Sustained Tumor Growth Inhibition Research Articles

Bcl-XL represents a druggable molecular vulnerability during aurora B inhibitor-mediated polyploidization

Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This "addiction" can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility.

Sustained inhibition of tumor growth and prolonged survival following sequential administration of doxorubicin and zoledronic acid in a breast cancer model

Combination therapy, using agents that target the microenvironment as well as the cancer cells, is common in the treatment of advanced breast cancer. Here, we show that a 6-week course of weekly sequential administration of the cytotoxic drug doxorubicin (2 mg/kg), followed 24 hr later by the antiresorptive agent zoledronic acid (100 microg/kg), causes substantial inhibition of subcutaneous MDA-MB-436 breast tumor growth in immunocompromised mice, leading to significantly increased survival. Tumor growth did not resume following withdrawal of treatment after 6 weeks, with 60% of the animals in this group surviving for more than 160 days. In comparison, animals receiving single-agent therapy all died within 50 days. Molecular analysis of the tumors showed no effect on cell cycle or apoptosis following administration of 100 microg/kg zoledronic acid or 2 mg/kg doxorubicin alone. When doxorubicin was administered 24 hr before zoledronic acid, tumors displayed decreased expression of CYCLINS E1, B, D1 and D3 as well as CDK2, CDC2, CDK4 and CDK7, indicative of cell-cycle inhibition. Tumors from animals receiving sequential treatment also showed induction of both intrinsic- and extrinsic-apoptotic pathways, with increased expression of BAX, decreased expression of BCL-2 and activation of CASPASE 3, 8 and 9. Accumulation of the unprenylated form of RAP1a, a surrogate marker for uptake of zoledronic acid, was only detected in tumors from animals treated with doxorubicin 24 hr before zoledronic acid. Our data are the first to show a sustained antitumor effect in vivo following a limited course of sequential administration of doxorubicin followed by zoledronic acid.

Rapid Suppression of Plasma Testosterone Levels and Tumor Growth in the Dunning Rat Model Treated with Degarelix, a New Gonadotropin-Releasing Hormone Antagonist

Degarelix (FE 200486) is a member of a new class of water-soluble (>50 mg/ml) gonadotropin-releasing hormone (GnRH) antagonists in clinical development for prostate cancer. Upon subcutaneous administration, degarelix forms a gel that results in a sustained release of the compound into the circulation, immediately blocking GnRH receptors in the pituitary and inducing a fast and sustained suppression of gonadotrophin secretion in rats and primates. One of the few animal models of prostate adenocarcinoma is the Dunning R-3327H rat carcinoma transplanted into Copenhagen rats. The growth of the Dunning tumor can be inhibited by various treatments reported to be effective in the clinic, such as GnRH superagonists, antiandrogens, 5-alphareductase inhibitors, tyrosine kinase inhibitors, and surgical castration. We report in this study that degarelix produces a fast and sustained suppression of the pituitary gonadal axis in rats and a similar inhibition of tumor growth compared with surgical castration in the Dunning R-3327H rat carcinoma model. First, degarelix as been compared with d-Trp(6)-luteinizing hormone-releasing hormone and surgical castration on a short-term study (2 months); and second, degarelix has been compared with leuprolide and surgical castration on a long-term study (12 months). In both studies, degarelix demonstrated a sustained inhibition of tumor growth at least comparable with surgical castration. These data provide a convincing profile of degarelix as a potential candidate for the clinical management of sex steroid-dependent pathologies, such as prostate cancer, where long-term reversible chemical castration is required.

106 POSTER Methylnaltrexone inhibits S1P, VEGF and PDGF-induced angiogenesis: role of receptor transativation

c-Met pathway is implicated in the resistance to anti-VEGF therapy in renal cell carcinoma (RCC). However, clinical translation of therapies targeting these pathways has been limited due to dose-limiting toxicities, feedback signaling, and low intratumoral drug accumulation. Here, we developed liposomes encapsulating a multi-receptor tyrosine kinase inhibitor (XL184) to explore the possibility of improving intratumoral concentration, enhancing antitumor efficacy and reducing toxicities. The liposomes showed increased cytotoxicity than XL184, and resulted in a sustained inhibition of phosphorylation of Met, AKT and MAPK pathways in RCC cells. In a RCC tumor xenograft model, the liposomes induced sustained inhibition of tumor growth as compared to XL184, consistent with higher inhibition of kinase signaling pathways. Biodistribution studies revealed higher accumulation of the liposomes in tumor, which translated into lower toxicities. This study shows the use of liposomes for effective inhibition of multi-kinase pathways, which can potentially emerge as a new treatment for RCC.

