Primary Human Tumor Xenografts Research Articles

Phosphoinositide 3'-Kinase (PI3K) Delta Inhibitor, GS-9820, Enhances Anti-Tumor Efficacy When Combined with Conventional Chemotherapeutic Agents in Primary Samples and Human Tumor Xenograft Model of Multiple Myeloma

AbstractAbstract 4017Phosphatidylinositide 3-kinases (PI3K) are a family of lipid kinases that are involved in signaling events which control a diverse number of cellular processes. The class I PI3Ks contain 4 isoforms designated p110a, b, d, g, and are activated by cell surface receptors. Aberrant regulation of the PI3K signaling pathway is frequently observed in a wide range of human malignancies including those of hematological origin and has been shown to play an important role in tumor progression. GS-9820 is a highly selective and potent p110d inhibitor (IC50 of 14 nM) with 114- to 400-fold selectivity over the other class I PI3K enzymes and no activity against Class II and III PI3K family members or other PI3K-related proteins including mTOR and DNA-PK. Although a role for PI3Kd has been shown in multiple myeloma (MM), GS-9820 has not been previously evaluated in combination with anti-MM agents in primary MM tumor cells. Hence, we determined the effects of GS-9820 as a single agent and in combination with melphalan, dexamethasone, doxorubicin, or bortezomib on primary MM cells. GS9820 alone inhibited MM tumor cell proliferation in a concentration-dependent fashion with a 50% growth inhibition (IC50) of primary MM cells between 1–10 μM. Notably, combinations of GS-9820 (100nM-1μM) and melphalan (10 μM) or dexamethasone (1 μM) significantly decreased cellular proliferation (P < 0.05), compared to either drug alone in all 6 MM BMMC samples. GS-9820 was synergistic when combined with doxorubicin on primary MM cells from two of these patients’ samples. Similarly, this PI3K inhibitor synergized when it was combined with bortezomib on primary MM cells from two of these patients. Using our LAGk-2 human tumor xenograft model, the combination of GS-9820 and melphalan provided greater growth inhibition than the individual drugs alone. Specifically on day 42 post-tumor implantation, LAGk-2-bearing mice treated with single agent GS-9820 did not show a significant reduction in tumor growth compared to vehicle-treated mice. In contrast, melphalan alone resulted in a significant decrease in tumor size when compared to vehicle-treated mice at this same time point (P = 0.0302). Importantly, the combination of GS-9820 plus melphalan resulted in statistically significantly smaller tumors when compared to melphalan alone (P = 0.0495), GS-9820 alone (P = 0.0061) and vehicle-control (P = 0.0025). In this study, we demonstrate that the combination of GS-9820 with melphalan or dexamethasone markedly inhibits primary MM cell proliferation in vitro. Furthermore, the combination of GS-9820 and melphalan shows enhanced anti-MM effects compared to single agent treatment in vivo using theLAGk-2 human xenograft MM model. The results from these studies suggest that the combination of the PI3K inhibitor, GS-9820, and other anti-MM drugs may be an effective treatment for MM patients. Disclosures:Johnson:Gilead Sciences: Employment. Lannutti:Gilead Sciences: Employment.

Combined Erlotinib and Cetuximab overcome the acquired resistance to epidermal growth factor receptors tyrosine kinase inhibitor in non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) cells with somatic mutations in epidermal growth factor receptors (EGFR) are initially susceptible to tyrosine kinase inhibitor (TKI); however, eventually resistance to TKI is developed in these cells, which leads to the failure of treatment. The most common mechanism of this acquired drug resistance is development of a secondary T790M mutation in EGFR. In this study, we investigated the effects of the combination of Erlotinib and Cetuximab on T790M and L858R mutation lung cancer cells lines (H1975), in the primary NSCLC cells with the T790M mutation and TKI-resistant EGFR mutations human tumor xenograft model (H1975). The effects of these two agents on cell proliferation, apoptosis, and EGFR-dependent signaling were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V staining, and Western blotting. Sensitivity of EGFR inhibitors was detected in the primary tumor cell suspension and human tumor xenograft model (H1975). Compared with single-agent treatment, the combination of Cetuximab and Erlotinib increased apoptosis of EGFR TKI-resistant NSCLC cells (H1975), resulting in more pronounced growth inhibition on cell proliferation and significant inhibition of EGFR-dependent signaling. These data suggest that treatment with a combination of Erlotinib and Cetuximab overcomes T790M-mediated drug resistance.

