapeutic resistance in patients.

Numerous studies have associated poly [ADP-ribosolose] polymerase 1 (PARP1) with DNA-damage repair. PARP1 rapidly localizes to DNA damage sites where it alters target proteins, (e. g. histones, topoisomerase I and DNA dependent protein kinase (DNA-PK)), through the addition of poly-ADP ribose side chains. By this mechanism, PARP1 plays a role in diverse repair pathways including homologous recombination/non-homologous end joining (HRR/NHEJ). Presently, third generation PARP inhibitors are in use clinically. These drugs have resulted in meaningful clinical responses and an increase in survival in metastatic breast and ovarian cancer patients bearing BRCA-deficient or triple negative tumors. The Abbott lead compound, ABT-888, is a potent PARP1 inhibitor that sensitizes many cancer cells in-vitro and in-vivo to temozolomide. In the present work, we hypothesized that colon cancers would be sensitized to the DNA damaging chemotherapeutic agent, irinotecan by ABT-888. Since PARP1 is involved in DNA repair, we propose that its inhibition would increase irinotecan-induced DNA damage and cell death in colon cancer cells lines. Using the sulforhodamine B assay (SRB), significant synergy (I Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4692. doi:1538-7445.AM2012-4692

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: The success of personalized medicine in oncology relies on translational research efforts to identify biomarkers that will influence clinical management. The discovery and validation of biomarkers is a concerted effort requiring an organizational framework that is often underestimated. The Quebec Clinical Research Organization in Cancer (Q-CROC) consortium is a multi-disciplinary and multi-institutional group of scientists and clinicians devoted to integrating and enhancing translational and clinical research capacity in Quebec. We describe here the organizational framework driving a multicenter, prospective study to identify biomarkers of clinical resistance to first-line therapy in metastatic colorectal cancer ([NCT00984048][1], Q-CROC-01). Results: The Q-CROC consortium has put in place an organizational infrastructure to support the activities and operations of its translational projects. We identified and addressed several critical issues during the course of the Q-CROC-01 translational project that were also common to our subsequent biomarker-driven trial in lymphoma (Q-CROC-02, [NCT01238692][2]) and breast cancer (Q-CROC-03, [NCT01276899][3]). Examples of these issues include: (i) feasibility and burden of tissue collection at participating sites, (ii) limiting pre-analytical variability in blood and tissue specimens for functional downstream applications, (iii) verification of tumor content on biopsy specimens, (iv) tracking sample flow, (v) integration of clinical data with discovery platforms, and (vi) engaging participation throughout all steps of the project. In part to address the above issues, we established five operational Cores: clinical, biobank, biospecimen processing, bioanalytical and bioinformatic. A further challenge was the integration between these Cores, who for the most part operated in silos. We observed that a critical element to unify all components of the consortium was a scientific project management team, consisting of dedicated individuals regularly interacting with each Core to ensure that objectives were aligned and deliverables were met. This academic framework for translational research may be comparable to that of multicenter clinical trials undertaken by industry, but some challenges, including financial and time constraints, data sharing and IP agreements, and engagement of its members, may be more palpable in the academic setting. Conclusion: Infrastructure science is underestimated and under-reported in translational cancer research and is crucial to the success of any large-scale biomarker discovery effort. Our experience with three multi-institutional biomarker-driven trials is that progress hinges upon the availability of an infrastructure that is not only the sum of its parts but that provides a concrete link between each component. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5534. doi:1538-7445.AM2012-5534 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00984048&atom=%2Fcanres%2F72%2F8_Supplement%2F5534.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01238692&atom=%2Fcanres%2F72%2F8_Supplement%2F5534.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01276899&atom=%2Fcanres%2F72%2F8_Supplement%2F5534.atom

