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31 N-Myc amplification sensitizes tumor cells to inhibition by Danusertib, an Aurora kinase inhibitor
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Wednesday 19 November 2014
30 POSTER The fungal-derived cyclohexadepsipeptide Destruxin E exerts multifaceted anticancer and antiangiogenic activities R. Dornetshuber-Fleiss1 , P. Heffeter2 , T. Mohr2 , P. Hazemi3 , K. Kryeziu4 , C. Seger5 , W. Berger2 , R. Lemmens-Gruber3 . 1 University of Vienna and Medical University of Vienna, Institute of Pharmacology and Toxicology and the Institute of Cancer Research Department of Medicine I and Comprehensive Cancer Center of the Medical University, Vienna, Austria; 2 Medical University of Vienna, Institute of Cancer Research Department of Medicine I and Comprehensive Cancer Center of the Medical University, Vienna, Austria; 3 University of Vienna, Department of Pharmacology and Toxicology, Vienna, Austria; 4 Medical University of Vienna, Institute of Cancer Research Department of Medicine I and Comprehensive Cancer Center of the Medical University, Vienna, Austria; 5 Leopold-Franzens University Innsbruck, Institute of Pharmacy Department of Pharmacognosy, Vienna, Austria Background: Destruxins − a group of secondary metabolites of the entomopathogenic fungus Metarhizium anisopliae − recently came into focus of interest as anticancer therapeutics for colorectal cancer. However, the knowledge on their anticancer effects is fragmentary. Consequently, the present study aimed to investigate the impact of the representative derivative Destruxin E (Dtx E) on human colon cancer cell growth and survival. Materials and Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and Western blot analyses were used to assess the cytostatic/cytotoxic activity of Dtx E on Caco-2 and HCT116 cells. Impact of cell cycle distribution was evaluated using PI-stainings and FACS analyses. Apoptotic effects were determined with DAPI stainings and Western blot analyses. Angiogenesis inhibition was evaluated using scratch and tube-formation assays. To investigate the impact on the intracellular redox potential flow cytometry with dihydroxyethidium (DHE) and combination studies with the radical scavenger N-acetyl cysteine (NAC) and the glutathione inhibitor BSO were performed. Results: The experimental data showed that Dtx E exerts potent antiproliferative activity in the nanomolar range in CaCo-2 and HCT116 cells. Moreover, Dtx E caused an imbalance of cell cycle distribution and the cytostatic/cytotoxic effects were shown to be widely p53-independent but reduced by p21- and bax-deletion, respectively. Cytotoxicity is based on intrinsic apoptosis induction and associated with phosphoinositide-3kinase (PI3K)/Akt pathway inhibition. Additionally, anticancer activity of Dtx E involves disturbance of the intracellular redox balance and finally, Dtx E inhibits the migration and tube formation of human endothelial cells indicating antiangiogenic potential. Conclusion: The data of the present study indicate that Dtx E represents a feasible, multifunctional anticancer drug candidate for preclinical development against colorectal cancer. 31 POSTER N-Myc amplification sensitizes tumor cells to inhibition by Danusertib, an Aurora kinase inhibitor P. Carpinelli1 , R. Ceruti2 , R. Alzani2 , C. Re1 , D. Ballinari1 , S. Cribioli2 , M. Russo2 , A. Degrassi2 , G. Texido2 , M. Ciomei2 , E. Pesenti2 , A. Montagnoli1 , A. Galvani1 . 1 Nerviano Medical Sciences Srl, Cell Biology, Nerviano (Milano), Italy; 2 Nerviano Medical Sciences Srl, Pharmacology, Nerviano (Milano), Italy Amplification of N-Myc is a driving mutational event in a subset of tumor types, particularly those of neural origin and neuroendocrine tumors such as neuroblastoma (NB), neuroendocrine prostate cancer (NEPC), small cell lung cancer (SCLC) and others. Thus, inhibition/reduction of N-Myc protein levels may be of therapeutic benefit for such tumor types but to date small molecules that specifically target this oncogene are not clinically available. Aurora A kinase has been shown to interact directly with N-Myc in neuroblastoma and to induce its stabilization by preventing binding of the Fbxw7 ubiquitin ligase, which mediates its ubiquitination and subsequent proteasomal degradation. Moreover, there is clear evidence that pharmacological intervention with small molecule inhibitors which target Aurora A represents a promising therapeutic approach for this tumor subtype. Danusertib (PHA-739358) is a small molecule ATP competitor that inhibits Aurora A, B and C kinases. Aurora kinases inhibition by Danusertib results in cell cycle block with concomitant inhibition of cell proliferation. The compound is in clinical investigation in both solid tumor and hematological malignancy settings. Here we report the in vitro and in vivo activity of Danusertib in N-Myc amplified tumor models. We demonstrate that in vitro, NB cell lines bearing amplified N-Myc display greater sensitivity to Danusertib than
