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African Americans have a higher risk of developing lung cancer compared with all other ethnic groups in the USA. Previous studies based on a small panel of markers suggested that certain circulating cytokines were associated with lung cancer. Given the complexity of the immune response in lung cancer and the multitude of cell types involved, we reasoned that examining a broad panel of inflammatory markers—including cytokines, chemokines, angiogenic and other pro-inflammatory factors—might identify a diagnostic signature of lung cancer for African American patients. Differences in 30 inflammatory marker serum levels were measured in 316 African-American cases and 508 African American controls from the National Cancer Institute-Maryland (NCI-MD) case-control study using a Mesoscale (MSD) multiplex platform. The panel of 30 serum inflammatory markers included chemokines, cytokines and other inflammatory related proteins. Logistic regression analyses were conducted to investigate the association between levels of inflammatory marker expression and lung cancer risk. Expression levels above the median threshold defined within the control samples were considered high. Statistical models were also adjusted for potential confounding factors such as age, gender, pack-years and smoking status. Nineteen inflammatory markers (CRP, IFN-ϒ, IL-10, IL-15, IL-17A, IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-4, MIP-1α, TARC, TNF-α, TNF-β and VEGF) were significantly different among African American cases compared with African American controls (P Serum cytokine levels vary by race and might contribute to lung cancer differently in African Americans and European Americans. The findings presented here build upon recent work that identified associations between elevated levels of IL-10, IL-1β, IL-6, IL-8, TNF-α and lung cancer specifically among African Americans. By analyzing a broader spectrum of inflammatory measures, we have identified additional markers of lung cancer for African American patients. Further to this, we have integrated GWAS and MSD data for 137 cases and 203 controls that allows us to examine the genetic basis to the relationship between risk of lung cancer and modulated inflammatory marker expression. Markers identified here as significant for lung cancer risk will be further analyzed using case-control samples from an independent African American validation cohort. Citation Format: Claire L. Meaney, Khadijah A. Mitchell, Adriana Zingone, Derek Brown, Wei Tang, Yunkai Yu, Liang Cao, Angela S. Wenzlaff, Ann G. Schwartz, Brid M. Ryan. Inflammatory based diagnostic markers of lung cancer in African Americans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4658. doi:10.1158/1538-7445.AM2017-4658

Lung cancer is the leading cause of cancer-related mortality worldwide. Low-dose CT (LDCT) imaging is now recommended to screen high-risk lung cancer individuals in the USA. The sensitivity of LDCT has resulted in increased detection of stage I lung cancer and a 20% reduction in lung cancer mortality. The current standard of care for stage 1 lung cancer patients is surgery alone. However, between 20% and 30% of these patients will develop recurrence and therefore are in need of further treatment upon diagnosis. This study aims to explore and validate biomarkers to identify patients at high-risk of mortality so that additional treatment modalities can be offered at time of diagnosis. Our recent work on a small panel of circulating cytokines identified elevated levels of IL-6, a pro-inflammatory cytokine, as an indicator of poor survival. To further examine the potential of inflammatory biomarkers as prognostic indicators, 125 stage I lung adenocarcinoma cases were selected from the National Cancer Institute-Maryland lung cancer case-control study. This is an on-going prospective study of non-small cell lung cancer based in the greater Baltimore region of the USA. A panel of 33 inflammatory markers was measured for each case using the Mesoscale V-Plex assay. The magnitude of association between serum inflammatory marker expression levels and lung cancer-specific survival was tested using multivariable Cox proportional hazards regression modeling (Stata 12.0 statistical software). All calculations were adjusted for age, gender, stage and smoking status. Maximum follow up time was 15 years. Five analytes were significantly associated with shorter survival. In concordance with the previous study, IL-6 was again associated with shorter survival (HR, 2.63; 95% CI, 1.25-5.56). Other cytokines associated with shorter survival included CRP (HR, 2.25; 95% CI, 1.09-4.65), Eotaxin-3 (HR, 2.20; 95% CI, 0.97-4.86), IL-12p40 (HR 1.98; 95% C.I. 1.00 - 3.91), and IL-17 (HR, 2.22; 95% CI, 1.08-4.58). Although IL6 and CRP were positively correlated (Rho = 0.451, P These results support the potential of cytokine markers as a prognostic tool to further classify stage I lung cancer and thus identify patients in need of additional treatment. The associations identified here justify further investigation of a novel, combined cytokine prognostic classifier to serve as a robust predictor of stage I lung adenocarcinoma survival. Citation Format: Claire L. Meaney, Adriana Zingone, Derek Brown, Yunkai Yu, Liang Cao, Brid M. Ryan. The identification of serum cytokine inflammatory markers as classifiers of lung cancer mortality for stage I lung adenocarcinoma: a retrospective cohort study. [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 3406.

