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Background Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we developed a strategy to curtail these drawbacks through inhibition of an mTORC1 target, the eukaryotic initiation factor 4A (eIF4A). Methods We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival and apoptosis, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). Results eIF4i exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. eIF4Ai reduces mitochondrial membrane potential (MMP), the rate of ATP synthesis from mitochondrial respiration and glycolysis, and the expression of the antiapoptotic factors BCL2 and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity. Conclusions We discovered that AML cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. Our work indicates that eIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis. Furthermore, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.

Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted laser desorption ionization (MALDI)] enables the development of highly sensitive and specific assays for low-abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. This is typically achieved through external calibration, often using surrogate matrices, which has inherent limitations for the analysis of clinical specimens as there are often substantial variations in the sample matrix, and sample amounts are typically limited. We have previously introduced the use of two peptide isotopologues for generating external calibration curves in plasma. Here, we present a two-point internal calibration (2-PIC) strategy using two isotopologues for immuno-MS assays and demonstrate its flexibility and robustness. Quantification of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI using 2-PIC and external calibration yielded very similar results (relative standard deviation between 2-PIC and external calibration: 4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for a surrogate matrix or additional patient material for calibration, while concurrently reducing the instrument time and cost. Although our PTEN immuno-MRM and immuno-MALDI assays can be considered to be orthogonal as they utilized entirely different sample preparation and MS analysis workflows, targeted different PTEN peptides, and were performed in different laboratories, the endogenous Colo-205 PTEN levels determined with 2-PIC showed a good correlation (r2 = 0.9966) and good agreement (0.48 ± 0.01 and 0.29 ± 0.02 fmol/μg of total protein) between immuno-MRM and immuno-MALDI.

Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the western world. CLL consists of the accumulation of malignant B-cells in the blood stream and homing tissues. Although treatable, this disease is not curable, and resistance or relapse is often present. In many cancers, the study of metabolic reprograming has uncovered novel targets that are already being exploited in the clinic. However, CLL metabolism is still poorly understood. The ability of CLL lymphocytes to adapt to diverse microenvironments is accompanied by modifications in cell metabolism, revealing the challenge of targeting the CLL lymphocytes present in all different compartments. Despite this, the study of CLL metabolism led to an ongoing clinical trial using glucose uptake and mitochondrial respiration inhibitors. In contrast, glutamine and fatty acid metabolism remain to be further exploited in CLL. Here, we summarize the present knowledge of CLL metabolism, as well as the metabolic influence of Myc, ATM and p53 on CLL lymphocytes.

Buparlisib is an orally available pan-Class I PI3K inhibitor, that is more potent than idelalisib in vitro. Its distinct toxicities include hyperglycemia, hypertension, and mood disturbance. IND216...

PARP-3 is member of the PARP family of poly (ADP-ribose) polymerases involved in ADPribosylation. PARPs are involved in the basic mechanisms of DNA repair. PARP3, a critical player for efficient mitotic progression, is required for the stabilization of the mitotic spindle by regulation of the mitotic components, NuMA and Tankyrase 1. The sensitization effect of vinorelbine on PARP3 inhibition-induced cytotoxicity was assessed by the SRB assay. The contribution of programed cell death and cell cycle arrest to the sensitization effect were determined by assessing changes in Annexin V, a marker of apoptosis. Alterations in cell cycle progression were assessed by cell cycle analysis. We used immunofluorescence to assess the effect of vinorelbine and/or PARP3 inhibitors on tubulin and microtubule depolarization. The PARP3 chemiluminescent assay kit was used for PARP3 activity. PARP3 inhibitors sensitize breast cancer cells to vinorelbine, a vinca alkaloid used in the treatment of metastatic breast cancer. Olaparib which was originally described as a PARP1 and 2 inhibitor has recently been shown to be a potent PARP3 inhibitor while ME0328 is a more selective PARP3 inhibitor. The combination of vinorelbine with nontoxic concentrations of ME0328 or olaparib reduces vinorelbine resistance by 10 and 17 fold, respectively, potentiating vinorelbine-induced arrest at the G2/M boundary. In addition, PARP3 inhibition potentiates vinorelbine interaction with tubulin. Furthermore, olaparib or ME0328 potentiates vinorelbine-induced PARP3 inhibition, mitotic arrest, and apoptosis. Our results indicated this approach with PARP3 inhibitors and vinorelbine is unique and promising for breast cancer patients with metastases. This combination could significantly increase the survival of breast cancer patients with metastases.

e12561 Background: Ki-67 is a marker of proliferating cells. The recurrence score based on the 21-gene breast cancer assay also called Oncotype Dx provides prognostic and predictive information for recurrence in early stage breast cancer patients. We previously showed that there is a moderate correlation between Ki67 and oncotype Dx recurrence score. In this retrospective study, we aimed to examine whether high Ki67 could predict the distant recurrence in early stage breast cancer with low oncotype Dx scores ( < 25). Methods: This retrospective study included 278 consecutive cases of hormone receptor-positive, HER2 negative (T1-2 N0 M0) breast cancer who were diagnosed between 2008 and 2015 with low oncotype Dx ( < 25). Patients’ clinical outcome in terms of distant recurrence after breast surgery was determined up to December 2020 (median follow-up of 7 years). Patients were divided in to low risk (Ki67 < 15%) and high risk (Ki67 > = 15%) groups. Results: Of 278 cases with average and median age of 59 and 60 respectively, 148 (53%) were in Ki67 low risk and 130 (47%) were in Ki67 high risk group. Average and median oncotype Dx were 13.86 and 15 respectively in Ki67 low risk versus 15.23 and 16 respectively in Ki67 high risk group. 13 patients (4%) experienced distant metastasis in lung, liver, bone and skin. Of these 13 cases with average and median oncotype Dx 15.84 and 19 respectively, 12 (92%) were in the Ki67 high risk group and only 1 (8%) belonged to the low risk category. High Ki67 patients were overrepresented in group with recurrent distant metastasis compare to group without recurrent disease (Pearson Chi-Square = 51.18 with 1 degree of freedom and P = < 0.001). Conclusions: Ki67 high patients in the low risk oncotype Dx group are relapsing at a significantly higher rate suggesting that Ki67 combined with low oncotype Dx further refines the risk of distant relapse.