Poster Session – Cytotoxics non-amplified cell lines. In such sensitive NB cell lines, inhibition of histone H3 phosphorylation, a marker of Aurora kinase inhibition, is associated with decreased levels of N-Myc and induction of apoptosis. In NB tumor xenograft models, Danusertib induced significant in vivo tumor growth inhibition, including tumor regression, accompanied by robust reductions of N-Myc protein levels, again concomitant with inhibition of phospho-histone H3 and induction of apoptosis. Additionally, Danusertib also displayed significant in vivo activity against the TRAMP, a transgenic mouse prostate carcinoma model, which displays several molecular and pathological features of NEPC. Here again, treatment with Danusertib induced decreased levels of N-Myc, associated with inhibition of histone H3 phosphorylation. Thus, Danusertib destabilizes N-Myc both in vitro and in vivo and therefore merits further investigation as a potential option for the therapy of N-Myc amplified neuroendocrine tumors, as well as other settings which are dependent on this oncogene. 32 POSTER Replication stress is a determinant of synergy between gemcitabine and Chk1 inhibition S.B. Koh1 , A. Courtin1 , R. Boyce2 , B. Boyle2 , F.M. Richards1 , D.I. Jodrell1 . 1 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom; 2 Sentinel Oncology Limited, Cambridge, United Kingdom Background: Chk1 plays multiple regulatory roles within the cell cycle and this study interrogates the mechanism of synergy between Chk1 inhibition and gemcitabine in cancer cells. Methodology: We evaluated the cytotoxicity of FS105, a Chk1-specific inhibitor (IC50 2.8nM), with gemcitabine in human and murine cancer cells. We then employed established mathematical models to quantitatively identify drug concentrations that yielded synergistic growth inhibition. Subsequent mechanistic studies, achieved by quantitative high-content imaging, immunofluorescence microscopy, flow cytometry and immunoblotting, were performed at the identified synergistic drug concentrations. Results: Synergy with FS105 was found at sub-GI50 concentrations of gemcitabine in all tested cell lines, e.g. Emax 91±3% growth inhibition at 3uM FS105+30nM gemcitabine in Panc-1 cells compared to Bliss predicted 33±1% growth inhibition. At these concentrations, we did not observe substantial premature mitotic entry, contrary to the prevailing model that inhibiting Chk1 abrogates the G2/M checkpoint. Downstream checkpoint effector CDK1 Y15 was not inhibited by FS105 at synergistic concentrations with gemcitabine. In agreement with this, significant S phase arrest with hallmarks of elevated replication stress and DNA damage such as ùH2AX was observed. For example, 63±6% MIA PaCa-2 cells were in S phase 24 hours after the combination treatment, compared to control (33±2%), FS105 alone (35±4%) and gemcitabine alone (43±4%). The induction of S phase arrest and ùH2AX by the combination was inhibited when the activity of replication initiator CDC7 was suppressed by PHA-767491. This observation is consistent with Chk1 role in regulating origin firing and our hypothesis that Chk1 inhibition synergises with gemcitabine via destabilisation of the replication machinery. Conclusions: This study elucidates how Chk1 inhibition with gemcitabine at synergistic concentrations perturbs predominantly the S-phase homeostasis in cancer cells. It also provides a molecular rationale to reassess the current scheduling paradigm of Chk1 inhibitors with DNA-damaging agents. 33 POSTER Combining the long-acting topoisomerase 1-inhibitor etirinotecan pegol with the PARP inhibitor rucaparib to provide anti-tumor synergy without increased toxicity U. Hoch1 , D. Charych2 . 1 Nektar Therapeutics, Development, San Francisco CA, USA; 2 Nektar Therapeutics, Research, San Francisco CA, USA Background: PARP inhibition sensitizes cells to DNA-damaging agents, particularly topoisomerase 1-inhibitors, where the combination shows synergy in nonclinical studies. However, in the clinic, the combination of either topotecan or irinotecan with PARP inhibitors showed severe hematologic toxicities, requiring dose reductions beyond optimal therapeutic effects. Etirinotecan pegol (NKTR-102) is the first long-acting Topoisomerase 1-inhibitor providing continuous exposure to active metabolite throughout the entire chemotherapy cycle with reduced peak concentrations, resulting in better efficacy and safety, including a low rate of Grade 3 neutropenia (11%). Here we present in vivo combination data of etirinotecan pegol and rucaparib in the MX-1 breast cancer model. Methods: Mice bearing BRCA1-deficient MX-1 breast cancer tumors (~100 mm3 ) received either vehicle, PO rucaparib, IV etirinotecan pegol, or a combination of etirinotecan pegol and rucaparib as follows: 30 or