Introduction: CD105 is involved in normal vascular development. It is over expressed on the surface of proliferating vascular endothelial cells and is implicated in tumor angiogenesis. In hypoxic conditions, CD105 is upregulated through induction of hypoxia-inducible factor 1-α. TRC105 is a chimeric IgG1 antibody specific for CD105 and the agent for this phase I trial. Methods: 20 patients with metastatic prostate cancer were treated with TRC105 at six dose levels in a phase I trial. Blood samples were analyzed for CD105 antigen depletion, VEGF as a marker for systemic hypoxia, and PSA. Results: Maximum tolerated dose of 20 mg/kg every two weeks was reached. Significant plasma CD105 reduction was observed at high dose levels. The reduction of CD105 was associated with induction of plasma VEGF. Ten patients had stable disease, and the reduction of CD105 is associated with PSA stabilization. Conclusion: A significant induction of VEGF was associated with CD105 reduction at three high dose levels, suggesting the anti-angiogenic activity of TRC105. Exploratory analysis showed a tentative correlation between the reduced CD105 and a decreased PSA velocity, suggestive of potential antitumor activity of TRC105 in metastatic prostate cancer. Citation Format: Liang Cao, Fatima Karzai, Andrea Apolo, Ravi Madan, Yunkai Yu, James Gulley, William Figg, William Dahut. Pharmacodynamic biomarker studies of TRC105 anti-endoglin (CD105) antibody revealed anti-angiogenic activity associated with CD105 depletion. [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 2048.

Insulin-like growth factor I receptor (IGF-IR) and its ligands are overexpressed by tumors, mediating proliferation and protecting against stress-induced apoptosis. Accordingly, there has been a considerable amount of interest in developing therapeutic agents against IGF-IR. IGF-IR is believed to be ubiquitously expressed without detectable mutation or amplification in cancer. We explored the determinants of cellular response to a humanized anti–IGF-IR antibody. Our results showed a large variation in IGF-IR levels in rhabdomyosarcoma tumor specimens that were comparable with those in rhabdomyosarcoma cell lines. In vitro analysis revealed a direct and very significant correlation between elevated IGF-IR levels and antiproliferative effects of the antibody and defined a receptor number that would predict sensitivity. Our data further suggested a strong dependence on IGF-IR for AKT signaling in cells with elevated IGF-IR. The sensitivity of the high IGF-IR–expressing cells was blocked with a constitutively active AKT. The extracellular signal-regulated kinase pathway was not affected by the antibody. In vivo studies showed that anti–IGF-IR had single-agent antitumor activity; furthermore, predictions of responses based on IGF-IR levels were accurate. In vivo biomarker analysis suggested that h7C10 down-regulated both IGF-IR and p-AKT initially, concordant with antitumor activity. Subsequent progression of tumors was associated with reactivation of p-AKT despite sustained suppression of IGF-IR. These results identified the first predictive biomarker for anti–IGF-IR therapies in cancer. [Cancer Res 2008;68(19):8039–48]

Despite good initial response to cytotoxic chemotherapy, approximately 30% of Ewing's sarcoma (EWS) patients who present with localized tumors develop recurrent disease, which is associated with poor prognosis. Addressing this clinical challenge requires the development of targeted therapies. This study shows that many EWS cell lines are selectively sensitive to a death receptor DR5 antibody and are more resistant to a DR4 antibody. Preclinical evaluation of these cell lines indicates their sensitivity to human DR5 agonist antibody conatumumab in vitro, which induces rapid activation of caspase-8 and apoptosis. Further analysis reveals the correlation of sensitivity to conatumumab with the expression of caspase-8, with its catalytic activity both necessary and sufficient to confer such sensitivity. In vivo, conatumumab has active against both EWS cell lines and a patient-derived xenograft with higher caspase-8 expression, but is not effective against another patient-derived xenograft with lower caspase-8 expression. These studies suggest the potential of conatumumab as a therapeutic agent against EWS and caspase-8 as a biomarker for correlative studies. Citation Format: Zhigang Kang, D Seth Goldstein, Yunkai Yu, Paul S. Meltzer, David M. Loeb, Liang Cao. Caspase-8 expression is predictive of tumor response to death receptor 5 agonist antibody conatumumab in Ewing's sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2917. doi:10.1158/1538-7445.AM2015-2917

