nkai Yu

Addiction to Elevated Insulin-like Growth Factor I Receptor and Initial Modulation of the AKT Pathway Define the Responsiveness of Rhabdomyosarcoma to the Targeting Antibody

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.

Genome-Wide Identification of PAX3-FKHR Binding Sites in Rhabdomyosarcoma Reveals Candidate Target Genes Important for Development and Cancer

The PAX3-FKHR fusion protein is present in a majority of alveolar rhabdomyosarcomas associated with increased aggressiveness and poor prognosis. To better understand the molecular pathogenesis of PAX3-FKHR, we carried out the first, unbiased genome-wide identification of PAX3-FKHR binding sites and associated target genes in alveolar rhabdomyosarcoma. 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 (a) mostly distal to transcription start sites, (b) conserved, (c) enriched for PAX3 motifs, and (d) strongly associated with genes overexpressed in PAX3-FKHR-positive rhabdomyosarcoma cells and tumors. There is little evidence in our data set for PAX3-FKHR binding at the promoter sequences. The genome-wide analysis further illustrates a strong association between PAX3 and E-box motifs in these binding sites, suggestive of a common coregulation for many target genes. We also provide the first direct evidence that FGFR4 and IGF1R are the targets for PAX3-FKHR. The map of PAX3-FKHR binding sites provides a framework for understanding the pathogenic roles of PAX3-FKHR, as well as its molecular targets to allow a systematic evaluation of agents against this aggressive rhabdomyosarcoma.

Rapamycin Pharmacokinetic and Pharmacodynamic Relationships in Osteosarcoma: A Comparative Oncology Study in Dogs

Background Signaling through the mTOR pathway contributes to growth, progression and chemoresistance of several cancers. Accordingly, inhibitors have been developed as potentially valuable therapeutics. Their optimal development requires consideration of dose, regimen, biomarkers and a rationale for their use in combination with other agents. Using the infrastructure of the Comparative Oncology Trials Consortium many of these complex questions were asked within a relevant population of dogs with osteosarcoma to inform the development of mTOR inhibitors for future use in pediatric osteosarcoma patients. Methodology/Principal Findings This prospective dose escalation study of a parenteral formulation of rapamycin sought to define a safe, pharmacokinetically relevant, and pharmacodynamically active dose of rapamycin in dogs with appendicular osteosarcoma. Dogs entered into dose cohorts consisting of 3 dogs/cohort. Dogs underwent a pre-treatment tumor biopsy and collection of baseline PBMC. Dogs received a single intramuscular dose of rapamycin and underwent 48-hour whole blood pharmacokinetic sampling. Additionally, daily intramuscular doses of rapamycin were administered for 7 days with blood rapamycin trough levels collected on Day 8, 9 and 15. At Day 8 post-treatment collection of tumor and PBMC were obtained. No maximally tolerated dose of rapamycin was attained through escalation to the maximal planned dose of 0.08 mg/kg (2.5 mg/30kg dog). Pharmacokinetic analysis revealed a dose-dependent exposure. In all cohorts modulation of the mTOR pathway in tumor and PBMC (pS6RP/S6RP) was demonstrated. No change in pAKT/AKT was seen in tumor samples following rapamycin therapy. Conclusions/Significance Rapamycin may be safely administered to dogs and can yield therapeutic exposures. Modulation pS6RP/S6RP in tumor tissue and PBMCs was not dependent on dose. Results from this study confirm that the dog may be included in the translational development of rapamycin and potentially other mTOR inhibitors. Ongoing studies of rapamycin in dogs will define optimal schedules for their use in cancer and evaluate the role of rapamycin use in the setting of minimal residual disease.

Insulin-like growth factor 1 receptor antibody induces rhabdomyosarcoma cell death via a process involving AKT and Bcl-x L

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.

A phase I study of TRC105 anti-endoglin (CD105) antibody in metastatic castration-resistant prostate cancer

TRC105 is a chimeric immunoglobulin G1 monoclonal antibody that binds endoglin (CD105). This phase I open‐label study evaluated the safety, pharmacokinetics and pharmacodynamics of TRC105 in patients with metastatic castration‐resistant prostate cancer (mCRPC).

Elevated Serum Megakaryocyte Potentiating Factor as a Predictor of Poor Survival in Patients with Mesothelioma and Primary Lung Cancer

Background There is an urgent need for a companion assay to work with mesothelin-targeted therapeutic agents and for noninvasive and accurate prognostication of malignant mesothelioma (MM) patients. We report the development and validation of a blood-based assay for megakaryocyte potentiating factor (MPF) and the evaluation of its effectiveness for prognosis in MM and lung cancer patients. Methods Using electrochemiluminescence technology, we developed a sensitive MPF assay and performed both analytical and clinical validations. Further, the effectiveness of the MPF assay in predicting prognosis was evaluated for 95 MM and 272 lung cancer patients. Results We performed comprehensive analytical and clinical validation, including precision and accuracy, interference, preanalytical variables, sensitivity, and specificity for mesothelioma. In MM patients, increased serum MPF is a predictor of poor survival with a hazard ratio (HR) = 2.46 (log-rank P = 0.003; n = 95). In refractory MM patients, increased MPF is a strong predictor of poor outcome with an HR = 6.12 (log-rank P = 0.0007; n = 57). In a lung cancer patient cohort, increased MPF is a predictor of poor survival, with an HR = 1.57 (log-rank P = 0.003; n = 272). Conclusions The MPF assay has robust technical characteristics, with strong analytic and clinical validation. Clinical studies indicate that increased serum MPF is a predictor of poor survival for MM patients, throughout the course of the disease. Increased MPF is also associated with poor overall survival for patients with newly diagnosed lung cancer.

Abstract 2048: Pharmacodynamic biomarker studies of TRC105 anti-endoglin (CD105) antibody revealed anti-angiogenic activity associated with CD105 depletion

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.

Abstract 2917: Caspase-8 expression is predictive of tumor response to death receptor 5 agonist antibody conatumumab in Ewing's sarcoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Despite good initial response to cytotoxic chemotherapy, approximately 30% of Ewings 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 Ewings 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

Abstract 366: A novel transcript variant of androgen receptor identified in circulating tumor cells from castration-resistant prostate cancer patients as a potentially prognostic biomarker

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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<0.0001). In vitro functional studies showed this novel ARv is constitutively active in the absence of testosterone. Our findings suggest that this AR variant might contribute to the late stage progression of the diseases and could be used as a potential indicator for alternative therapies. In summary, our new fluidic device is capable of isolating CTC to enable molecular marker analysis, providing value in prognosis and guidance to therapy. 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

Abstract 1719: Down-regulation of IGFBP2 is associated with resistance to IGF1R therapy in rhabdomyosarcoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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