Wednesday 19 November 2014
30 POSTER The fungal-derived cyclohexadepsipeptide Destruxin E exerts multifaceted anticancer and antiangiogenic activities R. Dornetshuber-Fleiss1 , P. Heffeter2 , T. Mohr2 , P. Hazemi3 , K. Kryeziu4 , C. Seger5 , W. Berger2 , R. Lemmens-Gruber3 . 1 University of Vienna and Medical University of Vienna, Institute of Pharmacology and Toxicology and the Institute of Cancer Research Department of Medicine I and Comprehensive Cancer Center of the Medical University, Vienna, Austria; 2 Medical University of Vienna, Institute of Cancer Research Department of Medicine I and Comprehensive Cancer Center of the Medical University, Vienna, Austria; 3 University of Vienna, Department of Pharmacology and Toxicology, Vienna, Austria; 4 Medical University of Vienna, Institute of Cancer Research Department of Medicine I and Comprehensive Cancer Center of the Medical University, Vienna, Austria; 5 Leopold-Franzens University Innsbruck, Institute of Pharmacy Department of Pharmacognosy, Vienna, Austria Background: Destruxins − a group of secondary metabolites of the entomopathogenic fungus Metarhizium anisopliae − recently came into focus of interest as anticancer therapeutics for colorectal cancer. However, the knowledge on their anticancer effects is fragmentary. Consequently, the present study aimed to investigate the impact of the representative derivative Destruxin E (Dtx E) on human colon cancer cell growth and survival. Materials and Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and Western blot analyses were used to assess the cytostatic/cytotoxic activity of Dtx E on Caco-2 and HCT116 cells. Impact of cell cycle distribution was evaluated using PI-stainings and FACS analyses. Apoptotic effects were determined with DAPI stainings and Western blot analyses. Angiogenesis inhibition was evaluated using scratch and tube-formation assays. To investigate the impact on the intracellular redox potential flow cytometry with dihydroxyethidium (DHE) and combination studies with the radical scavenger N-acetyl cysteine (NAC) and the glutathione inhibitor BSO were performed. Results: The experimental data showed that Dtx E exerts potent antiproliferative activity in the nanomolar range in CaCo-2 and HCT116 cells. Moreover, Dtx E caused an imbalance of cell cycle distribution and the cytostatic/cytotoxic effects were shown to be widely p53-independent but reduced by p21- and bax-deletion, respectively. Cytotoxicity is based on intrinsic apoptosis induction and associated with phosphoinositide-3kinase (PI3K)/Akt pathway inhibition. Additionally, anticancer activity of Dtx E involves disturbance of the intracellular redox balance and finally, Dtx E inhibits the migration and tube formation of human endothelial cells indicating antiangiogenic potential. Conclusion: The data of the present study indicate that Dtx E represents a feasible, multifunctional anticancer drug candidate for preclinical development against colorectal cancer. 31 POSTER N-Myc amplification sensitizes tumor cells to inhibition by Danusertib, an Aurora kinase inhibitor P. Carpinelli1 , R. Ceruti2 , R. Alzani2 , C. Re1 , D. Ballinari1 , S. Cribioli2 , M. Russo2 , A. Degrassi2 , G. Texido2 , M. Ciomei2 , E. Pesenti2 , A. Montagnoli1 , A. Galvani1 . 1 Nerviano Medical Sciences Srl, Cell Biology, Nerviano (Milano), Italy; 2 Nerviano Medical Sciences Srl, Pharmacology, Nerviano (Milano), Italy Amplification of N-Myc is a driving mutational event in a subset of tumor types, particularly those of neural origin and neuroendocrine tumors such as neuroblastoma (NB), neuroendocrine prostate cancer (NEPC), small cell lung cancer (SCLC) and others. Thus, inhibition/reduction of N-Myc protein levels may be of therapeutic benefit for such tumor types but to date small molecules that specifically target this oncogene are not clinically available. Aurora A kinase has been shown to interact directly with N-Myc in neuroblastoma and to induce its stabilization by preventing binding of the Fbxw7 ubiquitin ligase, which mediates its ubiquitination and subsequent proteasomal degradation. Moreover, there is clear evidence that pharmacological intervention with small molecule inhibitors which target Aurora A represents a promising therapeutic approach for this tumor subtype. Danusertib (PHA-739358) is a small molecule ATP competitor that inhibits Aurora A, B and C kinases. Aurora kinases inhibition by Danusertib results in cell cycle block with concomitant inhibition of cell proliferation. The compound is in clinical investigation in both solid tumor and hematological malignancy settings. Here we report the in vitro and in vivo activity of Danusertib in N-Myc amplified tumor models. We demonstrate that in vitro, NB cell lines bearing amplified N-Myc display greater sensitivity to Danusertib than