In this work we explored metabolic aspects of human primary leukemic lymphocytes that hold a potential impact on the treatment of Bruton tyrosine kinase (BTK)-driven diseases. Our results suggest that there is crosstalk between Bruton tyrosine kinase (BTK) signaling and bioenergetic stress responses. In primary chronic lymphocytic leukemia (CLL) lymphocytes, pharmacological interference with mitochondrial ATP synthesis or glucose metabolism affects BTK activity. Conversely, an inhibitor of BTK used clinically (ibrutinib) induces bioenergetic stress responses that in turn affect ibrutinib resistance. Although the detailed molecular mechanisms are still to be defined, our work shows for the first time that in primary B cells, metabolic stressors enhance BTK signaling and suggest that metabolic rewiring to hyperglycemia affects ibrutinib resistance in TP53 deficient chronic lymphocytic leukemia (CLL) lymphocytes.

Currently, there is no marker in use in the clinical management of colon cancer to predict which patients will respond efficiently to 5‐fluorouracil (5‐FU), a common component of all cytotoxic therapies. Our aim was to develop and validate a multigene signature associated with clinical outcome from 5‐FU therapy and to determine if it could be used to identify patients who might respond better to alternate treatments. Using a panel of 5‐FU resistant and sensitive colon cancer cell lines, we identified 103 differentially expressed genes providing us with a 5‐FU response signature. We refined this signature using a clinically relevant DNA microarray‐based dataset of 359 formalin‐fixed and paraffin‐embedded (FFPE) colon cancer samples. We then validated the final signature in an external independent DNA microarray‐based dataset of 316 stage III FFPE samples from the PETACC‐3 (Pan‐European Trails in Alimentary Tract Cancers) clinical trial. Finally, using a drug sensitivity database of 658 cell lines, we generated a list of drugs that could sensitize 5‐FU resistant patients using our signature. We confirmed using the PETACC‐3 dataset that the overall survival of subjects responding well to 5‐FU did not improve with the addition of irinotecan (FOLFIRI; two‐sided log‐rank test p = 0.795). Conversely, patients who responded poorly to 5‐FU based on our 12‐gene signature were associated with better survival on FOLFIRI therapy (one‐sided log‐rank test p = 0.039). This new multigene signature is readily applicable to FFPE samples and provides a new tool to help manage treatment in stage III colon cancer. It also provides the first evidence that a subgroup of colon cancer patients can respond better to FOLFIRI than 5‐FU treatment alone.

Poly (ADP-ribose) polymerase (PARP) resulting poly (ADP-ribosyl)ation is an early player in modification of proteins detected at single strand breaks and double strand breaks contributing to DNA repair. PARP1, 2 and 3 are involved in this process. However, PARP3 is unique in that, it is also required for efficient mitotic progression, specifically in stabilization of the mitotic spindle. The vinca alkaloids such as vinorelbine, arrest cells in metaphase due to inhibition of the assembly and dynamics of microtubules. Nontoxic concentrations of 2 PARP3 inhibitors, ME-0328 and olaparib, sensitized various her-2 negative and triple negative breast cancer cell lines greater than or equal to 10 fold to vinorelbine associated with potentiation of vinorelbine’s interaction with tubulin and also vinorelbine-induced PARP3 inhibition, mitotic arrest, and apoptosis without evidence of a DNA repair mechanism involved in this sensitization (Sharif-Askari B et al. Breast Cancer Res Treat. 2018 Nov;172(1):23-32). Eribulin is a Halichondrin B analogue, an antitubulin drug, with a unique mechanism of action; it inhibits microtubule dynamics via a novel mechanism of action which seems to involve binding to a unique site on tubulin resulting in the suppression of microtubule polymerization without affecting depolymerization along with sequestration of tubulin into nonfunctional aggregate. After impressive results in phase III trials (including one of the rare trials to show prolongation of survival in comparison to standard of care in heavily pretreated metastatic breast cancer patients) the U.S. Food and Drug Administration approved eribulin for treatment of patients with metastatic breast cancer. PARP3 inhibitors again sensitized at least 5 fold her-2 negative and triple negative breast cancer cell lines to eribulin. We will do similar studies with eribulin as we have done with vinorelbine including in-vivo studies. These studies could define a new therapy in the treatment of metastatic triple negative and ER+/her-2 negative breast cancer. The inhibition of PARP3 may increase the sensitivity of tumor cells to mitotic spindle poison chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use. Citation Format: Bahram Sharif-Askari, Raquel Aloyz, Lawrence Panasci. A new combination therapy to treat metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 300.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. Although promising new therapies for this incurable disease are being tested in clinical trials, the therapeutic relevance of metabolic rewiring in chronic lymphocytic leukemia (CLL) is poorly understood. The aim of this study was to identify targetable metabolic differences in primary CLL lymphocytes by the use of Dasatinib. Dasatinib is a multi-tyrosine kinase inhibitor used to treat chronic myelogenous leukemia (CML) and is being tested in clinical trials for several cancers including CLL. This drug has been shown to be beneficial to CML patients suffering from diabetes by reducing their glucose plasma levels. In keeping with this previous observation, we report that Dasatinib induced glucose use while reducing lactate production, suggesting that this tyrosine kinase inhibitor decreases aerobic glycolysis and shifts glucose use in primary CLL lymphocytes. Our results suggest that primary CLL lymphocytes (independently of traditional prognostic factors) can be stratified in two subsets by their sensitivity to Dasatinib in vitro. Increased glucose use induced by Dasatinib or by inhibition of mitochondrial respiration was not sufficient to sustain survival and ATP levels in CLL samples sensitive to Dasatinib. The two subsets of primary CLL lymphocytes are characterized as well by a differential dependency on mitochondrial respiration and the use of anabolic or catabolic processes to cope with induced metabolic/energetic stress. Differential metabolic reprogramming between subsets is supported by the contrasting effect on the survival of Dasatinib treated CLL lymphocytes with pharmacological inhibition of two master metabolic regulators (mTorc1 and AMPK) as well as induced autophagy. Alternative metabolic organization between subsets is further supported by the differential basal expression (freshly purified lymphocytes) of active AMPK, regulators of glucose metabolism and modulators of AKT signaling. The contrasting metabolic features revealed by our strategy could be used to metabolically target CLL lymphocyte subsets creating new therapeutic windows for this disease for mTORC1 or AMPK inhibitors. Indeed, we report that Metformin, a drug used to treat diabetes was selectively cytotoxic to Dasatinib sensitive samples. Ultimately, we suggest that a similar strategy could be applied to other cancer types by using Dasatinib and/or relevant tyrosine kinase inhibitors.