Abstract 854: Patient stratification strategies for NVP-BGJ398, a selective pan-FGFR inhibitor in phase I clinical trials

Abstract The mammalian fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) family comprises 22 FGF ligands, which exert their action through 4 highly conserved transmembrane tyrosine kinase receptors (FGFR1, FGFR2, FGFR3 and FGFR4). This highly complex signaling network regulates fundamental developmental pathways and physiological processes in the adult organism. A growing body of evidence linking deregulation of the FGF/FGFR system, as a consequence of genetic alterations, to the pathogenesis of cancer makes these receptors attractive targets for therapeutic intervention. To this purpose we have developed NVP-BGJ398, an orally bioavailable, selective pan-FGFR kinase inhibitor which is currently in clinical Phase I dose escalation trial. In order to preclinically identify and validate patient stratification biomarkers that accurately predict for response to NVP-BGJ398 in the clinic, we have utilized the Cancer Cell Line Encyclopedia (CCLE). The CCLE is a collection of almost 1000 cancer cell lines representing multiple tumor types that, in a collaborative effort between The Novartis Institutes for BioMedical Research and the Broad Institute, has been comprehensively annotated in terms of genome-scale mRNA expression, gene copy number alterations and gene mutations (http:/www.broadinstitute.org/ccle). In addition, over half of these cell lines were subjected to high-throughput cell viability assays in the presence NVP-BGJ398, with the aim of generating cell line sensitivity data. Here we show the validation of predictive biomarkers of response in primary human tumor xenografts, revealed through the analysis of the CCLE, as well as the identification of potentially new indications for NVP-BGJ398. 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 854. doi:1538-7445.AM2012-854

Abstract 3285: Hedgehog pathway inhibition delays regrowth of ovarian cancer following paclitaxel and carboplatinum only if initiated immediately after completion of chemotherapy

Abstract The high mortality associated with ovarian cancer is attributed to the lack of any reliable early detection method, unknown pathogenesis of the disease, and the development of recurrent and chemoresistant tumors. Current efforts have focused on the identification of therapeutics that may be used independently or in combination with current chemotherapeutic regimens to reduce tumor volume. To date, limited research has focused on preventing or delaying disease recurrence. The Hedgehog (Hh) signal transduction pathway is inactive in most adult cells. Malignant activation of the Hh pathway through the signaling protein Smoothened (Smo) occurs in a broad range of cancers, including ovarian. IPI-926 is a potent orally delivered small molecule that targets the Hh pathway by inhibiting Smo. Recent studies from our laboratory provide evidence that IPI-926 slows serous ovarian cancer growth in a primary human tumor xenograft model. More importantly, IPI-926 delays the resurgence of tumor growth typically observed after cytoreduction with paclitaxel and carboplatinum (T/C) treatment. Our current objective was to assess whether this effect of IPI-926 required that the Smo inhibitor be administered during a critical window following T/C treatment to prevent the resurgence of tumor growth. To test our hypothesis, mice bearing human ovarian cancer xenografts were treated with vehicle or T/C. T/C treatment was withdrawn following significant reduction (30-50%) in tumor volume. The original vehicle treated cohort was divided into 2 arms which then either received IPI-926 or continued on vehicle for the duration of the experiment. Mice in the T/C cohort were divided into 3 groups. Group 1 received vehicle alone (T/C-Vehicle), group 2 received IPI-926 immediately following the last T/C dose (T/C-IPI-926) and group 3 received no vehicle or IPI treatment for 14 days following the last T/C dose (Window). Group 3 was then maintained on IPI-926 treatment starting at day 15 post-T/C withdrawal. The withdrawal of T/C led to a dramatic increase in tumor volume in the T/C-Vehicle group. As previously observed, tumor growth inhibition was maintained in mice receiving IPI-926 immediately following the cessation of T/C treatment. In contrast, delaying the administration of IPI-926 following T/C prevented the suppression of tumor growth as evidenced by the increased tumor volume. Our data suggest that blocking Hh pathway activity immediately following chemotherapy maintains and prolongs the inhibitory effect of chemotherapy on ovarian tumor growth. The absence of an IPI-926-mediated inhibition of tumor resurgence following a 14-day delay in treatment supports the concept that there is a critical period for tumor re-establishment. Our data, along with those of others, suggest that the early stages of ovarian tumor re-growth may be dependent on Hh pathway signaling. 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 3285. doi:1538-7445.AM2012-3285