Antitumor Activity of ZD6474, a Vascular Endothelial Growth Factor-2 and Epidermal Growth Factor Receptor Small Molecule Tyrosine Kinase Inhibitor, in Combination with SC-236, a Cyclooxygenase-2 Inhibitor

Abstract Purpose: The epidermal growth factor receptor (EGFR) autocrine pathway plays an important role in cancer cell growth. Vascular endothelial growth factor (VEGF) is a key regulator of tumor-induced endothelial cell proliferation and vascular permeability. Enhanced cyclooxygenase-2 (COX-2) expression has been linked to cancer cell proliferation, EGFR activation, VEGF secretion, and tumor-induced angiogenesis. ZD6474 is an orally available, small molecule, dual VEGF receptor-2 (VEGFR-2) and EGFR tyrosine kinase inhibitor. We investigated the activity of ZD6474 in combination with SC-236, a selective COX-2 inhibitor, to determine the antitumor activity of the simultaneous blockade of EGFR, COX-2, and VEGF functions. Experimental Design: The antitumor activity in vitro and in vivo of ZD6474 and/or SC-236 was tested in human cancer cell lines with a functional EGFR autocrine pathway. Results: The combination of ZD6474 and SC-236 determined supra-additive growth inhibition in all cancer cell lines tested. In nude mice bearing established human colon (GEO) or lung adenocarcinoma (A549) cancer xenografts and treated with ZD6474 and/or SC-236 for 3 weeks, a reversible tumor growth inhibition was seen with each agent, whereas a more prolonged growth inhibition that lasted for 3 to 5 weeks following the end of treatment resulted from the combination of the two agents. A long-term, 10-week treatment with ZD6474 plus SC-236 resulted in sustained tumor growth inhibition in all mice with tumor eradication in 3 of 10 GEO tumor–bearing mice and in 4 of 10 A549 tumor–bearing mice. Conclusions: This study provides a rationale for evaluating the simultaneous blockade of EGFR, COX-2, and VEGF signaling as cancer therapy in a clinical setting.

Preclinical validation of anti-TMEFF2-auristatin E–conjugated antibodies in the treatment of prostate cancer

Current treatments for advanced stage, hormone-resistant prostate cancer are largely ineffective, leading to high patient mortality and morbidity. To fulfill this unmet medical need, we used global gene expression profiling to identify new potential antibody-drug conjugate (ADC) targets that showed maximal prostate cancer-specific expression. TMEFF2, a gene encoding a plasma membrane protein with two follistatin-like domains and one epidermal growth factor-like domain, had limited normal tissue distribution and was highly overexpressed in prostate cancer. Immunohistochemistry analysis using a specific monoclonal antibody (mAb) to human TMEFF2 showed significant protein expression in 74% of primary prostate cancers and 42% of metastatic lesions from lymph nodes and bone that represented both hormone-naïve and hormone-resistant disease. To evaluate anti-TMEFF2 mAbs as potential ADCs, one mAb was conjugated to the cytotoxic agent auristatin E via a cathepsin B-sensitive valine-citrulline linker. This ADC, Pr1-vcMMAE, was used to treat male severe combined immunodeficient mice bearing xenografted LNCaP and CWR22 prostate cancers expressing TMEFF2. Doses of 3 to 10 mg/kg of this specific ADC resulted in significant and sustained tumor growth inhibition, whereas an isotype control ADC had no significant effect. Similar efficacy and specificity was shown with huPr1-vcMMAE, a humanized anti-TMEFF2 ADC. No overt in vivo toxicity was observed with either murine or human ADC, despite significant cross-reactivity of anti-TMEFF2 mAb with the murine TMEFF2 protein, implying minimal toxicity to other body tissues. These data support the further evaluation and clinical testing of huPr1-vcMMAE as a novel therapeutic for the treatment of metastatic and hormone-resistant prostate cancer.

