Michael D. Curley

CD133 Expression Defines a Tumor Initiating Cell Population in Primary Human Ovarian Cancer

Evidence is accumulating that solid tumors contain a rare phenotypically distinct population of cells, termed cancer stem cells (CSC), which give rise to and maintain the bulk of the tumor. These CSC are thought to be resistant to current chemotherapeutic strategies due to their intrinsic stem‐like properties and thus may provide the principal driving force behind recurrent tumor growth. Given the high frequency of recurrent metastasis associated with human ovarian cancer, we sought to determine whether primary human ovarian tumors contain populations of cells with enhanced tumor‐initiating capacity, a characteristic of CSC. Using an in vivo serial transplantation model, we show that primary uncultured human ovarian tumors can be reliably propagated in NOD/SCID mice, generating heterogeneous tumors that maintain the histological integrity of the parental tumor. The observed frequency of tumor engraftment suggests only certain subpopulations of ovarian tumor cells have the capacity to recapitulate tumor growth. Further profiling of human ovarian tumors for expression of candidate CSC surface markers indicated consistent expression of CD133. To determine whether CD133 expression could define a tumor‐initiating cell population in primary human ovarian tumors, fluorescence‐activated cell sorting (FACS) methods were employed. Injection of sorted CD133+ and CD133− cell populations into NOD/SCID mice established that tumor‐derived CD133+ cells have an increased tumorigenic capacity and are capable of recapitulating the original heterogeneous tumor. Our data indicate that CD133 expression defines a NOD/SCID tumor initiating subpopulation of cells in human ovarian cancer that may be an important target for new chemotherapeutic strategies aimed at eliminating ovarian cancer. STEM CELLS 2009;27:2875–2883

Mammalian target of rapamycin is a therapeutic target for murine ovarian endometrioid adenocarcinomas with dysregulated Wnt/β-catenin and PTEN.

Despite the fact that epithelial ovarian cancers are the leading cause of death from gynecological cancer, very little is known about the pathophysiology of the disease. Mutations in the WNT and PI3K pathways are frequently observed in the human ovarian endometrioid adenocarcinomas (OEAs). However, the role of WNT/β-catenin and PTEN/AKT signaling in the etiology and/or progression of this disease is currently unclear. In this report we show that mice with a gain-of-function mutation in β-catenin that leads to dysregulated nuclear accumulation of β-catenin expression in the ovarian surface epithelium (OSE) cells develop indolent, undifferentiated tumors with both mesenchymal and epithelial characteristics. Combining dysregulated β-catenin with homozygous deletion of PTEN in the OSE resulted in development of significantly more aggressive tumors, which was correlated with inhibition of p53 expression and cellular senescence. Induced expression of both mTOR kinase, a master regulator of proliferation, and phosphorylation of its downstream target, S6Kinase was also observed in both the indolent and aggressive mouse tumors, as well as in human OEA with nuclear β-catenin accumulation. Ectopic allotransplants of the mouse ovarian tumor cells with a gain-of-function mutation in β-catenin and PTEN deletion developed into tumors with OEA histology, the growth of which were significantly inhibited by oral rapamycin treatment. These studies demonstrate that rapamycin might be an effective therapeutic for human ovarian endometrioid patients with dysregulated Wnt/β-catenin and Pten/PI3K signaling.

Systems biology driving drug development: from design to the clinical testing of the anti-ErbB3 antibody seribantumab (MM-121)