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.

B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and there is no cure for the disease. Although dasatinib is cytotoxic to primary CLL lymphocytes in vitro, the drug has been shown to be active in a small percent of CLL patients. Our previous results suggest that dasatinib targets del17 CLL lymphocytes which are the CLL patients with the worst prognosis. Here we present mechanistic evidence that dasatinib induces endoplasmic reticulum stress and autophagy in CLL lymphocytes. Furthermore we provide evidence suggesting that autophagy mediates resistance to the drugs, process that is modulated by p53.

The tyrosine kinase inhibitor, imatinib, has the potential to indirectly inhibit DNA repair. This mechanism of action has been shown to mediate sensitization to chlorambucil in chronic lymphocytic leukemia (CLL). To evaluate this effect in vivo, we performed a phase I study of chlorambucil combined with imatinib in relapsed CLL patients.The three dose levels studied included imatinib at 300, 400, or 600 mg/day. Imatinib was given on days 1-10, and chlorambucil (8 mg/m(2) daily) was given on days 3-7 of a 28-day cycle (up to 6 cycles).Eleven patients participated in this study. Low-grade gastrointestinal toxicities were observed in a dose-dependent manner. Forty-five percent of patients responded (two unconfirmed CRs and three PRs). Two responding patients were fludarabine refractory. The in vitro IC(50) of chlorambucil alone or in the presence of 5 μM imatinib in CLL lymphocytes correlated with the decrease in lymphocyte counts on day 15. Imatinib plasma concentrations achieved in patients were in the range of those effective in in vitro sensitization studies.The combination of chlorambucil and imatinib in patients with previously treated CLL was well tolerated and showed evidence of clinical efficacy. Based on our results, we recommend the 400 mg daily dose of imatinib on days 1-10 with 8 mg/m(3) chlorambucil on days 3-7 every 28 days as the phase II dose. This represents the first clinical trial examining the potential synergy between a tyrosine kinase inhibitor and a conventional alkylating agent for the treatment of CLL.