Identification and validation of anti-TMEFF2-Auristatin E conjugated antibodies in the treatment of prostate cancer

2557 Background: Current treatments for advanced stage prostate cancer are largely ineffectual, resulting in high mortality and morbidity. Experimental strategies to fulfill this unmet medical need include the use of toxin-based antibody-drug conjugates (ADCs) to specifically target cancer cells in vivo. Using gene expression profiling, TMEFF2 was identified as being selective to prostate cancer and was validated as a target suitable for an ADC approach using pre-clinical xenograft models in vivo. Methods: A panel of antibodies specific to TMEFF2 protein was generated. One antibody was selected on the basis of affinity and in vitro functionality as an ADC to undergo further analysis in vivo. Results: TMEFF2, a gene encoding a plasma membrane protein with 2 follistatin-like domains and one EGF-like domain, was found to exhibit a restricted normal tissue expression profile and was determined to be highly over-expressed in prostate cancer. A monoclonal antibody (mAb) raised against TMEFF2 was used in immunohistochemistry (IHC) of clinical specimens confirming significant TMEFF2 protein expression in 73% of primary prostate tumors. The exquisite tissue specificity of TMEFF2 followed by the over expression in prostate cancer, allowed for the use of an ADC based therapeutic strategy. We used an Auristatin-E conjugated anti-TMEFF2 mAb, Pr1-vcMMAE, to evaluate the effect on human prostate cancer xenograft tumors grown in male severe combined immunodeficient (SCID) mice. Treatment of SCID mice bearing LNCaP and CWR22 tumors, both of which express TMEFF2, with doses of 3 to 10 mg/kg of ADC resulted in significant and sustained tumor growth inhibition. No overt in vivo toxicity was observed with either murine or humanized ADC, despite significant cross-reactivity of anti-TMEFF2 mAb with murine TMEFF2 protein, indicating a high safety profile. Conclusions: The TMEFF2 protein exhibits the desired expression profile for an ADC target. Our data on significant tumor growth inhibition in the in vivo treatment models support humanized Pr1-vcMMAE as a novel therapeutic for the treatment of prostate cancer. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Protein Design Labs, Inc. Protein Design Labs, Inc. Protein Design Labs, Inc.

Identification and validation of anti-TMEFF2-Auristatin E conjugated antibodies in the treatment of prostate cancer

2557 Background: Current treatments for advanced stage prostate cancer are largely ineffectual, resulting in high mortality and morbidity. Experimental strategies to fulfill this unmet medical need include the use of toxin-based antibody-drug conjugates (ADCs) to specifically target cancer cells in vivo. Using gene expression profiling, TMEFF2 was identified as being selective to prostate cancer and was validated as a target suitable for an ADC approach using pre-clinical xenograft models in vivo. Methods: A panel of antibodies specific to TMEFF2 protein was generated. One antibody was selected on the basis of affinity and in vitro functionality as an ADC to undergo further analysis in vivo. Results: TMEFF2, a gene encoding a plasma membrane protein with 2 follistatin-like domains and one EGF-like domain, was found to exhibit a restricted normal tissue expression profile and was determined to be highly over-expressed in prostate cancer. A monoclonal antibody (mAb) raised against TMEFF2 was used in immunohistochemistry (IHC) of clinical specimens confirming significant TMEFF2 protein expression in 73% of primary prostate tumors. The exquisite tissue specificity of TMEFF2 followed by the over expression in prostate cancer, allowed for the use of an ADC based therapeutic strategy. We used an Auristatin-E conjugated anti-TMEFF2 mAb, Pr1-vcMMAE, to evaluate the effect on human prostate cancer xenograft tumors grown in male severe combined immunodeficient (SCID) mice. Treatment of SCID mice bearing LNCaP and CWR22 tumors, both of which express TMEFF2, with doses of 3 to 10 mg/kg of ADC resulted in significant and sustained tumor growth inhibition. No overt in vivo toxicity was observed with either murine or humanized ADC, despite significant cross-reactivity of anti-TMEFF2 mAb with murine TMEFF2 protein, indicating a high safety profile. Conclusions: The TMEFF2 protein exhibits the desired expression profile for an ADC target. Our data on significant tumor growth inhibition in the in vivo treatment models support humanized Pr1-vcMMAE as a novel therapeutic for the treatment of prostate cancer. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Protein Design Labs, Inc. Protein Design Labs, Inc. Protein Design Labs, Inc.

