Raquel Aloyz

Small Molecules, Inhibitors of DNA-PK, Targeting DNA Repair, and Beyond.

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.

Chlorambucil Cytotoxicity in Malignant B Lymphocytes Is Synergistically Increased by 2-(Morpholin-4-yl)-benzo[h]chomen-4-one (NU7026)-Mediated Inhibition of DNA Double-Strand Break Repair via Inhibition of DNA-Dependent Protein Kinase

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 (γH2AX) 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 G2/M phase arrest, and NU7026 increases this CLB-induced G2/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 γH2AX, 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.

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

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

Homologous recombinational repair vis-à-vis chlorambucil resistance in chronic lymphocytic leukemia

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.

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

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

Intrathecal trastuzumab and thiotepa for leptomeningeal spread of breast cancer

A 31-year-old woman underwent a right segmental mastectomy in October 1999 for a pT2, pN2 invasive ductal carcinoma, negative for estrogen receptor/progesterone receptor, positive for amplification of human epidermal growth factor receptor-2 (HER-2). She received adjuvant chemotherapy (doxorubicin + cyclophosphamide, followed by paclitaxel) and local radiotherapy. Two and a half years after surgery, she developed multiple liver metastases. She received i.v. carboplatin, paclitaxel, and trastuzumab, followed by capecitabine and trastuzumab, with a radiological complete response. Approximately 14 months later (April 2004), a single brain lesion was detected on magnetic resonance imaging (MRI), in the right occipital lobe. The lesion was surgically resected, followed by external beam whole-brain radiation. In August 2006, the patient presented with visual impairment, ptosis of the right eye, right hemi-facial hypoesthesia, left foot drop, and paralysis of the left facial nerve. An MRI exam revealed a right occipital brain lesion (1.5 cm), diffuse leptomeningealmetastases (LM), a cervical extramedullary intradural enhancing lesion (C1– C3), an intramedullary lesion centered at T12, and a nodular area of enhancement on the roots of the cauda equina. Only a suspicious 6-mm lung nodule was found on systemic restaging. A diagnostic lumbar puncture was carried out, and cytology of the cerebro-spinal fluid (CSF) was positive for cancer cells (5%of malignant cells). CSF glucose, lactate dehydrogenase, and protein levels were always normal. After placement of an Ommaya reservoir, she was started on intrathecal (i.t.) trastuzumab, initially on a weekly schedule, with gradual dose increase from 20 to 30 mg (Table 1), later combined with i.t. methotrexate (10 mg) for two doses. To reduce the risk of anaphylaxis and neurotoxicity from the preservative agent [1], i.t. trastuzumab was administered without the diluent provided. Interestingly, after the first dose of i.t. trastuzumab, all subsequent CSF cytologies were negative for malignant cells. After the two first doses of i.t. trastuzumab, we also started i.v. trastuzumab, that was never interrupted afterwards, and i.v. pegylated liposomal doxorubicin (PLD; letters to the editor Annals of Oncology

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

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

p53 and autophagy contribute to dasatinib resistance in primary CLL lymphocytes

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.

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

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

Magnetotactic bacteria penetration into multicellular tumor spheroids for targeted therapy

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.