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR/ErbB1), human EGFR 2 (HER2/ErbB2), ErbB3/HER3, and ErbB4/HER4. The first two members of this family, EGFR and HER2, have been implicated in tumorigenesis and cancer progression for several decades, and numerous drugs have now been approved that target these two proteins. Less attention, however, has been paid to the role of this family in mediating cancer cell survival and drug tolerance. To better understand the complex signal transduction network triggered by the ErbB receptor family, we built a computational model that quantitatively captures the dynamics of ErbB signaling. Sensitivity analysis identified ErbB3 as the most critical activator of phosphoinositide 3-kinase (PI3K) and Akt signaling, a key pro-survival pathway in cancer cells. Based on this insight, we designed a fully human monoclonal antibody, seribantumab (MM-121), that binds to ErbB3 and blocks signaling induced by the extracellular growth factors heregulin (HRG) and betacellulin (BTC). In this article, we present some of the key preclinical simulations and experimental data that formed the scientific foundation for three Phase 2 clinical trials in metastatic cancer. These trials were designed to determine if patients with advanced malignancies would derive benefit from the addition of seribantumab to standard-of-care drugs in platinum-resistant/refractory ovarian cancer, hormone receptor-positive HER2-negative breast cancer, and EGFR wild-type non-small cell lung cancer (NSCLC). From preclinical studies we learned that basal levels of ErbB3 phosphorylation correlate with response to seribantumab monotherapy in mouse xenograft models. As ErbB3 is rapidly dephosphorylated and hence difficult to measure clinically, we used the computational model to identify a set of five surrogate biomarkers that most directly affect the levels of p-ErbB3: HRG, BTC, EGFR, HER2, and ErbB3. Preclinically, the combined information from these five markers was sufficient to accurately predict which xenograft models would respond to seribantumab, and the single-most accurate predictor was HRG. When tested clinically in ovarian, breast and lung cancer, HRG mRNA expression was found to be both potentially prognostic of insensitivity to standard therapy and potentially predictive of benefit from the addition of seribantumab to standard of care therapy in all three indications. In addition, it was found that seribantumab was most active in cancers with low levels of HER2, consistent with preclinical predictions. Overall, our clinical studies and studies of others suggest that HRG expression defines a drug-tolerant cancer cell phenotype that persists in most solid tumor indications and may contribute to rapid clinical progression. To our knowledge, this is the first example of a drug designed and clinically tested using the principles of Systems Biology.

Seribantumab, an anti-ERBB3 antibody, delays the onset of resistance and restores sensitivity to letrozole in an estrogen receptor-positive breast cancer model

Heregulin-driven ERBB3 signaling has been implicated as a mechanism of resistance to cytotoxic and antiendocrine therapies in preclinical breast cancer models. In this study, we evaluated the effects of seribantumab (MM-121), a heregulin-blocking anti-ERBB3 monoclonal antibody, alone and in combination with the aromatase inhibitor letrozole, on cell signaling and tumor growth in a preclinical model of postmenopausal estrogen receptor–positive (ER+) breast cancer. In vitro, heregulin treatment induced estrogen receptor phosphorylation in MCF-7Ca cells, and long-term letrozole-treated (LTLT-Ca) cells had increased expression and activation levels of EGFR, HER2, and ERBB3. Treatment with seribantumab, but not letrozole, inhibited basal and heregulin-mediated ERBB receptor phosphorylation and downstream effector activation in letrozole-sensitive (MCF-7Ca) and -refractory (LTLT-Ca) cells. Notably, in MCF-7Ca–derived xenograft tumors, cotreatment with seribantumab and letrozole had increased antitumor activity compared with letrozole alone, which was accompanied by downregulated PI3K/MTOR signaling both prior to and after the development of resistance to letrozole. Moreover, the addition of an MTOR inhibitor to this treatment regimen did not improve antitumor activity and was not well tolerated. Our results demonstrate that heregulin-driven ERBB3 signaling mediates resistance to letrozole in a preclinical model of ER+ breast cancer, suggesting that heregulin-expressing ER+ breast cancer patients may benefit from the addition of seribantumab to antiendocrine therapy. Mol Cancer Ther; 14(11); 2642–52. ©2015 AACR.

Dual Inhibition of IGF-1R and ErbB3 Enhances the Activity of Gemcitabine and Nab-Paclitaxel in Preclinical Models of Pancreatic Cancer

