Citations

Abstract Background: The five year survival rate for women with ovarian cancer (OC) is less than 40%. Crucial to improving the outcomes for ovarian cancer patients is the development of accurate pre-clinical models of human epithelial OC, which can be used to unravel the mechanisms underlying its evolution and behaviour. Methods: We have generated a novel xenograft model of human high-grade serous OC (HG-SOC) in order to enable preclinical molecular and drug response assessment of individual HG-SOC: this includes a comparison of standard OC xeno-transplantion approaches (intra-peritoneal, subcutaneous and sub renal capsular), with the novel use of the rodent ovarian bursa, the orthotopic site. Utilising the rodent bursa may better replicate the relevant microenvironment and increase the success of EOC xeno-transplantion. Other aspects designed to optimize the model include the use of NOD-SCID-IL-2rg recipient mice, systemic estrogen supplementation and transplantation of fresh human HG-SOC fragments which have had no prior in vitro culture. Histological, functional and molecular analysis of the novel xenograft cohort (at baseline and following xenotransplantation) includes histological review; documentation of in vitro Homologous Recombination (HR) DNA repair and drug response capabilities (using novel α-irradiation and explant drug assays); classification according to molecular subtype (Tothill classfier); documentation of NHEJ pathway status, BRCA1/2 status and other DNA repair gene status. In vivo drug treatment studies are being performed, with the choice of treatment targeted to the specific molecular characteristics of the HG-SOC in question. Results: Fourteen consecutive chemotherapy naive potentially HG-SOC samples have been collected. Corresponding clinical data has been collected. Data concerning DNA repair capability and response to DNA damaging agents will be presented, including IHC for markers of DNA damage (γH2AX), DNA repair (RAD51) and apoptosis (capsase 3 cleavage). HG-SOC in this cohort have been classified according to Tothilll (Tothill et al 2008). Eight appropriate HG-SOC have been transplanted and 5 of the first 6 have successfully xenografted, with phenotyping of xenografts underway. In vivo analysis of response to cisplatin treatment and other relevant therapeutics will be presented. Conclusions: A novel xenograft model has been developed of human HG-SOC, which includes comparative characterization of important prognostic features both in the baseline panel of fresh human HG-SOC and in subsequent xenografts. This clinically, functionally and molecularly annotated consecutive xenograft cohort of HG-SOC will provide outstanding utility for the development of improved therapeutic approaches. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3276. doi:1538-7445.AM2012-3276

Abstract Human Sulfatase1 enzyme (HSulf-1) is down-regulated in a majority of ovarian cancer cell lines and primary tumors, but the functional consequence of this downregulation remains unclear. Here, we show that down-regulation of HSulf-1 by RNA interference resulted in an elevated level of phosphorylated Erk and phosphorqylated Akt as well as decreased Bim level in OV202 ovarian cancer cells. Treatment with PD98059 (p-Erk inhibitor) but not LY90024 (PI3K inhibitor) resulted in increased Bim level in Hsulf-1 knockdown cells, suggesting that increased Erk pathway plays a more important role in modulating Bim level after loss of HSulf-1. Consistently, the results of real-time PCR confirmed that there was no significant change in the mRNA level of Bim after knockdown of HSulf-1 expression. When treated with CDDP, OV202 cells with knockdown of HSulf-1 displayed more resistance to drug-induced apoptosis, with IC50 value 3-fold higher than cells with non-targeting shRNA (NTC), while SKOV3 cells with overexpression of HSulf-1 were more sensitive to CDDP-induced apoptosis compared to the control cells. Moreover, HSulf-1 downregulated OV202 cells showed remarkable growth of colonies in soft agar compared to no colony growing with NTC cells. When injected subcutaneously, HSulf-1 ShRNA clones but not NTC cells formed xenogrfts in nude mice. Importantly, rescuing Bim expression in HSulf-1 shRNA cells not only endowed cells sensitivity to CDDP treatment, but also significantly decreased numbers of colony formation in soft agar and significantly retarded tumor growth in nude mice. Collectively, these results strongly suggest that down-regulation of HSulf-1 promotes chemoresistance and tumorigenicity in ovarian cancer cells through post-translational downregulation of Bim level. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4179. doi:1538-7445.AM2012-4179

Abstract Purpose: Previous studies have shown that the replication checkpoint, which involves the kinases ataxia telangiectasia mutated and Rad3 related (ATR) and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical acute myelogenous leukemia (AML) during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro. Experimental design: AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA, and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays. Results: Immunoblotting revealed increased Chk1 phosphorylation, a marker of checkpoint activation, in more than half of Chk1-containing AMLs after 48 hours of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S-phase arrest but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the antiproliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors. Conclusions: These results not only provide evidence for cytarabine-induced S-phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S-phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted. Clin Cancer Res; 18(19); 5364–73. ©2012 AACR.