David Davidson

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.

Dual inhibition of the homologous recombinational repair and the nonhomologous end-joining repair pathways in chronic lymphocytic leukemia therapy.

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.

Simultaneous inhibition of ATR and PARP sensitizes colon cancer cell lines to irinotecan

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 1.4–3 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 4.5–27 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.

Adaptive metabolic rewiring to chronic SFK inhibition

Src family kinases (SFK) are key regulators of cellular proliferation, differentiation, survival, motility and angiogenesis. As such, SFK inhibitors are being tested in clinical trials to prevent metastasis as an alternative to current treatment regimens for a variety of cancers including breast cancer. To contribute to the development of molecular tools improving SFK-targeted therapies, we used the SFK inhibitor dasatinib and a well characterized triple negative breast cancer cell line (BT20). Comparison of the response of BT20 cells with acquired resistance to dasatinib and its’ parental counterpart suggest that chronic exposure to SFK inhibition results in increased dependency on TGFβ signaling for proliferation, both in the absence or the presence of dasatinib. In addition, we found that acquired (but not de novo) resistance to dasatinib was reduced by non-cytotoxic concentrations compounds hindering on PI3K, mTORC1 signaling, endoplasmic reticulum stress or autophagy.

Abstract 268: PARP3 inhibitors in cancer therapy

Poly-ADP-ribose-polymerase (PARP1) is an important regulator of DNA damage response (DDR). After induction of certain types of DNA damage, including nicks and double strand breaks (DSB)s, PARP1 is rapidly recruited to altered DNA sites were its catalytic activity produces protein-conjugated long-branched-poly-ADP-ribose (PAR) chains. These protein modifications result in the recruitment and activation of multiple proteins involved in DNA repair. Although there is considerable research describing the characteristics and activity of PARP1/2, there are 15 other PARP family members that are less well characterized. Of these PARP3 has been shown to have a distinct role in DNA repair. This protein has been shown to have a critical role in DSB resolution and to interact with partner proteins know to function in classical nonhomologous end joining (NHEJ) such as DNA-PKcs, Ku70, and Ku80. In order to examine the role of PARP3 vis-a-vis chemotherapy, we utilized the specific PARP3 inhibitor, ME-0328 in combination with activated cyclophosphamide (4HC) in the chronic lymphocytic leukaemia (CLL) cell lines, MEC1 and MEC2. Results from MTT cytotoxicity assays showed that the 50% inhibitory concentration (IC 50 ) (50% of control) of 4HC in MEC-1 cells was 12.9μM. When used in combination with a nontoxic concentration (2μM) of the specific PARP3 inhibitor ME-0328 the IC 50 of 4HC was reduced to 3.3μM. In MEC-2 cells the IC 50 of 4HC was 10.8μM and was reduced to 2.1μM in the presence of ME-0328 at 2μM. ABT-888, a specific PARP1/2 inhibitor in clinical trials had a minimal effect on 4HC cytotoxicity in these cell lines. 4HC results in interstrand crosslinks (ICLs) which are believed to be repaired by homologous recombination. Intriguingly, PARP3 negatively regulates class switch recombination via activation-induced cytidine deaminase in B-lymphocytes. This may be involved in ME-0328 sensitization of 4HC in B-lymphocytic malignancies. Given these preliminary results we hypothesize that the PARP3 inhibitor, ME-0328 will sensitize B-lymphocytic cancers to chemotherapies. To explore this hypothesis this project will pursue the following objectives: 1. Characterize the selective sensitization of ME-0328 with 4HC and/or bendamustine (an ICL-inducing drug utilized in the treatment of B-lymphocyte malignancies) in B-lymphoma and CLL cell lines plus CLL clinical samples. 2. Determine the mechanisms by which ME-0328 sensitizes CLL cells to 4HC and/or bendamustine (i.e. reduction of PARP1 or 3 activity and/or altered activity of known DNA repair proteins (Rad51, DNA-PK, H2AX) and/or altered activation-induced cytidine deaminase). 3. Determine the in-vivo activity of 4HC and/or bendamustine with or without ME-0328 in a Rag2 /-γcc-/- xenograft model using the MEC1-CLL cell line. The inhibition of PARP3 may increase the sensitivity of tumor cells to DNA damaging chemotherapies. Our results should stimulate development of specific PARP3 inhibitors for clinical use. Citation Format: Bahram Sharif-Askari, David Davidson, Lilian Amrein, Lawrence Panasci. PARP3 inhibitors in cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 268.