Growth inhibition of an orthotopic glioblastoma in immunocompetent mice by cationic lipid-DNA complexes.

The effect of a cationic liposome non-coding plasmid DNA complex on the growth of an intracerebral glioblastoma in an immunocompetent syngeneic mouse strain was evaluated. Previous studies of extraneural tumors in mice have demonstrated that such complexes containing plasmid DNA are capable of stimulating a potent Th-1 cytokine immune-mediated response with a dramatic inhibition of tumor growth. A DOTIM-cholesterol cationic liposome complexed to non-coding plasmid DNA (EV-CLDC) was administered intravenously (i.v.) at weekly intervals to 6-week-old male mice of the B6D2F1 strain at either 3, 10 or 17 days post-inoculation (DPI) of 4C8 glioblastoma cells. Tumor growth was monitored by volumetric image analysis obtained from sequential weekly magnetic resonance imaging studies of the brain. Experiments were terminated between 30 to 38 DPI. Terminal tumor volumes calculated from histological sections directly correlated with tumor volumes from corresponding MR images. The EV-CLDC administered at 3 DPI resulted in a statistically significant (P<0.0001) sustained inhibition of tumor growth compared with tumors in mice administered only individual components of the EV-CLDC. The EV-CLDC similarly inhibited growth of longer established glioblastomas. Histopathologic evaluation of terminal tumors did not find any hemorrhage, edema or necrosis in either the EV-CLDC-treated or control tumors. The results indicate that an i.v.-administered EV-CLDC can significantly inhibit the growth of a brain tumor in immunocompetent syngeneic mice.

Anti-tumor effect of intraperitoneal administration of cisplatin-loaded microspheres to human tumor xenografted nude mice

This study evaluates the anti-tumor effect of cisplatin-loaded microspheres (CDDP-MS) against peritoneal carcinomatosis using human tumor xenografts. The incorporated CDDP was released from CDDP-MS for 3 weeks in vivo as well as in vitro. CDDP-MS at a dose of 35 mg/kg (at maximal tolerable dose (MTD)) showed effective anti-tumor activity (tumor growth inhibition rate (IR)=70.3%) against Li-7 (human liver cancer) xenografts transplanted into the peritoneal cavity. This procedure also resulted in increased life span (ILS (%)=47.2%), whereas CDDP dissolved in saline solution (CDDP–SOL) at a dose of 8 mg/kg (at MTD) was ineffective (IR=15.7%, ILS=2.6%). Likewise, CDDP-MS (35 mg/kg) significantly prolonged the mean survival time (ILS=50.8%) compared with a CDDP–SOL group (8 mg/kg) (ILS=13.1%) in the mice with Li-7 xenografts transplanted into the spleen. Furthermore, CDDP-MS showed markedly effective anti-tumor activity (IR=82.2%) against H-154 (human stomach cancer) xenografts, in which CDDP–SOL was effective (IR=69.5%) at the MTDs. The suppressive effect of CDDP-MS on accumulation of malignant ascites was intimately related to unchanged CDDP concentration in ascites. These results demonstrated that the administration of CDDP-MS resulted in an unchanged CDDP concentration in ascites, and induced a sustained tumor growth inhibition along with a prolonged survival time.

Antisense DNA-targeting protein kinase A-RIA subunit: a novel approach to cancer treatment.

Enhanced expression of the RIa subunit of cAMP-dependent protein kinase type I (PKA-I) has been shown during carcinogenesis, in human cancer cell lines and in primary tumors. We demonstrate that the sequence-specific inhibition of RIa gene expression by antisense oligonucleotides results in the differentiation of leukemia cells and growth arrest of cancer cells of epithelial origin and tumors in mice. The loss of RI by the antisense results in rapid increase in the half-life of the competitor molecule, RII protein, via its stabilization in a holoenzyme complex (PKA-II) that insures depletion of PKA-I and sustained inhibition of tumor growth. RI antisense, which restrains tumor cell growth by turning on the signals for blockade of tumor cell survival, namely blockade of the tyrosine kinase signaling, cell cycle deregulation and apoptosis, provides a single gene-targeting approach to treatment of cancer.