Circulating tumor cells (CTCs) are rare cancer cells in the bloodstream thought to have a key role in cancer metastasis. There are enormous interests in their analysis for castration-resistant prostate cancer (CRPC) due to their tendency to metastasize to bone. In this study, we applied a surface marker-independent microfluidic device for CTCs capture and retrieval that is based on cell size and deformability. Our device was able to capture 90% and recover 30% of tumor cells in spike experiments. We also developed and validated assays capable of detecting the transcript levels of selected molecular markers in single cells prepared with C1TM Single-Cell AutoPrep System. Blood samples drawn from 34 CRPC patients and 10 primary prostate cancer patients (PPC) were preceded for CTC isolation and molecular characterization. We found that 73% CRPC patients and 10% PPC patients were positive for EpCAM, and only about half of the EpCAM-positive CRPC cases were PSA or AR positive. It is interesting that while there is a strong association between AR and PSA expression in CTC samples, there is no correlation between AR or PSA transcripts in CTCs and serum PSA protein levels in CRPC patients. The positive detection of AR or PSA, but not EpCAM is statistically associated with poor prognosis of the CRPC patients. Very intriguingly, we identified a novel androgen receptor (AR) transcript variant -ARv from four CRPC patients’ CTCs. This ARv lacks a 554bp sequence region in the ligand binding domain (LBD), resulting in a loss of the entire LBD. Clinical data analysis revealed that the ARv is significantly associated with worse survival of CRPC patients (p Citation Format: Zhigang Kang, Avani Shah, Yunkai Yu, Yuelin Zhu, Ali Asgar Bhagat, Kyra Zhao, Andrew Wu, James Gao, Ravi Madan, James Gulley, William Dahut, Paul Meltzer, Liang Cao. A novel transcript variant of androgen receptor identified in circulating tumor cells from castration-resistant prostate cancer patients as a potentially prognostic biomarker. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 366. doi:10.1158/1538-7445.AM2015-366

Circulating tumor cells (CTCs) are rare cancer cells circulating in the bloodstream that are thought to have a key role in cancer metastasis. They have been associated with worse prognosis in several major cancer types. There is an intense interest in the molecular analysis of CTC to gain information that would assist treatment decisions and potentially improve the disease outcome. There are enormous interests in the analysis of CTCs for castration-resistant prostate cancer (CRPC) due to the nature of the tumor that tends to metastasize to bone, making them generally inaccessible to obtain biopsies. In this study, we applied a surface marker independent, label-free cell trap microfluidics device for CTCs isolation and retrieval based on cell size and deformability. A total of 47 blood samples drawn from 36 patients with CRPC were preceded for CTC isolation followed by molecular characterization using epithelial tumor markers and prostate cancer specific markers. We identified a novel androgen receptor (AR) splice variant from four patients' CTCs, and confirmed by DNA sequencing that this variant lacks a 554bp region in the ligand binding domain of the receptor in comparison with wild type human AR. Patients with this splice variant were found to be associated with poor survival (p Note: This abstract was not presented at the meeting. Citation Format: Zhigang Kang, Yunkai Yu, Liang Cao, Paul S. Meltzer, Yuelin Zhu, Avan Shah, James Gulley, William Duht, Ravi Madan, Ali Bhagat, Kyra Zhao. A novel AR splice variant identified in circulating tumor cells from castration-resistant prostate cancer patient blood as a potentially prognostic biomarker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4815. doi:10.1158/1538-7445.AM2014-4815