Purpose: Insulin-like growth factor receptor 1 (IGF-1R) is critically involved in pancreatic cancer pathophysiology, promoting cancer cell survival and therapeutic resistance. Assessment of IGF-1R inhibitors in combination with standard-of-care chemotherapy, however, failed to demonstrate significant clinical benefit. The aim of this work is to unravel mechanisms of resistance to IGF-1R inhibition in pancreatic cancer and develop novel strategies to improve the activity of standard-of-care therapies. Experimental Design: Growth factor screening in pancreatic cancer cell lines was performed to identify activators of prosurvival PI3K/AKT signaling. The prevalence of activating growth factors and their receptors was assessed in pancreatic cancer patient samples. Effects of a bispecific IGF-1R and ErbB3 targeting antibody on receptor expression, signaling, cancer cell viability and apoptosis, spheroid growth, and in vivo chemotherapy activity in pancreatic cancer xenograft models were determined. Results: Growth factor screening in pancreatic cancer cells revealed insulin-like growth factor 1 (IGF-1) and heregulin (HRG) as the most potent AKT activators. Both growth factors reduced pancreatic cancer cell sensitivity to gemcitabine or paclitaxel in spheroid growth assays. Istiratumab (MM-141), a novel bispecific antibody that blocks IGF-1R and ErbB3, restored the activity of paclitaxel and gemcitabine in the presence of IGF-1 and HRG in vitro. Dual IGF-1R/ErbB3 blocking enhanced chemosensitivity through inhibition of AKT phosphorylation and promotion of IGF-1R and ErbB3 degradation. Addition of istiratumab to gemcitabine and nab-paclitaxel improved chemotherapy activity in vivo. Conclusions: Our findings suggest a critical role for the HRG/ErbB3 axis and support the clinical exploration of dual IGF-1R/ErbB3 blocking in pancreatic cancer. Clin Cancer Res; 24(12); 2873–85. ©2018 AACR.

Abstract 521: Dual-targeting of IGF-1R and ErbB3 pathways in Ewing’s Sarcoma cellular models with istiratumab (MM-141), a bispecific, tetravalent monoclonal antibody

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Abstract 1209: Istiratumab (MM-141), a bispecific antibody targeting IGF-1R and ErbB3, inhibits pro-survival signaling in vitro and potentiates the activity of standard of care chemotherapy in vivo in ovarian cancer models

Insulin-like growth factor receptor 1 (IGF-1R) signaling has been implicated in the pathogenesis of ovarian cancer. However, clinical trials evaluating monospecific IGF-1R inhibitors have demonstrated limited clinical efficacy. Our data indicate that ErbB3, a member of the ErbB receptor tyrosine kinase family, can activate pro-survival AKT signaling in response to IGF-1R blockade and may represent a potential escape route in the development of resistance to therapy. Istiratumab (MM-141), an IGF-1R and ErbB3 directed bispecific antibody, inhibits ligand activation of these signaling pathways and degrades IGF-1R and ErbB3 receptor-containing complexes, leading to inhibition of downstream pro-survival signaling. Here we tested the activity of istiratumab, alone and in combination with chemotherapy, in in vitro and in vivo models of ovarian cancer. Anti-proliferative activity of istiratumab monotherapy was evaluated in a panel of ovarian cancer cell lines in vitro. The effects of istiratumab and the ligands IGF-1 and heregulin on IGF-1R- and ErbB3-mediated survival signaling were tested by ELISA and immunoblotting. Co-treatment assays with istiratumab and chemotherapy investigated mechanisms of synergy and additivity. Anti-tumor activity of istiratumab, alone and in combination with chemotherapy, was tested in in vivo ovarian xenograft tumor models. Our results indicated that istiratumab monotherapy inhibits ovarian cancer cell line proliferation in vitro. In addition, istiratumab blocked ligand-mediated resistance to chemotherapy. Co-treatment of istiratumab, ligands or chemotherapy indicated a strong correlation between drug activity and IGF-1R expression. Furthermore, co-treatment of chemotherapies and ligands potentiated AKT activation, which was inhibited by istiratumab. In vivo studies showed that istiratumab potentiates the activity of chemotherapy in ovarian xenograft tumor models. Our findings demonstrate that co-inhibition of IGF-1R and ErbB3 signaling with istiratumab can potentiate standard of care chemotherapies in ovarian tumor models and warrant further investigation of istiratumab as a potential therapy for ovarian cancer patients. Citation Format: Michael D. Curley, Gege Tan, Isabel Yannatos, Adam Camblin, Sergio Iadevaia, Chrystal Louis, Alexey Lugovskoy. Istiratumab (MM-141), a bispecific antibody targeting IGF-1R and ErbB3, inhibits pro-survival signaling in vitro and potentiates the activity of standard of care chemotherapy in vivo in ovarian cancer models. [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 1209.

Abstract 1072: MM-121/SAR256212, an anti-ErbB3 antibody, restores sensitivity to letrozole and delays the onset of resistance in an ER+ breast cancer model.