e11055 Background: Most newly diagnosed patients with invasive breast cancer have node negative disease. A key question in management of these patients is the risk/benefit ratio of adjuvant chemotherapy. Several microarray tests have been developed and are currently used to help identifying the patients who will benefit from adjuvant chemotherapy. Ki 67 proliferation index is less expensive and has been used as a prognostic and predictive factor in node positive breast cancer and neoadjuvant setting. Methods: Forty five cases of T1-2 N0 M0 (ER positive, HER2/neu negative) breast cancer were reviewed. Oncotype Dx results were available for all patients. Tumor specimens were stained for Ki 67. Results: The median of Ki 67 index was 17 (range 2-90). The median Oncotype recurrence score was 17 (range 7-60). There was a strong linear correlation between Ki67 index and Oncotype recurrence score (correlation coefficient= 0.74, P value= 0.000). This correlation was stronger in tumors with Ki 67 proliferation inde...

1093 Background: Sorafenib is an oral inhibitor of Raf and VEGFR/PDGFR. In preclinical models, favorable effects were noted combining sorafenib and vinorelbine. Both vinorelbine and sorafenib undergo hepatic metabolism. We aimed to determine the recommended doses in a phase 1b study. Methods: Patients were eligible with HER2-negative metastatic breast cancer, measurable (RECIST), not previously treated with chemotherapy for advanced stage; ECOG PS 0-1, adequate renal, liver and bone marrow functions, and LVEF ≥50%. Treatment was in two subsequent cohorts of 6 patients each: vinorelbine (30 mg/m2 days 1, 8 every 21) + sorafenib 200 mg BID continuously (cohort 1); vinorelbine (same as cohort 1) + sorafenib 400 mg BID continuously (cohort 2). Patients still benefiting after 8 cycles of treatment were allowed to continue on single-agent sorafenib. Results: Six patients in each cohort (n=12) were evaluated. Patients received a median of 8 cycles of treatment. Dose-limiting toxicities with the first cycle inclu...

B- Cell chronic leukemia (CLL) is the most common leukemia in adults. It is clinically very heterogeneous and has a highly variable clinical course. Telomere length and telomerase activity have been shown to be excellent prognostic factors in CLL. Moreover, telomere maintenance is deficient in CLL lymphocytes. These studies have prompted the assessment of the telomerase inhibitor GRN163L in a phase I-II clinical trial in CLL and lymphomas. Here we report that telomerase activity is not required for the survival of quiescent primary CLL lymphocytes. In contrast telomerase activity seems to be required for the survival of quiescent primary CLL lymphocytes after treatment with fludarabine (FLU) in vitro. Furthermore, the effect of fludarabine on telomerase activity was associated with the basal expression of the telomere shelterin protein TRF2. In summary, our results suggest that GRN163L in combination with FLU may be useful to decrease the tumor burden in CLL. To our knowledge this is the first report assessing the effect of telomerase inhibition in combination with a chemotherapeutic agent in non-stimulated primary CLL lymphocytes. 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 3207.

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.

16513 Background: Chronic lymphocytic leukemia (CLL) is an indolent leukemia in which there is an accumulation of immature malignant B-lymphocytes. While treatment with chlorambucil, a nitrogen mustard analogue or fludarabine can control the disease, eventually all patients become resistant to chlorambucil/fludarabine. Chlorambucil’s cytotoxicity is mediated by the interstrand crosslinks (ICLs) introduced into DNA. In mammalian cells, two main pathways are known to be involved in double strand break(DSB) repair: homologous recombinational repair (HRR) and nonhomologous endjoining (NHEJ). Methods: Our laboratory has demonstrated that resistance to chlorambucil involved accelerated repair of the ICLs associated with enhanced DNA repair, specifically enhanced nonhomologous endjoining (NHEJ) and homologous recombinational repair (HRR). We also recently demonstrated that gleevec inhibition of c-abl with resulting inhibition of Rad51-related HRR sensitizes CLL lymphocytes in vitro to chlorambucil. This exciting result has stimulated the development of a clinical protocol to test this combination in CLL. Furthermore, our laboratory has demonstrated that wortmannin, a nonspecific inhibitor of DNA-PK (a key component of NHEJ) sensitizes CLL lymphocytes to chlorambucil. Results: We are actually evaluating the effect of NU7026, a relatively specific DNA-PK inhibitor, in the sensitivity of CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and primary CLL-lymphocytes chlorambucil plus NU7026 have a synergistic cytotoxic effect. Noteworthy, the NU7026 doses used in combination with chlorambucil were not toxic to the cells when used alone. Moreover, chlorambucil treatment induced DNA-PK nuclear foci which were inhibited by NU7026 suggesting that the synergy of both drugs is mediated by Nu7026-inhibition of DNA-PK. Conclusion: NU7026 plus chlorambucil should be a useful combination to treat CLL. No significant financial relationships to disclose.