Abstract A225: Dose scheduling and efficacy analysis of the SMAC mimetic TL32711 in primary melanoma tumor xenotransplant models.

Abstract Inhibitor of Apoptosis Proteins (IAPs) are cellular proteins that prevent cell death by apoptosis and are frequently overexpressed in cancer cells resulting in elevated levels leading to tumor survival and progression. SMAC mimetics are a new class of apoptosis-inducing agents that relieve the effects of IAPs including XIAP, cIAP1 and cIAP2 resulting in apoptosis of sensitive tumor cells from a broad range of tumor types. TL32711 is a potent SMAC mimetic currently in Phase 1 clinical trials. To date, a weekly IV infusion schedule has proved to be well tolerated in patients, resulting in sustained cIAP1 suppression throughout the dosing interval. Initial pharmacokinetics modeling of TL32711 in mice bearing the MDA-MB-231 tumor indicated a potential efficacy benefit may be possible with a biweekly dosing schedule. The objectives of the current study were to 1) evaluate the efficacy of TL32711 as a single agent in primary human melanoma tumor xenograft models, 2) assess the efficacy and tolerability of TL32711 in combination with carboplatin and paclitaxel and 3) determine if a biweekly dosing schedule is more effective than weekly administration. Significant tumor growth inhibition was observed in 5 of 6 of the primary melanoma tumor xenografts evaluated following treatment with single agent TL32711 (30 mg/kg, IP). Combining TL32711 with carboplatin and paclitaxel resulted in a further enhancement in anti-tumor efficacy with tumor regressions noted in 4 of the 6 models without any marked changes in tolerability (&amp;lt;14% reduction in bodyweight). Based on the initial PK modeling a follow up study was conducted to assess the activity of TL32711 in a primary melanoma model when the dose was fractionated (15 mg/kg twice/week versus 30 mg/kg once/week). Surprisingly, the biweekly dosing schedule did not result in enhanced anti-tumor activity and demonstrated equivalent suppression of cIAP1 in tumors compared to the weekly dosing schedule. Pharmacokinetic analysis of the TL32711 in tumor tissue at 15, 30 and 60 mg/kg revealed that TL32711 exhibits a greater than dose proportional relationship in that a 4-fold increase in dose, resulted in a 14-fold increase in exposure. This increase in exposure led to a change in the TL32711 tumor half-life from 56 to 166 hrs, possibly due to the saturation of an efflux transporter at higher dose levels. Together, these data show that TL32711 is highly active in primary human melanoma xenografts and that efficacy can be enhanced by combination therapy with carboplatin and paclitaxel without reducing tolerability. These data also demonstrate that biweekly dosing confers no advantage over the current clinical weekly dosing regimen due to the dose dependent changes in TL32711 half-life and exposure observed in tumor tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A225.

Abstract A236: Human liposarcoma growth inhibition by novel CDK4/6 inhibitor LEE011.