Chronic lymphocytic leukaemia (CLL) is characterized by the accumulation of mature quiescent B-lymphocytes in the G0/G1 phase of the cell cycle. B-lymphocyte accumulation is likely to be a consequence of an undefined defect in the apoptotic machinery rather than an increased proliferation of leukaemic cells (Hamblin & Oscier, 1997). The prolonged survival of CLL lymphocytes has also been linked to deregulated expression and/or activity of related non receptor tyrosine kinases including members of the SRC family kinases (SFK) and ABL1. Inhibition of either c-abl (Aloyz et al, 2004) or SFK (Contri et al, 2005) results in CLL lymphocyte death in vitro. Previous results from our laboratory suggest that dasatinib cytotoxicity in CLL lymphocytes is associated with c-abl rather than SFK inhibition (Amrein et al, 2008a). Recent results of phase I–II clinical trials using dasatinib in CLL suggest that the drug might be beneficial in only a small subset (≤10%) of previously treated patients (Amrein et al, 2008b). In agreement with these clinical results, we have recently reported that dasatinib is cytotoxic to primary CLL lymphocytes in vitro but mainly at clinically unobtainable concentrations (Amrein et al, 2008a). Although dasatinib resistance was associated with the basal expression of c-abl, our study suggested that wild type TP53 is important in CLL lymphocyte homeostasis and/or survival in the presence of dasatinib (Amrein et al, 2008a). To test this hypothesis we assessed: (i) dasatinib cytotoxicity in p53 proficient (wild type) CLL lymphocytes treated with dasatinib in the presence or absence of pifithrin-α, a small molecule inhibitor of p53 transcriptional activity (Steele et al, 2008) and 2) dasatinib cytotoxicity in primary CLL lymphocytes with impaired TP53 signalling from patients diagnosed with del 17p13.1. In the eleven samples we verified the functionality of p53 signalling as described before by examining changes in p53 and its downstream target p21Cip1 (p21) after treatment with chlorambucil (Willmore et al, 2008). Briefly, the lymphocytes were treated with equivalent cytotoxic concentrations of chlorambucil (50% inhibitory concentrations [IC50’s]) for 24 h and induced p53 and p21 protein levels were monitored by Western blotting. As expected p53 and p21 protein levels were induced by chlorambucil only in TP53 wild type CLL lymphocytes (data not shown). As previously reported, dasatinib IC50s in vitro in CLL lymphocytes expressing wild type TP53 were not in the clinically attainable range (mean value of 30 μmol/l, Table I) (Amrein et al, 2008a). In contrast, the dasatinib IC50s in del 17p13.1 lymphocytes were significantly lower (up to 100 times) than in the wild type TP53 lymphocytes. Moreover, in agreement with previous reports demonstrating that del 17p13.1 is associated with chemoresistance in CLL lymphocytes, we found that del 17p13.1 lymphocytes were significantly more resistant to chlorambucil that TP53 wild type CLL lymphocytes [Table I, Fig 1A (Zenz et al, 2008)]. Table I Clinical characteristic of the patients and IC50 concentrations of dasatinib and chlorambucil in primary CLL-lymphocytes in vitro. Fig 1 Dasatinib decreases p53 basal expression levels in primary CLL lymphocytes expressing wild type p53. Dasatinib and chlorambucil IC50s were significantly different between CLL lymphocytes expressing wild type TP53 or del 17 (*P = 0.012). The bars represent ... In available wild type TP53 CLL samples, pifithrin-α sensitized the lymphocytes of 2 out 3 patients (1·3 and 12-fold) (Table I). Importantly, pifithrin-α was not toxic to CLL lymphocytes when used alone. Treatment with dasatinib for twenty four hours resulted in a dose dependent reduction of p53 and p21 basal expression levels in the lymphocytes of patients expressing wild type TP53 (Fig 1B, C). In contrast, p53 levels were not affected in del 17 lymphocytes. Importantly, p21 was not detected in del 17p13.1 lymphocytes suggesting that TP53 is not functional (Fig 1D). These results are in agreement with previous reports suggesting that in the majority of CLL patients with malignant lymphocytes displaying del 17p13.1, the remaining TP53 allele is mutated (Zenz et al, 2008). In addition, we found that dasatinib resistance (higher IC50) in CLL lymphocytes expressing wild type TP53 correlated with residual p53 protein levels (in respect to control) after dasatinib treatment (r = 0·8, P = 0·02, Fig 1E). As ATM is a key regulator of p53 functionality, we assessed del 11q22-23 status in del 17p13.1 lymphocytes (Table I) (Pettitt et al, 2001). Two of the three del 17p13.1 samples tested were positive for del 11q22–23. Although del 17p13.1 lymphocytes were hypersensitive when compared to p53 proficient lymphocytes, we did not find a correlation between the percentage of del 17p13.1 or del 11q22–23 in the lymphocytes and the IC50 of dasatinib. Studies regarding the role of p53 signalling (and its regulators e.g. ATM) in dasatinib sensitivity in a larger cohort of CLL samples should be informative. Taken together, our results suggest that p53 is important to maintain CLL lymphocyte homeostasis following exposure to dasatinib and suggest that dasatinib may be effective to treat del 17p13.1 CLL patients. The recent report of an excellent clinical response to dasatinib of a CLL patient with lymphocytes displaying del 17p13.1 supports this hypothesis (Pitini et al, 2009).

Enhanced DNA damage repair is one mechanism involved in 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 DNA repair inhibitors utilized alone at nontoxic single agent concentrations resulted in sensitization to SN38 producing a 1.4 to 3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 in three colon cancer cell lines. When combined together, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5 to 27 fold reduction in the IC50 of SN38 with the HCT-116 colon cancer cells demonstrating the highest sensitization as compared to LoVo and HT-29 colon cancer cells. Furthermore, the combination of all three agents was associated with maximal G2-M arrest and enhanced DNA-damage (γH2AX) in all three colon cancer cell lines. The mechanism of this enhanced sensitization was associated with: (a) maximal suppression of SN38 induced PARP activity in the presence of both inhibitors and (b) ABT-888 producing partial abrogation of the VE-821 enhancement of SN38 induced DNA-PK phosphorylation, resulting in more unrepaired DNA damage; these alterations were only present in the HCT-116 cells which have reduced levels of ATM. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help produce sensitization to this drug in colon cancer.