The association of endogenous synenkephalin and met-enkephalin containing peptides with the membrane of bovine chromaffin granules and physicochemical characteristics of this association were studied. The associated materials were only released at a non physiological pH range and this effect was enhanced with growing salt concentrations (0.5, 1.0 and 2.0 M KSCN). A higher peptide dissociation occurred with membrane solubilizing agents (SDS greater than Triton X-100 greater than digitonin). In microsomes the materials dissociated with 2 M KSCN (pH 7.4) corresponded to peptides larger than 12.0 kDa, while in granules corresponded to molecules smaller than 8.5 kDa, displaying synenkephalin and met-enkephalin immunoreactivities. These data suggest that some sequence of the C-terminal portion of synenkephalin may be responsible for the association of proenkephalin derived peptides with microsome and granule membranes.

Adrenal enkephalin and enkephalin-containing peptides were studied during postnatal development in normotensive (WKY) and spontaneously hypertensive rats (SHR). The effect of chronic treatment with the ganglionic blocker chlorisondamine (5 mg/kg) was also assessed. Free enkephalin immunoreactivity and total enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin containing fragments, were quantitated in the adrenal glands at 11 days and 7, 16, and 24 weeks of age. Both total and free metenkephalin were significantly diminished in the adrenal of SHR when compared to WKY at all ages tested. The analysis of the chromatographic profile showed that SHR displayed reduced levels of high and low molecular weight materials at 11 days and 16 weeks of age; however intermediate compounds were high in the glands of these animals. Similar increased values for free met-enkephalin were found in adrenals of WKY and SHR after ganglionic blocker treatment, which means that the relative increase was larger in SHR than WKY; while for total enkephalin the relative increase and the concentration reached in SHR was about half of those presented in WKY. These and other results presented suggest that the basic alteration of the adrenal proenkephalin system of SHR may be due to a genetic reduction of proenkephalin levels. Otherwise, the free enkephalin decrease could be related to changes in nervous input to the adrenal gland.

Recent studies have indicated that exogenously administered neurotrophins produce antidepressant-like behavioral effects. We have here investigated the role of endogenous brain-derived neurotrophic factor (BDNF) and its receptor trkB in the mechanism of action of antidepressant drugs. We found that trkB.T1-overexpressing transgenic mice, which show reduced trkB activation in brain, as well as heterozygous BDNF null (BDNF(+/)(−)) mice, were resistant to the effects of antidepressants in the forced swim test, indicating that normal trkB signaling is required for the behavioral effects typically produced by antidepressants. In contrast, neurotrophin-3(+/)(−) mice showed a normal behavioral response to antidepressants. Furthermore, acute as well as chronic antidepressant treatment induced autophosphorylation and activation of trkB in cerebral cortex, particularly in the prefrontal and anterior cingulate cortex and hippocampus. Tyrosines in the trkB autophosphorylation site were phosphorylated in response to antidepressants, but phosphorylation of the shc binding site was not observed. Nevertheless, phosphorylation of cAMP response element-binding protein was increased by antidepressants in the prefrontal cortex concomitantly with trkB phosphorylation and this response was reduced in trkB.T1-overexpressing mice. Our data suggest that antidepressants acutely increase trkB signaling in a BDNF-dependent manner in cerebral cortex and that this signaling is required for the behavioral effects typical of antidepressant drugs. Neurotrophin signaling increased by antidepressants may induce formation and stabilization of synaptic connectivity, which gradually leads to the clinical antidepressive effects and mood recovery.