Abstract Background: Well-differentiated and de-differentiated liposarcomas (WD/DDLPS) are characterized by high-level chromosomal amplification of 12q13–15, a region including the cyclin-dependent kinase 4 (CDK4) oncogene. In this study, we explored the effects of LEE011, a novel selective inhibitor of CDK4/CDK6 on human liposarcoma cell lines and primary tumor xenografts. Materials and Methods: We examined the expression of cell cycle regulatory proteins in human liposarcoma cell lines and human normal preadipocytes/adipocytes. CDK4 small interfering RNA (siRNA) or LEE011 was applied to liposarcoma cell lines and the biological consequences were determined by phospho-RB immunoblot, cell cycle assay and cell enumeration. In addition, liposarcoma cells were transfected with RB siRNA to determine the specificity of the effects of LEE011. Nude mice implanted with human liposarcoma cell lines or primary tumors were treated with LEE011 or vehicle by oral gavage. After 3 daily doses, in vivo BrdU incorporation assay, immunostains for phospho-RB, and 18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) scan were performed. Tumor size and animal weight were serially measured during 3 weeks of dosing. Results: CDK4 and p-Rb are highly expressed in human liposarcoma cell lines in comparison with normal preadipocytes/adipocytes. Both CDK4 siRNA and LEE011 inhibited RB phosphorylation at the CDK4/6-specific sites Ser780 and Ser807/811 in a dose-dependent manner and dramatically inhibited liposarcoma cell growth. Cell cycle analysis demonstrated arrest at G0/G1. siRNA-mediated knockdown of RB1 rescued the inhibitory effects of LEE011, suggesting that LEE011 inhibited proliferation as expected through the RB pathway. Oral administration of LEE011 to mice bearing human liposarcoma xenografts reduced tumor 18F-FDG uptakes by approximately 50% and dramatically decreased biomarkers including RB phosphorylation and BrdU incorporation in vivo. Continued LEE011 treatment inhibited growth of established cell line xenografts as well as primary human liposarcoma tumor xenografts without detrimental effects on mouse weight. Conclusions: LEE011 decreases cell cycle progression, hypermetabolism, and proliferation of human liposarcomas in an RB-dependent manner, consistent with inhibition of CDK4. Clinical trials of LEE011 in patients with liposarcoma are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A236.

Abstract A25: Phase 1 PK/PD analysis of the Smac-mimetic TL32711 demonstrates potent and sustained cIAP1 suppression in patient PBMCs and tumor biopsies.