Endocrine therapies directed against estrogen action are initially effective in estrogen-receptor positive (ER+) breast cancer, though most patients will eventually develop resistance to these treatments. It has recently been shown that growth factor signaling can activate ER through the AKT and MAPK signaling pathways. Moreover, both heregulin and ErbB3 signaling have been implicated as potential escape routes in the development of resistance to anti-estrogen therapies. Here we have used an in vivo model of ER+ breast cancer to investigate the activity of MM-121/SAR256212, alone and in combination with the aromatase inhibitor letrozole. Tumor xenografts of human breast cancer cells (MCF-7) engineered to express aromatase (MCF7-Ca) were generated in ovariectomized athymic nude mice. Mice were injected daily with androstenedione and randomized to treatment groups once the average tumor volume per group reached ∼300 mm3. Mice received vehicle, letrozole, MM-121, or both MM-121 and letrozole. Following development of resistance to letrozole, mice receiving letrozole alone were re-distributed into three treatment groups - letrozole, MM-121, or letrozole plus MM-121. Tumors were harvested at 24 h post-treatment initiation, at development of letrozole resistance, and at the end of study. Total and phosphorylated levels of certain signaling proteins in the ErbB network were measured by quantitative Western blotting. MM-121 in combination with letrozole significantly inhibited tumor growth (p In conclusion, MM-121 delayed the onset of resistance to letrozole in this in vivo model, and re-sensitized tumors to letrozole upon development of resistance. These data indicate a potential role for combining MM-121 with letrozole in first and second line treatment of ER+ breast cancer. Citation Format: Michael D. Curley, Gauri Sabnis, Lucia Wille, Gabriela Garcia, Victor Moyo, Armina Kazi, Gavin MacBeath, Angela Brodie. MM-121/SAR256212, an anti-ErbB3 antibody, restores sensitivity to letrozole and delays the onset of resistance in an ER+ breast cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1072. doi:10.1158/1538-7445.AM2013-1072

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 4302: Human endometrial cancer cell CD133+ cell fractions are regulated by methylation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Like other solid tumors, endometrial tumors have been shown to contain a subset of tumor initiating cells although little is known about how these rare sub-fractions are regulated. Our primary objective was to analyze potential epigenetic regulation of such a tumor initiating cell population in human endometrial cancer cells. To accomplish this, we demonstrated by flow cytometry that primary endometrial tumors contain CD133+ cells. To assess their tumor initiating capacity, serially transplanted endometrial human tumor explants generated in NOD/SCID mice were harvested, enzymatically dissociated, depleted of H-2Kd+ mouse cells and sorted via flow cytometry to generate relatively pure (> 98.8 %) CD133+ and CD133− fractions. These positive and negative fractions were serially diluted and subcutaneously injected into immunocompromised mice. The CD133+ fractions had a significantly increased capacity for tumor formation relative to the CD133− fractions and this difference was more pronounced as the number of injected cells decreased. Interestingly, the level of CD133+ tumor cells appeared to be enriched following serial transplantation as evidenced by flow cytometric and immunohistochemical analyses. It has been proposed that methylation may play a role in regulation of tumor initiating cells. To investigate this possibility in endometrial cancer, we isolated DNA from serially transplanted tumors and analyzed the methylation status of CpG islands located upstream of the CD133 transcription start site. This region was shown to be a target of methylation, which led us to determine whether changing the methylation status would alter CD133 expression in endometrial cancer cells. We treated 4 individual human endometrial cancer cell lines with either vehicle or 5 μM 5-aza-2′-deoxyctidine (5-aza-DC), for 72 hours, and evaluated post-treatment levels of CD133 expression by RT-PCR and flow cytometry. In the tested cell lines, CD133 mRNA levels, as measured by RT-PCR, were increased following treatment with 5-aza-DC suggesting that methylation of the CD133 promoter was suppressing its expression. To extend this finding, we analyzed the percentage of CD133 expressing cells in either vehicle treated or 5-aza-DC by flow cytometry. Consistent with the RT-PCR results, the frequency of CD133-expressing cells was increased in 3 of the 4 cell lines following treatment with 5-aza-DC. It is not clear however, if the 5-aza-DC mediated demethylation and subsequent shift in the percentage of CD133+ cells correlates with a shift in the frequency of cells that have increased tumor initiating capacity. Nevertheless, CD133 expression in human endometrial cancer cells is regulated at least in part by methylation and altering this may cause these tumor cells to become more sensitive to standard chemotherapy regimens. 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 4302.