The effect of imatinib on chlorambucil (CLB) cytotoxicity in chronic lymphocytic leukemia (CLL) lymphocytes was examined in vitro. Imatinib sensitizes the WSU and I83 human CLL cell lines, 10- and two-fold, respectively, to CLB. Furthermore, in primary cultures of malignant B-lymphocytes obtained from 12 patients with CLL (seven patients were untreated and five treated with CLB), imatinib synergistically sensitized these lymphocytes from two- to 20-fold to CLB. This synergistic effect was observed at concentrations of imatinib (

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 p0.05). 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 objective of this study was to further define the role of homologous recombinational repair (HRR) in resistance to the nitrogen mustards in B-cell chronic lymphocytic leukemia (B-CLL). We have demonstrated previously that increased chlorambucil (CLB)-induced HsRad51 nuclear foci formation correlated with a CLB-resistant phenotype in B-CLL lymphocytes. In this report, we measured the protein levels of HsRad51 and Xrcc3 (an HsRad51 paralog) and correlated them with the in vitro CLB cytotoxicity ( ld 50) in lymphocytes from seventeen B-CLL patients. Both HsRad51 (r=0.75, P=0.0005) and Xrcc3 (r=0.52, P=0.03) protein levels correlated with the in vitro CLB ld 50. In addition, multiple linear regression analysis showed a significant correlation between Xrcc3 and Rad51 protein levels versus the CLB ld 50 (r=0.78, P=0.0014), suggesting that both proteins influence CLB cytotoxicity. Moreover, since HsRad51 expression varies in cell lines during the cell cycle, we determined proliferating cell nuclear antigen (PCNA) protein levels to assess possible differences in cell cycle progression. There was no correlation between PCNA protein levels and the CLB ld 50 (r=0.042, P=0.87) or with HsRad51/Xrcc3 protein levels. Our data suggest that HsRad51 and Xrcc3 protein expression may be predictive of the response in B-CLL patients to treatment with nitrogen mustards.

In this report, we provide evidence that NGF and BDNF have functionally antagonistic actions on sympathetic neuron growth and target innervation, with NGF acting via TrkA to promote growth and BDNF via p75NTR to inhibit growth. Specifically, in cultured sympathetic neurons that themselves synthesize BDNF, exogenous BDNF inhibits and function-blocking BDNF antibodies enhance process outgrowth. Both exogenous and autocrine BDNF mediate this effect via p75NTR because (1) BDNF does not inhibit growth of neurons lacking p75NTR, (2) function-blocking p75NTR antibodies enhance NGF-mediated growth, and (3) p75NTR−/−sympathetic neurons grow more robustly in response to NGF than do their wild-type counterparts. To determine the physiological relevance of this functional antagonism, we examined the pineal gland, a well defined sympathetic target organ. BDNF is present in the pineal gland during target innervation, and incoming sympathetic axons are p75NTR positive. Moreover, the pineal glands of BDNF+/−and BDNF−/−mice are hyperinnervated with sympathetic fibers, and tyrosine hydroxylase (TH) levels are elevated. Increased tyrosine hydroxylase is also observed in the BDNF+/−carotid artery, another sympathetic neuron target. Thus, BDNF, made by sympathetic neurons and/or their target organs, acts via p75NTR to antagonize NGF-mediated growth and target innervation, suggesting that sympathetic target innervation is determined by the balance of positively and negatively acting neurotrophins present in developing and potentially mature targets.

The neurotrophic factor hypothesis, which is largely based on the neurotrophin family of growth factors (for review, see Levi-Montalcini 1987; Thoenen and Barde 1989; Snider 1994), postulates that trophic factors produced and released by target neurons regulate the survival, differentiation, and morphological growth of their innervating neurons (for review, see Oppenheim 1991; Majdan and Miller 1999). However, recent studies suggest that at least one member of the neurotrophin family, brain-derived neurotrophic factor (BDNF) (Barde et al. 1982; Leibrock et al. 1989), acts as an anterograde trophic factor that is derived from afferent neurons (von Bartheld et al. 1996; Altar et al. 1997; Conner et al. 1997; Fawcett et al. 1998). In particular, BDNF has been localized to both axons and terminals of peripheral (Zhou and Rush 1996; Michael et al. 1997) and central neurons (Conner et al. 1997; Fawcett et al. 1997, 1998), and the TrkB/BDNF receptor (Klein et al. 1991; Soppet et al. 1991) has been localized to neuronal dendrites in both the hippocampus and cortex (Fryer et al. 1996; Yan et al. 1997a), where at least a subpopulation of these receptors is present in postsynaptic densities (Wu et al. 1996; Lin et al. 1998). Moreover, this anterogradely trafficked BDNF has the potential to affect the survival and differentiation of target CNS neurons, at least during development (Fawcett et al. 1998). A number of recent studies also indicate that BDNF is localized to vesicles in presynaptic terminals in vivo (Fawcett et al. 1997; Michael et al. 1997), that it may be released in an activity-dependent fashion (Goodman et al. 1996; Mowla et al. 1999), and that following intense neural activity such as during kindling, Trk receptors are autophosphorylated (Binder et al. 1999), raising the interesting possibility that BDNF secretion in the mature nervous system could be regulated in a manner similar to neuropeptides (Mowla et al. 1999). The consequences of activity-dependent release of BDNF in the mature nervous system might be several. First, BDNF could play a more traditional role in regulating the morphology and, potentially, the survival of mature target neurons, a role analogous to that proposed for anterogradely transported BDNF during development (Fawcett et al. 1998). Second, BDNF could play a novel role for a trophic factor, modulating neuronal excitability either directly and/or by modification of the phosphorylation state of postsynaptic neurotransmitter receptors (Jarvis et al. 1997; Suen et al. 1997; Lin et al. 1998). Finally, postsynaptic signaling events resulting from BDNF-mediated TrkB receptor activation could synergize with signaling events caused by neurotransmitter receptor activation and/or calcium influx (Meyer-Franke et al. 1995; McAllister et al. 1996; Vaillant et al. 1999), raising the possibility that presynaptic corelease of a neurotransmitter and BDNF could have more dramatic effects on the postsynaptic neuron than the release of either of these stimuli alone. Such activity-dependent release of BDNF at central synapses could play an essential role both during development and in the adult. For example, during development, appropriate formation of ocular dominance columns is absolutely dependent on appropriate afferent activity (for review, see McAllister et al. 1999), and either application of exogenous BDNF (Cabelli et al. 1995) or disruption of endogenous BDNF (Cabelli et al. 1997) is sufficient to perturb this developmental process. Moreover, in the mature hippocampus, BDNF can modulate the strength of synaptic transmission at both the presynaptic and postsynaptic neuron (Kang and Schuman 1995, 1996; Levine et al. 1995, 1998; Gottschalk et al. 1998), and elimination of one BDNF allele in the BDNF+/− mice is sufficient to dampen long-term potentiation (LTP), an effect that can be rescued by the addition of exogenous BDNF (Korte et al. 1996; Patterson et al. 1996). On the basis of these considerations, we have hypothesized that BDNF may function, at least in part, by activity-dependent release from presynaptic terminals and subsequent activation of postsynaptic TrkB receptors. In this paper we have tested this hypothesis and demonstrate that elevated activity leads to rapid activation of synaptic TrkB receptors in the cortex and that pharmacological activation of brain-stem noradrenergic neurons, which synthesize and anterogradely traffic BDNF, is sufficient to cause rapid TrkB receptor activation on postsynaptic cortical neurons.