Abstract The Smac mimetic TL32711 differentially targets members of the inhibitor of apoptosis proteins (cIAP1, cIAP2 and XIAP) involved in the blockade of apoptosis. TNFα-activated pro-survival signaling pathway is maintained by the cIAPs. TL32711 causes rapid autoubiquitinylation and proteosomal degradation of the cIAP1 converting TNFα signaling from pro-survival to pro-apoptotic and potently inhibiting TNFα driven canonical NF-kB signaling. TL32711 also relieves inhibition of caspase-3 and -7 by XIAP to further potentiate apoptosis. The pharmacokinetics (PK) and pharmacodynamics (PD) of TL32711 have been studied in human tumor xenografts, patient plasma/PBMCs and Phase 1 tumor biopsy samples. In mice bearing the MDA-MB-231 xenograft, TL32711 is rapidly and extensively taken up into the tumor (tumor/plasma AUC ratio &amp;gt;22) and is eliminated slowly with a half-life of 96 hrs (20 hrs in plasma). A PK/PD link model was used to characterize the relationship between TL32711 tumor concentrations and cIAP1 suppression. cIAP1 suppression was dose and time dependent with cIAP1 levels reduced to &amp;lt;20% baseline within 30 minutes and with &amp;gt;70% inhibition maintained 7–14 days post treatment following a single IV bolus dose (5 mg/kg). TL32711 had a potent effect on tumor cIAP1 levels (EC50 24 ng/g) and caused significant tumor growth inhibition and regressions at doses ≥2.5 mg/kg q3D. Efficacy has also been evaluated in primary human melanoma tumors, recently derived from patients and transplanted into nude mice. Significant tumor growth inhibition was observed in 5/6 primary melanoma tumor xenografts with mean Day 7 tumor concentrations of 187, 579 and 2658 ng/g at 15, 30 and 60 mg/kg respectively. TL32711 PK/PD (drug concentration analysis and cIAP1 degradation in PBMCs and tumor biopsies) has also been investigated in patients as part of the single agent Phase I study. Following weekly, 30 min IV infusions TL32711 plasma PK was dose proportional and non-accumulating (0.18 to 47 mg/m2). Plasma PK was tri-exponential with a long terminal t1/2 (73–79 hrs). The target AUC in plasma for therapeutic activity (71 h.ng/mL) based on the MDA-MB-231 model was achieved in patients at dose &amp;gt;2.88 mg/m2 (Mean AUC 86 h.ng/mL). This exposure was associated with marked uptake and retention in PBMCs (t1/2 = 29–35hrs) and resulted in prolonged cIAP1 suppression over 7 days. A dose related increase in PBMC PARP cleavage and plasma caspase-3 activity was also observed indicative of apoptosis pathway activation. TL32711 PK/PD was also assessed in tumor biopsy samples from patients 4 hrs to 6 days post treatment (11.5 to 17.2 mg/m2). TL32711 is extensively taken up into the tumor with levels &amp;gt;350 ng/g on day 6, significantly in excess of the EC50 for cIAP1 inhibition. Estimated tumor exposure at 35 to 47 mg/m2 was also in excess of the measured drug levels observed at 15 to 30 mg/kg in the primary human tumor xenograft models in mice. Together these PK/PD data show that TL32711 results in potent and sustained cIAP1 suppression over 7 days at tolerable dose levels with evidence of apoptosis pathway activation and promising early signs of efficacy in patients with solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A25.

Abstract LB-58: Spontaneous Epstein Barr virus-associated lymphomagenesis in primary human solid tumor xenografts

Abstract Xenotransplantation of primary human solid tumors into immunodeficient mice is widely utilized as a tool for the study of human cancer biology. We have generated subcutaneous xenografts from a large number of human hepatocellular carcinoma (HCC) resection specimens, and have observed that subcutaneous implantation of tumor fragments or bulk cell suspensions results in a much higher rate of primary engraftment than implantation of cells sorted to exclude CD45+ leukocytes in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. While the majority of xenografts arising from tumor fragments or bulk tumor cells retain typical characteristics of parent HCCs, we have noted the frequent development of very rapidly-growing tumors that do not share these attributes, and sought to further characterize these tumors. Histopathological analysis of these tumors revealed monomorphic populations of lymphoid cells demonstrating nuclear atypia, high mitotic index, and invasion into surrounding tissues. Flow cytometry and immunohistochemistry revealed that these tumors consisted almost completely of cells expressing the human leukocyte antigen CD45 and the human B-cell antigen CD20. In situ hybridization demonstrated strong expression of the Epstein-Barr virus (EBV)-encoded small RNA (EBER) in all tumors. Staining of tumors for mouse H2K expression was negative in all cases. These findings suggest that the originally implanted human HCC tumor fragments or bulk tumor cells were replaced by human B-cell lymphomas. No lymphomas arose from the implantation of human HCC cells that were sorted to exclude human CD45+ leukocytes. Histopathological re-examination of the parent tumors in all cases confirmed a diagnosis of HCC with no evidence of lymphoma. Analogous to post-transplant lymphoproliferative disorder (PTLD) in humans, our data suggest that these lymphomas spontaneously developed in xenografts through EBV-mediated transformation of EBV-infected passenger lymphocytes harbored in patient tumors in the permissive context of an immunodeficient host environment. Recognition of this phenomenon is important for those utilizing primary human tumor tissue in xenotransplantation assays, because the development of lymphomas may confound accurate analysis of tumor biology and may out-compete the tumor type of interest resulting in the loss of valuable experimental samples. Our observations highlight an important potential pitfall of human solid tumor xenotransplantation assays in models where primary engraftment requires the implantation of bulk cells or tumor fragments which may contain passenger lymphocytes. In such circumstances, our observations underscore the critical importance of routinely quantifying the degree of human lymphocyte “contamination” in xenograft-derived data and implementing strategies to deplete or eliminate human lymphocytes from these assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-58. doi:10.1158/1538-7445.AM2011-LB-58