Poster Session – Cytotoxics non-amplified cell lines. In such sensitive NB cell lines, inhibition of histone H3 phosphorylation, a marker of Aurora kinase inhibition, is associated with decreased levels of N-Myc and induction of apoptosis. In NB tumor xenograft models, Danusertib induced significant in vivo tumor growth inhibition, including tumor regression, accompanied by robust reductions of N-Myc protein levels, again concomitant with inhibition of phospho-histone H3 and induction of apoptosis. Additionally, Danusertib also displayed significant in vivo activity against the TRAMP, a transgenic mouse prostate carcinoma model, which displays several molecular and pathological features of NEPC. Here again, treatment with Danusertib induced decreased levels of N-Myc, associated with inhibition of histone H3 phosphorylation. Thus, Danusertib destabilizes N-Myc both in vitro and in vivo and therefore merits further investigation as a potential option for the therapy of N-Myc amplified neuroendocrine tumors, as well as other settings which are dependent on this oncogene. 32 POSTER Replication stress is a determinant of synergy between gemcitabine and Chk1 inhibition S.B. Koh1 , A. Courtin1 , R. Boyce2 , B. Boyle2 , F.M. Richards1 , D.I. Jodrell1 . 1 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom; 2 Sentinel Oncology Limited, Cambridge, United Kingdom Background: Chk1 plays multiple regulatory roles within the cell cycle and this study interrogates the mechanism of synergy between Chk1 inhibition and gemcitabine in cancer cells. Methodology: We evaluated the cytotoxicity of FS105, a Chk1-specific inhibitor (IC50 2.8nM), with gemcitabine in human and murine cancer cells. We then employed established mathematical models to quantitatively identify drug concentrations that yielded synergistic growth inhibition. Subsequent mechanistic studies, achieved by quantitative high-content imaging, immunofluorescence microscopy, flow cytometry and immunoblotting, were performed at the identified synergistic drug concentrations. Results: Synergy with FS105 was found at sub-GI50 concentrations of gemcitabine in all tested cell lines, e.g. Emax 91±3% growth inhibition at 3uM FS105+30nM gemcitabine in Panc-1 cells compared to Bliss predicted 33±1% growth inhibition. At these concentrations, we did not observe substantial premature mitotic entry, contrary to the prevailing model that inhibiting Chk1 abrogates the G2/M checkpoint. Downstream checkpoint effector CDK1 Y15 was not inhibited by FS105 at synergistic concentrations with gemcitabine. In agreement with this, significant S phase arrest with hallmarks of elevated replication stress and DNA damage such as ùH2AX was observed. For example, 63±6% MIA PaCa-2 cells were in S phase 24 hours after the combination treatment, compared to control (33±2%), FS105 alone (35±4%) and gemcitabine alone (43±4%). The induction of S phase arrest and ùH2AX by the combination was inhibited when the activity of replication initiator CDC7 was suppressed by PHA-767491. This observation is consistent with Chk1 role in regulating origin firing and our hypothesis that Chk1 inhibition synergises with gemcitabine via destabilisation of the replication machinery. Conclusions: This study elucidates how Chk1 inhibition with gemcitabine at synergistic concentrations perturbs predominantly the S-phase homeostasis in cancer cells. It also provides a molecular rationale to reassess the current scheduling paradigm of Chk1 inhibitors with DNA-damaging agents. 33 POSTER Combining the long-acting topoisomerase 1-inhibitor etirinotecan pegol with the PARP inhibitor rucaparib to provide anti-tumor synergy without increased toxicity U. Hoch1 , D. Charych2 . 1 Nektar Therapeutics, Development, San Francisco CA, USA; 2 Nektar Therapeutics, Research, San Francisco CA, USA Background: PARP inhibition sensitizes cells to DNA-damaging agents, particularly topoisomerase 1-inhibitors, where the combination shows synergy in nonclinical studies. However, in the clinic, the combination of either topotecan or irinotecan with PARP inhibitors showed severe hematologic toxicities, requiring dose reductions beyond optimal therapeutic effects. Etirinotecan pegol (NKTR-102) is the first long-acting Topoisomerase 1-inhibitor providing continuous exposure to active metabolite throughout the entire chemotherapy cycle with reduced peak concentrations, resulting in better efficacy and safety, including a low rate of Grade 3 neutropenia (11%). Here we present in vivo combination data of etirinotecan pegol and rucaparib in the MX-1 breast cancer model. Methods: Mice bearing BRCA1-deficient MX-1 breast cancer tumors (~100 mm3 ) received either vehicle, PO rucaparib, IV etirinotecan pegol, or a combination of etirinotecan pegol and rucaparib as follows: 30 or