Agents targeting the insulin-like growth factor-1 receptor (IGF1R) are in clinical development, but, despite some initial success of single agents in sarcoma, response rates are low with brief durations. Thus, it is important to identify markers predictive of response, to understand mechanisms of resistance, and to explore combination therapies. In this study, we found that, while associated with PAX3-FKHR translocation, increased IGF1R level is an independent prognostic marker for worse overall survival, particularly in patients with PAX3-FKHR-positive rhabdomyosarcoma (RMS). IGF1R antibody-resistant RMS cells were generated using an in vivo model. Expression analysis indicated that IGFBP2 is both the most affected gene in the IGF signaling pathway and the most significantly down-regulated gene in the resistant lines, indicating that there is a strong selection to repress IGFBP2 expression in tumor cells resistant to IGF1R antibody. IGFBP2 is inhibitory to IGF1R phosphorylation and its signaling. Similar to antibodies to IGF1/2 or IGF2, the addition of exogenous IGFBP2 potentiates the activity of IGF1R antibody against the RMS cells, and it reverses the resistance to IGF1R antibody. In contrast to IGF1R, lower expression of IGFBP2 is associated with poorer overall survival, consistent with its inhibitory activity found in this study. Finally, blocking downstream AKT activation with PI3K- or mTOR-specific inhibitors significantly sensitized the resistant cells to the IGF1R antibody. These findings show that constitutive IGFBP2 down-regulation may represent a novel mechanism for acquired resistance to IGF1R therapeutic antibody in vivo and suggest various drug combinations to enhance antibody activity and to overcome resistance. Citation Format: Zhigang Kang, Yunkai Yu, Yuelin J. Zhu, Lee Helman, Paul Meltzer, Liang Cao. Down-regulation of IGFBP2 is associated with resistance to IGF1R therapy in rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1719. doi:10.1158/1538-7445.AM2014-1719

Agents targeting the insulin-like growth factor 1 receptor are in various stages of clinical development. Despite some initial response with single agent trials, the responses are limited and durations are short. It is important to understand the mechanics of innate and adaptive responses of the tumors that lead to resistance. An in vivo mouse model was used for the development of IGF1R antibody-resistant rhabdomyosarcoma tumor. All tumors, despite remarkable initial response, all eventually grow in the presence of the antibody. The isolated cell lines show cross-resistance to IGF1R antibodies, and are presented with increased AKT activity in the presence of an IGF1R antibody. Expression analysis indicates that IGFBP2 is the most affected gene in the IGF signaling process that is significantly down-regulated in two independently generated resistant lines. In addition, the recombinant IGFBP2 is capable of inhibiting of IGF1R phosphorylation and AKT signaling. Further, the inhibitory activity is amplified in the presence of an IGF1R antibody, against both sensitive and resistant RMS cells. Finally, the addition of antibodies against IGF1/2 or IGF2 enhances the activity of an IGF1R antibody against RMS cells. The findings suggest that IGFBP2 down-regulation may represent a novel mechanism to escape the blockage of IGF1R signaling by a therapeutic antibody. Citation Format: Yunkai Yu, Zhigang Kang, Lee Helman, Paul Meltzer, Liang Cao. Resistance to insulin-like growth factor 1 receptor antibody associated with downregulated IGFBP2. [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 4449. doi:10.1158/1538-7445.AM2013-4449

Circulation tumor cells are critically important to understand tumor metastasis and may have a potential to noninvasively evaluate the cancer for clinical management. The isolation and characterization of CTC represent a major technological challenge. While the CellSeach system by J&J has shown that CTC enumeration can be an independent predictor of survival for breast, prostate and colon cancer, little molecular information can be obtained from the CTC to have an impact on patient management or clinical outcome. Most hormone dependent castration-resistant prostate cancer (CRPC) becomes refractory after one to three years of treatment, many through the amplification or mutation of the androgen receptor gene. As multiple targeted agents become available for this cancer and no tumor material can be obtained due to bone metastasis, there is an urgent need to be able to evaluate biomarkers of CRPC for clinical management of the patients. In order to assess biomarkers that may influence the management of CRPC, we worked on a novel antibody-independent CTC purification technology for isolation of Live CTC from CRPC. Citation Format: Zhigang Kang, William Dahut, James Gulley, Yunkai Yu, Paul S. Meltzer, Liang Cao. Development of a novel circulating tumor cell technology for cancer predictive biomarker analysis. [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 736. doi:10.1158/1538-7445.AM2013-736