Abstract 2828: Antitumor activity of the investigational proteasome inhibitor MLN9708 in preclinical models of NSCLC

Abstract The successful development of VELCADE® (bortezomib) for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for hematological malignancies. The investigational drug MLN9708 is a modified dipeptidyl boronic acid and a potent, reversible and specific inhibitor of the proteasome. MLN9708 is currently being evaluated in Phase I trials for hematologic malignancies as well as solid tumors. In preclinical models, MLN9708 showed better antitumor activity and PD response compared to bortezomib.Upon exposure to aqueous solutions or plasma, MLN9708 immediately hydrolyzes to MLN2238, the biologically active form. MLN2238 was used for all preclinical studies described in this report. We evaluated the antitumor activity of MLN2238 in several non small cell lung cancer (NSCLC) xenografts. More than 70% tumor growth inhibition was observed in the HCC-827 xenograft model with 13 mg/kg, IV, twice weekly (BIW) dosing. In the H1650 NSCLC xenograft model, 40-50% tumor growth inhibition was found at 8 and 11 mg/kg, IV, BIW dosing. However, a synergistic effect on H1650 tumor growth inhibition was found when MLN2238 was combined with chemotherapeutic agents including doxorubicin 8 mg/kg IV, weekly (QW) dosing (90% tumor growth inhibition) and taxotere 5 and 10 mg/kg, IV, QW (80-100% tumor growth inhibition). Furthermore, the combination of MLN2238 (8 and 11 mg/kg, IV, BIW) and taxotere (10 mg/kg, IV, QW) resulted in tumor regression in the H1650 xenograft model, whereas no regressions were observed with either drug as a single agent. Similar single agent activity and combination effect with taxotere was observed We also evaluated single agent activity and combination with taxotere in an early-passage primary human tumor xenograft model, PHTX-132Lu. In an effort to understand the factors that may determinecontributing sensitivity vs resistance to proteasome inhibitors, we tested the ability of MLN2238 to inhibit anchorage independent growth and colony formation of NSCLC tumor cells in semi-solid medium. For these studies, tumor cells are derived from primary human tumor explants which have been maintained by serial passage in immunocompromised mice. Initial experiments show a range of sensitivity to MLN2238 among the primary tumors, Comparisons of genomic data, including gene expression profiles, copy number variation, and sequencing of commonly mutated genes, may illuminate factors contributing to sensitivity and resistance. In addition, in vivo studies will be performed on selected models to examine the value of this in vitro colony formation assay in predicting in vivo sensitivity. Data suggest that MLN2238 has antitumor activity alone and in combination with cytotoxic agents in preclinical models of NSCLC, and these results support the ongoing Phase I clinical investigation of MLN9708 in solid tumor types including NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2828. doi:10.1158/1538-7445.AM2011-2828

Significance of the hedgehog (Hh) pathway in ovarian cancer xenograft growth.

5058 Background: Initial immunohistochemical analysis revealed that about 50% of ovarian cancer samples expressed sonic Hh ligand in the tumor epithelium, suggesting this signaling pathway might be active in this disease. Thus we examined the activity of IPI-926, a potent inhibitor of the Hh pathway that blocks the activity of Smoothend, in a preclinical model of serous ovarian cancer in an adjuvant and maintenance setting. Methods: Primary human serous ovarian tumor xenograft tissue was utilized to generate tumor explants in NOD/SCID mice. Mice bearing tumors (250-1900 mm3) were randomized into four groups 1) IPI-926 PO gavage q 24 hours along with intraperitoneal (IP) vehicle; 2) paclitaxel and carboplatinum (T/C) IP q 7 days with vehicle by PO gavage q 24 hours; 3) IPI-926 by PO gavage q 24 hours + IP T/C; 4) vehicle by PO gavage q 24 hours + IP vehicle q 7 days. The adjuvant treatment period spanned 19 days. In the maintenance arms of the experiment, T/C treatment was stopped in the two groups receivi...