Proenkephalin peptides produced by endocrine and nervous tissues are involved in stress-induced immunosuppression. However, the role of peptides produced by immune cells remains unknown. The present study examines the effect of acute and chronic foot-shock stress on proenkephalin peptide content in bone marrow (BMMC), thymus (TMC), and spleen (SMC) rat mononuclear cells. Proenkephalin was not processed to met-enkephalin in BMMC, while in TMC and SMC met-enkephalin represented 10% and 26% of total met-enkephalin-containing peptides, respectively. Naive rats receiving a stress stimulus showed a significant decrease of proenkephalin derived peptides in BMMC, TMC and SMC. However, in chronically stressed rats that already showed basal low peptide levels, a new stress stimulus produced a differential response in each immune tissue. That is, in BMMC peptide levels reached control rats values; in TMC remained unmodified; and in SMC, although precursors content increased, met-enkephalin levels were even lower than those observed in acutely stressed rats. Free synenkephalin content paralleled met-enkephalin changes in SMC of acutely and chronically stressed rats. The in vitro release of met-enkephalin and free synenkephalin increased in SMC of stressed rats. Met-enkephalin produced in SMC and partially processed proenkephalin peptides detected in BMMC, were only found in macrophages. However, met-enkephalin only appeared in bone marrow macrophages after at least 4 h of cell culture. Altogether, these results suggest that a stress stimulus induced proenkephalin peptide release from immune tissue macrophages. The differential response observed in chronically stressed rats suggest an alternative activation of heterogeneous proenkephalin-storing macrophage subpopulations.

In this report, we have tested the hypothesis that brain-derived neurotrophic factor (BDNF) is an anterograde neurotrophic factor in the CNS and have focused on central noradrenergic neurons that synthesize BDNF. Double-label immunocytochemistry for BDNF and dopamine-β-hydroxylase (DBH), a marker for noradrenergic neurons, demonstrated that BDNF is partially localized to noradrenergic nerve fibers and terminals in the adult rat brain. To test the functional importance of this anterograde BDNF, we analyzed transgenic mice carrying a DBH–BDNF minigene. Increased synthesis of BDNF in noradrenergic neurons of DBH–BDNF mice caused elevated TrkB tyrosine kinase activation throughout postnatal life in the neocortex, a noradrenergic target region. This afferently regulated increase in TrkB receptor activity led to long-lasting alterations in cortical morphology. To determine whether noradrenergic neuron-expressed BDNF also anterogradely regulated neuronal survival, we examined a second noradrenergic target, neonatal facial motoneurons. One week after axotomy, 72% of facial motoneurons were lost in control animals, whereas only 30–35% were lost in DBH–BDNF transgenic mice. Altogether, these results indicate that BDNF is anterogradely transported to fibers and terminals of noradrenergic neurons, that anterogradely secreted BDNF causes activation of TrkB in target regions, and that this secretion has functional consequences for target neuron survival and differentiation. This presynaptic secretion of BDNF may provide a cellular mechanism for modulating neural circuitry, in either the developing or mature nervous system.

To determine whether the p75 neurotrophin receptor (p75NTR) plays a role in naturally occurring neuronal death, we examined neonatal sympathetic neurons that express both the TrkA tyrosine kinase receptor and p75NTR. When sympathetic neuron survival is maintained with low quantities of NGF or KCl, the neurotrophin brain-derived neurotrophic factor (BDNF), which does not activate Trk receptors on sympathetic neurons, causes neuronal apoptosis and increased phosphorylation of c-jun. Function-blocking antibody studies indicate that this apoptosis is due to BDNF-mediated activation of p75NTR. To determine the physiological relevance of these culture findings, we examined sympathetic neurons in BDNF−/− and p75NTR−/− mice. In BDNF−/− mice, sympathetic neuron number is increased relative to BDNF+/+ littermates, and in p75NTR−/− mice, the normal period of sympathetic neuron death does not occur, with neuronal attrition occurring later in life. This deficit in apoptosis is intrinsic to sympathetic neurons, since cultured p75NTR−/− neurons die more slowly than do their wild-type counterparts. Together, these data indicate that p75NTR can signal to mediate apoptosis, and that this mechanism is essential for naturally occurring sympathetic neuron death.