Introduction: MM is an aggressive cancer with limited treatment options and poor prognosis. Previous studies have shown that the interaction between IGF-1R and its ligands IGF-1 and IGF-2 play an important role in MM tumorogenesis. To exploit IGF-1R as a target for mesothelioma therapy we investigated the activity of IMC-A12 against early passage mesothelioma tumor cells, established MM cell lines and an in vivo human mesothelioma tumor xenograft model. Material and methods: Eight early passage mesothelioma tumor cells obtained from patients with mesothelioma and eight established MM cell lines were evaluated for IGF-1R expression by real time PCR, Western blotting, and a quantitative immunoassay. The cell surface IGF-IR expression (sites/cell) was analyzed by flow cytometry using QuantiBRITE-PE bead assay. The anti-proliferative effect of IMC-A12 and its ability to induce antibody mediated cell cytoxicity (ADCC) was evaluated in vitro using both early passage tumor cells as well as established cell lines. For our in vivo experiment, the mesothelioma cell line NCI-H226, stably transfected with the luciferase gene, was inoculated intraperitoneally (i.p) into athymic nude mice. Three days after tumor cell inoculation, mice received either IMC-A12 (50 mg/kg) or saline by i.p. injection twice weekly for 8 weeks and tumor growth was monitored using bioluminescence imaging. Results: All early passage as well as established MM cell lines showed IGF-1R expression at the mRNA and protein levels. Using a quantitative electrochemiluminescence immunoassay we determined that there is considerable variability of IGF-1R expression ranging from 1-12 ng/mg of cell lysate. Using flow cytometry we evaluated the number of IGF-1R cell surface receptors, which varied from ≈ 2,000-50,000 sites/cell. Cells expressing >10,000 sites/cell had greater than 10% (range, 10% -60%) growth inhibition when treated with IMC-A12 (100 μg/mL). Similarly, IMC-A12 treatment induced ADCC (> 10% specific lysis) in those cell lines with IGF-1R expression of > 10,000 sites/cell. In vivo IMC-A12 treatment delayed tumor growth in athymic nude mice bearing H226 tumors and improved the overall survival when compared to the control group (p< 0.05). Conclusion: Our results show that both early passage mesothelioma cells as well as established MM cell lines express IGF-1R but that there is a wide variability in the degree of IGF-1R expression. IMC-A12 exhibits anti-proliferative activity against these cell lines as well as induces ADCC, both of which are strongly co-related with IGF-1R cell surface expression. Single agent IMC-A12 also has significant in vivo anti-tumor activity. These results suggest IMC-A12 may be useful for treatment of MM. A phase II clinical trial of IMC-A12 has just opened for patient accrual. 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 646. doi:10.1158/1538-7445.AM2011-646

The paired box transcription factor (PAX) family consists of 9 highly related members with critical roles in organogenesis and tissue specification during development, including skeletal muscle, pancreas, central nervous system, kidney, and immune system. In various cell model systems, PAX genes have been implicated in the maintenance of the multipotent state, the initiation of differentiation programs, cell migration, proliferation and survival. Mutations and gene rearrangement of PAX genes are frequently associated with human diseases and cancers. In particular, PAX3 and PAX7 are required during early embryogenesis for the development of the neural-crest and skeletal muscle, and gene rearrangements with FKHR are frequently implicated with rhabdomyosarcoma (RMS). The PAX3-FKHR fusion protein is present in the majority of alveolar rhabdomyosarcoma (ARMS) associated with increased aggressiveness and poor prognosis. To better understand the molecular pathogenesis of PAX3-FKHR, we carried out the first and unbiased genome-wide identification of PAX3-FKHR binding sites and associated target genes in ARMS, with a novel monoclonal antibody specific for the fusion protein. The data shows that PAX3-FKHR binds to the same sites as PAX3 at both MYF5 and MYOD enhancers. The genome-wide analysis reveals that the PAX3-FKHR sites are: 1) mostly distal to transcription start sites; 2) conserved; 3) enriched for PAX3 motifs; and 4) strongly associated with genes over-expressed in PAX3-FKHR positive RMS cells and tumors. There is little evidence in our dataset for PAX3-FKHR binding at the promoters. The genome-wide analysis further illustrates a strong association between PAX3 and E-box motifs in these binding sites, suggestive of a common co-regulation for many target genes. We further provide the first direct evidence that FGFR4 and IGF1R are the targets for PAX3-FKHR. The study is the first genome-wide analysis of direct targets for a PAX protein. The map of PAX3-FKHR binding sites provides the frame work for the understanding of PAX3-FKHR pathogenic roles and molecular targets to allow a systematic evaluation of agents against this aggressive RMS. 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 5344. doi:10.1158/1538-7445.AM2011-5344