Abstract 4290: The smoothened antagonist NVP-LDE225 targets stroma and cancer stem cells in primary human pancreatic tumor xenografts

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality. Despite surgical intervention and treatment with Gemcitabine, the standard of care, the 5-year survival rate remains less than 5%. Because the aberrant activation of the Hedgehog (Hh) pathway in stroma cells and cancer stem cells in PDAC has become evident during the last few years, therapeutic agents able to inhibit Smoothened (Smo), the positive regulator of the pathway, are under vigorous investigation. We have developed a selective Smo antagonist, NVP-LDE225, which is orally bioavailable and potently inhibits Hh signaling in vitro and in vivo. Using a combination of in vitro and in vivo assays, we evaluated the effects of NVP-LDE225 alone or in combination with Gemcitabine in primary human tumor xenograft models. In these models, NVP-LDE225 dosed together with Gemcitibine resulted in a trend towards combination activity. Tumor samples collected at the end of the studies showed that the Hh pathway was completely inhibited in the stroma of the NVP-LDE225 treated animals. Moreover, analysis of the ratio of tumor to stroma area demonstrated no differences between the drug treated as compared to vehicle treated tumors. After subcutaneous implantation of the human tumors in mice, the mouse stroma replaces the human stroma. Using mouse and human Affymetrix analysis of tumors following in vivo treatment, we were able to differentiate the changes occurring in murine stromal cells versus human epithelial cells. This analysis revealed changes in the regulation of several key developmental and growth factor pathways in the mouse stroma. To study cancer stem cells, we isolated Aldehyde Dehydrogenase (ALDH) positive cells from untreated xenograft tumors and confirmed up regulation of the Hh pathway genes, and that ALDH positive cells were more tumorigenic than ALDH negative cells when implanted in animals. Furthermore, we used 2 different stem cell functional assays, in vitro sphere formation and in vivo serial transplantation to evaluate the effect of NVP-LDE225 on cancer stem cells. We observed that upon NVP-LDE225 treatment the cells had impaired ability to form spheres in vitro and similarly, tumor growth was delayed following in vivo serial transplantation. These experiments suggest that Smo inhibitors can have a high impact on the treatment of this very aggressive cancer. 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 4290.

Remarks on Suprapubic and Lateral Lithotomy and on Litholapaxy

Little is known about the role of epithelial membrane protein-2 (EMP2) in breast cancer development or progression. In this study, we tested the hypothesis that EMP2 may regulate the formation or self-renewal of breast cancer stem cells (BCSC) in the tumor microenvironment. In silico analysis of gene expression data demonstrated a correlation of EMP2 expression with known metastasis-related genes and markers of cancer stem cells (CSC) including aldehyde dehydrogenase (ALDH). In breast cancer cell lines, EMP2 overexpression increased and EMP2 knockdown decreased the proportion of stem-like cells as assessed by the expression of the CSC markers CD44+/CD24−, ALDH activity, or by tumor sphere formation. In vivo, upregulation of EMP2 promoted tumor growth, whereas knockdown reduced the ALDHhigh CSC population as well as retarded tumor growth. Mechanistically, EMP2 functionally regulated the response to hypoxia through the upregulation of HIF-1α, a transcription factor previously shown to regulate the self-renewal of ALDHhigh CSCs. Furthermore, in syngeneic mouse models and primary human tumor xenografts, mAbs directed against EMP2 effectively targeted CSCs, reducing the ALDH+ population and blocking their tumor-initiating capacity when implanted into secondary untreated mice. Collectively, our results show that EMP2 increases the proportion of tumor-initiating cells, providing a rationale for the continued development of EMP2-targeting agents.