Naturally occurring sympathetic neuron death is the result of two apoptotic signaling events: one normally suppressed by NGF/TrkA survival signals, and a second activated by the p75 neurotrophin receptor. Here we demonstrate that the p53 tumor suppressor protein, likely as induced by the MEKK-JNK pathway, is an essential component of both of these apoptotic signaling cascades. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75NTR activation. NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax. Functional ablation of p53 using the adenovirus E1B55K protein inhibits neuronal apoptosis as induced by either NGF withdrawal or p75 activation. Direct stimulation of the MEKK-JNK pathway using activated MEKK1 has similar effects; p53 and Bax are increased and the subsequent neuronal apoptosis can be rescued by E1B55K. Expression of p53 in sympathetic neurons indicates that p53 functions downstream of JNK and upstream of Bax. Finally, when p53 levels are reduced or absent in p53+/− or p53−/− mice, naturally occurring sympathetic neuron death is inhibited. Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.

In this report, we have examined the role of neuron-derived BDNF at an accessible synapse, that of preganglionic neurons onto their sympathetic neuron targets. Developing and mature sympathetic neurons synthesize BDNF, and preganglionic neurons express the full-length BDNF/TrkB receptor. When sympathetic neuron-derived BDNF is increased 2- to 4-fold in transgenic mice, preganglionic cell bodies and axons hypertrophy, and the synaptic innervation to sympathetic neurons is increased. Conversely, when BDNF synthesis is eliminated in BDNF −/− mice, preganglionic synaptic innervation to sympathetic neurons is decreased. Together these results indicate that variations in neuronal neurotrophin synthesis directly regulate neuronal circuitry by selectively modulating synaptic innervation density.

We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.

Programmed cell death is an ongoing process in both the developing and the mature nervous system. The tumor suppressor gene, p53, can induce apoptosis in a number of different cell types. Recently, the enhanced expression of p53 has been observed during acute neurological disease. To determine whether p53 overexpression could influence neuronal survival, we used a recombinant adenovirus vector carrying wild type p53 to transduce postmitotic neurons. A control consisting of the same adenovirus vector background but carrying the lacZ reporter expression cassette was used to establish working parameters for the effective genetic manipulation of sympathetic neurons. We have found that recombinant adenovirus can be used at titers sufficiently high (10 to 50 multiplicity of infection) to transduce the majority of the neuronal population without perturbing survival, electrophysiological function, or cytoarchitecture. Moreover, we demonstrate that overexpression of wild type p53 is sufficient to induce programmed cell death in neurons. The observation that p53 is capable of inducing apoptosis in postmitotic neurons has major implications for the mechanisms of cell death in the traumatized mature nervous system.

Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining a major mechanism for the repair of double-strand breaks (DSB) in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK). The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its' central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA-PK. Computer based drug design will not only assist in identifying novel functional moieties to replace the metabolically labile morpholino group but will also facilitate the design of molecules to target the DNA-PKcs/Ku80 interface or one of the autophosphorylation sites.

Preface.- Repair of DNA Interstrand Cross-links Produced by Cancer Chemotherapeutic Drugs.- DNA-PK, a pharmacological target in cancer chemotherapy and radiotherapy?.- Growth factor receptor signaling, DNA damage response, and cancer cell susceptibility to chemotherapy and relapses.- The relationship between DNA-repair genes, cellular radiosensitivity and the response of tumors and normal tissues to radiotherapy.- Important Roles of ERCC1 in DNA Repair and Targeted Therapy.- The role of BRCA1 and BRCA2 in anticancer drug therapy.- DNA-PK in CLL Chemotherapy.- Poly(ADP) ribose polymerase at the interface of DNA damage signalling and DNA repair.- Cellular protection against the antitumor drug bleomycin.- ATR as a Therapeutic Target.- Telomeres, Telomerase and DNA Damage Response in Cancer Therapy.- RAD51 is a key protein of DNA repair and homologous recombination in humans.- Index.

DNA is the principal target of many conventional anticancer agents, and inhibition of DNA repair is one of the most promising strategies in novel cancer therapy. Many studies demonstrated that nonhomologous end-joining (NHEJ) repair pathway proteins, and especially DNA-dependent protein kinase (DNA-PK), is an attractive and effective target for the sensitization of cancer cells, including the most common type of leukemia in western countries, chronic lymphocytic leukemia (CLL), to DNA double-strand break (DSB)-inducing agents used in conventional cancer therapy. Nevertheless, promising results obtained in vitro cannot be translated to the clinic yet due to the nature of the DNA-PK inhibitors which are either nonspecific, for the first class of inhibitors, or degraded/eliminated from the human body before reaching the tumor site for the newer specific DNA-PK inhibitors.

Poly [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 0.125 μM. The level of synergy observed correlated with the degree of PARP1 inhibition as measured biochemically in cell lysates. ABT-888 at concentrations of 0.5–4 μM resulted in synergy with oxaliplatin. Furthermore, 24 h 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 48 h 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.

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.