Abstract 1881: New therapeutic options for CLL treatment: Src/c-abl-directed molecular re-engineering of chlorambucil and bendamustine

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA B-cell chronic lymphocytic leukemia (CLL) is characterized by actively dividing B-lymphocytes in the lymph nodes and bone marrow, associated with the accumulation of quiescent lymphocytes in the peripheral blood of affected patients. Current treatments for this disease include chemotherapeutic (chlorambucil (CLB), cyclophosphamide, fludarabine) and immunotherapeutic agents (Rituximab, Alemtuzumab) or the combination of immunotherapy with chemotherapeutics drugs. In 2008, the FDA approved bendamustine, a bifunctional agent with a nitrogen mustard moiety and a purine analog, for the treatment of patients with CLL. Signals from the bone marrow and lymph node microenvironments protect CLL lymphocytes from drug-induced apoptosis. During treatment the enzyme-mediated repair of DNA damage can induce resistance to chemotherapeutic drugs. We have previously shown that small molecule inhibitors of c-abl (a key protein of the homologous recombinational repair pathway) such as imatinib, nilotinib or dasatinib, sensitize primary CLL lymphocytes to CLB. We also demonstrated that ZRF4, a combi-molecule designed to target c-abl (through an imatinib moiety) and to induce DNA damage (through a nitrogen mustard moiety), has a more potent cytotoxic effect than the combination of CLB plus imatinib in CLL lymphocytes. Here we report the anticancer effect of three different combi-molecules composed of a chlorambucil moiety and a dasatinib (a dual Src/c-abl inhibitor) moiety compare to the individual components in primary CLL lymphocytes in-vitro. Using MTT assays on CLL lymphocytes from 40 CLL patients, we found that the IC50 (concentration which kills 50% of the cells) of AL748, AL758 and AL816 are significantly lower than the IC50 of CLB when used alone and the combination of CLB with 0.1 μM dasatinib (median value = 1.7 μM, 0.6 μM, 0.9 μM, 11 μM and 3.1 μM respectively). Moreover, our biochemical results suggest that the mechanism of action of AL758, AL816, CLB and CLB plus dasatinib share common downstream targets including inhibition of Src kinase, chemotaxis, and cross-talk with the microenvironment along with induction of DNA damage (p53, p21, comet assay) and apoptosis (AnnexinV, cleaved caspase-3). We also determined, by the MTT assay, the cytotoxic effect of the combi-molecule AL887 composed of a bendamustine moiety and a dasatinib moiety, compare to the individual components in vitro in primary B-lymphocytes from 20 CLL patients. Our results demonstrated that this combi-molecule has a better anticancer activity than bendamustine in combination with 0.1 μM dasatinib (p<0.0005). Dose-limiting toxicity and drug pharmacokinetics are important and limiting factors to take into account for the development of chemotherapy and will be tested in a CLL mouse model. Our results suggest that the combi-molecules AL758, AL816, and AL887 may be useful alternatives for treatment of CLL patients. Citation Format: Lilian Amrein, Anne-Laure Larocque, David Davidson, Lisa Peyrard, Daniel Borrelli, Bertrand Jean-Claude, Lawrence Panasci. New therapeutic options for CLL treatment: Src/c-abl-directed molecular re-engineering of chlorambucil and bendamustine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1881. doi:10.1158/1538-7445.AM2014-1881

Abstract B13: ATR and PARP inhibition enhances topoisomerase I-dependent DNA damage in colon cancer cell lines

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.

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

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