Insulin-like growth factors (IGFs) and their receptor, IGF-1 receptor (IGF1R), have important roles in growth, development, stress response, aging and cancer. There are many agents that inhibit IGF1R in oncology clinical development, and in some cases, they have been associated with rapid tumor regression. However, it is not clear by which process these targeted agents induce cancer cell death and how to predict such tumor responses. Here, we showed that IGF1R antibody led to rapid cell death and tumor regression in some rhabdomyosarcoma (RMS) cells. Mechanistic analysis revealed a rapid onset of mitochondrial-dependent apoptosis, including mitochondrial depolarization, cytochrome C release and the activation of specific caspases. The antibody sensitive cells had greater dependence on AKT for maintaining downstream signaling and the expression of a constitutively active AKT, which restored AKT-signaling in these cells, inhibited anti-IGF1R induced cell death. Further analysis showed IGF1R antibody-induced hypophosphorylation of BAD and activation of downstream BAX. Interestingly, the examination of RMS cell lines and tumors revealed an inverse correlation between elevated IGF1R and Bcl-2 level (P=0.033), with the sensitive cells lacking Bcl-2 expression. The overexpression of BAD specific target, Bcl-xL, conferred resistance, whereas Bcl-xL knockdown sensitized cells lacking Bcl-2 to anti-IGF1R-induced cell death. We propose that RMS pathogenesis involves increased IGF1R expression that enhances AKT and Bcl-xL-mediated cell survival, and the blockage of IGF1R results in inhibition of survival signal from Bcl-xL and cell death in the sensitive Bcl-2 negative cells. 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 651. doi:10.1158/1538-7445.AM2011-651

Purpose: Rhabdomyosarcoma (RMS) is a common pediatric soft-tissue tumor. In this study, we evaluated the efficacy and selectivity of drozitumab, a death receptor DR5-targeted therapeutic antibody, in RMS preclinical models. Experimental design: A panel of 12 RMS cell lines was used for in vitro studies. The molecular marker predictive of response was interrogated. Selected RMS cell lines were injected into the gastrocnemius muscle of mice for in vivo assessment of both potency and selectivity of drozitumab. Results: We report that DR5, but not DR4, persisted at high levels and on the surface of all RMS cell lines. DR5 antibody drozitumab was effective in vitro against the majority of RMS cell lines. There was a strong correlation between caspase-8 expression and the sensitivity to drozitumab, which induced the rapid assembly of the death-induced signaling complex (DISC) and the cleavage of caspase-8 only in sensitive cells. More importantly, caspase-8 catalytic activity was both necessary and sufficient in mediating the sensitivity to drozitumab. Furthermore, drozitumab had potent anti-tumor activity against established RMS xenografts with specificity as predicted from in vitro analysis, and with tumor free status in half of the treated mice. Conclusion: Our study provides the first preclinical evaluation on the potency and selectivity of a death receptor antibody in sarcoma. Drozitumab is effective against the majority of RMS in vitro with caspase-8 expression predictive of sensitivity and, in vivo, drozitumab may provide long-term control and cure of RMS. 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 4094. doi:10.1158/1538-7445.AM2011-4094

Insulin-like growth factors and their receptor IGF1R play important roles in growth, development, stress response, aging, and cancer. Many antibodies targeting IGF1R are in varies stages of clinical development. It is important to understand the mechanism of the antibody-mediated cancer cell growth inhibition and death, and to identify biomarkers predictive of responses that can be implemented in clinical investigations. We previously reported that IGF1R mediated cell growth suppression of rhabdomyosarcoma (RMS) in vitro and in vivo, in which the anti-proliferative activity was correlated with IGF1R levels and AKT signaling (Cao, et al., Cancer Research 2008, 68:8039). Here, we show that in some RMS cells, treatment with a humanized IGF1R antibody results in a rapid onset of mitochondrial-dependent apoptosis, including mitochondrial depolarization, cytochrome C release, and the activation of specific caspases. The antibody-induced cell death is mediated in part via AKT-dependent pathway, and both the inhibition of AKT signaling and induction of cell death by anti-IGF1R can be rescued by the introduction of a constitutively active AKT. Further analysis reveals IGF1R antibody-induced hypophosphorylation (activation) of BAD and the activation of downstream BAX. Bcl-xL was previously shown to be a key target of activated BAD. Our data indicate that cells with the transgenic expression of Bcl-xL are completely resistant to antibody-induced cell death, while still susceptible to antibody-mediated growth inhibition. Moreover, siRNA against Bcl-xL enhances anti-IGF1R-induced apoptosis. Interestingly, the examination of RMS cell lines and tumors reveals an inverse correlation between elevated IGF1R and another pro-survival gene Bcl-2 (P=0.033). Thus, our results indicate that RMS pathogenesis may involve two strategies for cell survival: 1) increasing IGF1R expression that enhances Bcl-xL activity; or 2) up-regulating Bcl-2 expression. Our study suggests a particular subgroup of the cancer with elevated IGF1R and low Bcl-2 maybe more susceptible to anti-IGF1R treatment. 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 701.