PALB2/FANCN is a BRCA1- and BRCA2-interacting Fanconi Anemia (FA) protein crucial for key BRCA2 genome caretaker functions. Heterozygous germline mutations in PALB2 predispose to breast cancer and biallelic mutations cause FA. FA proteins play a critical role in the telomere maintenance pathway, with telomeric shortening observed in FA cells. Less is known about telomere maintenance in the heterozygous state. Here, we investigate the roles of PALB2 heterozygous mutations in genomic instability, an important carcinogenesis precursor. Patient-derived lymphoblastoid (LCL) and fibroblast (FCL) cell lines with monoallelic truncating PALB2 mutations were investigated using a combination of molecular imaging techniques including centromeric FISH, telomeric Q-FISH and spectral karyotyping (SKY). Mitomycin C and Cisplatin sensitivity was assayed via cellular metabolism of WST-1. The PALB2 c.229delT FCL showed increases in telomere counts associated with increased mean intensity compared with two wild-type FCLs generated from first-degree relatives (P =1.04E-10 and P =9.68E-15) and it showed evidence of chromosomal rearrangements. Significant differences in centromere distribution were observed in one of three PALB2 heterozygous FCLs analyzed when compared with PALB2 wild-type, BRCA1 and BRCA2 heterozygous FCLs. No significant consistently increased sensitivity to Mitomycin C or Cisplatin was observed in LCLs. Our results are suggestive of an altered centromere distribution profile and a telomere instability phenotype. Together, these may indicate critical nuclear organization defects associated with the predisposition to transformation and early stage development of PALB2-related cancers.

Background: Immunohistological assessment of Ki 67 expression is less expensive than Oncotype Dx, which is currently used to identify patients with lymph node-negative breast cancer, who will benefit from adjuvant chemotherapy. Methods: The relationship of immunohistologically measured Ki 67 to Oncotype DX recurrence score (RS) was examined in 53 cases of T1–2 N0 M0 (oestrogen receptor-positive, HER2/neu negative) breast cancer. Results: There was a strong linear correlation between Ki 67 value and the Oncotype Dx RS. All patients in the low Ki 67 group (Ki 67 of ⩽10%) had Oncotype Dx RSs of low or intermediate risk. The vast majority of patients (93.8%) in the high-Ki 67 group (Ki 67⩾25%) had oncotype RSs of high or intermediate risk. Conclusion: Ki 67 proliferation value is a major, but not the sole determinant of Oncotype Dx score.

ZRF4, a combi-molecule with increased efficacy as compared with the individual components in chronic lymphocytic leukemia lymphocytes in vitro

The combination of vinorelbine and trastuzumab (VH) is highly active and well tolerated in patients with metastatic HER2+ breast cancer. We assessed the efficacy and tolerability of VH as an alternative adjuvant treatment for patients with localized breast cancer refusing or ineligible for standard adjuvant trastuzumab-based chemotherapy. Twenty-eight patients with stage I-III breast cancer were treated only with VH as preoperative or postoperative chemotherapy. Fourteen patients received VH as adjuvant treatment for pT1a-b pN0 or eR+ pT1c pN0 cancers. VH was well tolerated, the only grade 3-4 toxicity being neutropenia with 2 cases of febrile neutropenia. At a median follow-up of 39 months, no breast cancer relapses were documented; moreover, overall and disease-free survival was 96.4%. In summary, our results indicate that VH is effective and well tolerated. VH should be prospectively tested as adjuvant treatment for pN0 pT1a-b breast cancer patients for which no standard treatment is well defined.

The 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.

Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3) and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion.

Preliminary experiments showed that MC-1 magnetotactic bacteria (MTB) could be used for the delivery of therapeutic agents to tumoral lesions. Each bacterium can provide a significant thrust propulsion force generated by two flagella bundles exceeding 4pN. Furthermore, a chain of single-domain magnetosomes embedded in the cell allows computer directional control and tracking using a magnetic resonance imaging (MRI) system. Although these embedded functionalities suggest that MTB when under the influence of an external computer could be considered as biological microrobots with the potential of targeting tumors, little is known about their level of penetration in tumoral tissues. In this paper, in vitro experiments were performed to assess the capability of these bacteria to penetrate tumor tissue for the delivery of therapeutic agents. Multicellular tumor spheroids were used since they reproduce many properties of solid tumors. The results show the ability of these MTB when submitted to a directional magnetic field to penetrate inside a 3D multicellular tumor spheroid through openings present in the tissue.

This 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 IC(50) values of SN38 were observed at 5 and 10 μM of DNA-PK inhibitors. Moreover, at 1-2 μM (attainable concentrations with ICC in mice) these DNA-PKcs inhibitors demonstrated synergistic reductions in the IC(50) of SN38. Flow cytometric data indicated that SN38 and SN38 in combination with DNA-PKcs inhibitors showed dramatic G2/M arrest at 24 h. 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.

a b s t r a c t Resistance to chlorambucil in chronic lymphocytic leukemia (CLL) has been associated with increased DNA repair. Specifically, inhibition of either c-abl, which modulates Rad51 directed homologous recombination or DNA-PK dependent nonhomologous end joining has been shown to sensitize primary CLL lymphocytes to chlorambucil. Here we report that inhibition of c-abl can result in a compensatory increase in DNA-PK and thus inhibition of both c-abl and DNA-PK optimally sensitizes CLL lymphocytes to chlorambucil. In this paper we report a drug-induced compensatory change between two DNA repair pathways with potential therapeutic implications in CLL therapy.