Mark R. Miglarese

Feedback Mechanisms Promote Cooperativity for Small Molecule Inhibitors of Epidermal and Insulin-Like Growth Factor Receptors

Epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR) can cooperate to regulate tumor growth and survival, and synergistic growth inhibition has been reported for combined blockade of EGFR and IGF-IR. However, in preclinical models, only a subset of tumors exhibit high sensitivity to this combination, highlighting the potential need for patient selection to optimize clinical efficacy. Herein, we have characterized the molecular basis for cooperative growth inhibition upon dual EGFR and IGF-IR blockade and provide biomarkers that seem to differentiate response. We find for epithelial, but not for mesenchymal-like, tumor cells that Akt is controlled cooperatively by EGFR and IGF-IR. This correlates with synergistic apoptosis and growth inhibition in vitro and growth regression in vivo upon combined blockade of both receptors. We identified two molecular aspects contributing to synergy: (a) inhibition of EGFR or IGF-IR individually promotes activation of the reciprocal receptor; (b) inhibition of EGFR-directed mitogen-activated protein kinase (MAPK) shifts regulation of Akt from EGFR toward IGF-IR. Targeting the MAPK pathway through downstream MAPK/extracellular signal-regulated kinase kinase (MEK) antagonism similarly promoted IGF-driven pAkt and synergism with IGF-IR inhibition. Mechanistically, we find that inhibition of the MAPK pathway circumvents a negative feedback loop imposed on the IGF-IR- insulin receptor substrate 1 (IRS-1) signaling complex, a molecular scenario that parallels the negative feedback loop between mTOR-p70S6K and IRS-1 that mediates rapamycin-directed IGF-IR signaling. Collectively, these data show that resistance to inhibition of MEK, mTOR, and EGFR is associated with enhanced IGF-IR-directed Akt signaling, where all affect feedback loops converging at the level of IRS-1.

Compensatory insulin receptor (IR) activation on inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for cotargeting IGF-1R and IR in cancer.

Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase–AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti–IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti–IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone. Mol Cancer Ther; 9(10); 2652–64. ©2010 AACR.

Reduced VEGF production, angiogenesis, and vascular regrowth contribute to the antitumor properties of dual mTORC1/mTORC2 inhibitors

The mammalian target of rapamycin (mTOR) pathway is implicated widely in cancer pathophysiology. Dual inhibition of the mTOR kinase complexes mTORC1 and mTORC2 decreases tumor xenograft growth in vivo and VEGF secretion in vitro, but the relationship between these two effects are unclear. In this study, we examined the effects of mTORC1/2 dual inhibition on VEGF production, tumor angiogenesis, vascular regression, and vascular regrowth, and we compared the effects of dual inhibition to mTORC1 inhibition alone. ATP-competitive inhibitors OSI-027 and OXA-01 targeted both mTORC1 and mTORC2 signaling in vitro and in vivo, unlike rapamycin that only inhibited mTORC1 signaling. OXA-01 reduced VEGF production in tumors in a manner associated with decreased vessel sprouting but little vascular regression. In contrast, rapamycin exerted less effect on tumoral production of VEGF. Treatment with the selective VEGFR inhibitor OSI-930 reduced vessel sprouting and caused substantial vascular regression in tumors. However, following discontinuation of OSI-930 administration tumor regrowth could be slowed by OXA-01 treatment. Combining dual inhibitors of mTORC1 and mTORC2 with a VEGFR2 inhibitor decreased tumor growth more than either inhibitor alone. Together, these results indicate that dual inhibition of mTORC1/2 exerts antiangiogenic and antitumoral effects that are even more efficacious when combined with a VEGFR antagonist.

Epithelial–Mesenchymal Transition Predicts Sensitivity to the Dual IGF-1R/IR Inhibitor OSI-906 in Hepatocellular Carcinoma Cell Lines

A growing body of data indicates that inhibiting the type 1 insulin-like growth factor receptor (IGF-1R) might be an effective treatment strategy for hepatocellular carcinoma (HCC). OSI-906 is a dual IGF-1R/IR kinase inhibitor currently in phase II clinical development for HCC. However, biomarkers are lacking to help identify patients with HCC who are more likely to benefit from OSI-906 treatment. We sought to determine the effect of OSI-906 on proliferation against a panel of 21 HCC cell lines and to investigate molecular determinants of responsiveness to OSI-906. We identified a subset of HCC cell lines that was sensitive to OSI-906, and sensitivity is associated with elevated phosphorylation levels of IGF-1R and IR and greater inhibition of AKT signaling. Dual targeting of both receptors seems to be important for maximal inhibition as treatment with a selective IGF-1R–neutralizing antibody was associated with increased IR signaling, whereas OSI-906 fully inhibited both phosphorylated IR and IGF-1R and resulted in greater inhibition of the IRS/AKT pathway. Epithelial–mesenchymal transition (EMT) seems to predict HCC cell sensitivity to OSI-906, as the epithelial phenotype is strongly associated with expression of IGF-2 and IR, activation of IGF-1R and IR, and sensitivity to OSI-906, alone or in combination with erlotinib. Induction of EMT upon treatment with TGFβ reduced sensitivity to OSI-906. Collectively, these data support the concept for dual IGF-1R/IR targeting in HCC, where EMT status and expressions of IGF-2 and IR may be used to identify those patients who are most likely to benefit from treatment with an IGF-1R/IR dual inhibitor. Mol Cancer Ther; 11(2); 503–13. ©2011 AACR.

Mechanisms of resistance to EGFR tyrosine kinase inhibitors: implications for patient selection and drug combination strategies

The receptor for epidermal growth factor (EGFR, ErbB1, HER1) supports the growth and maintenance of a broad range of human tumor types, and EGFR-targeting drugs are approved for the treatment of several advanced stage cancers, including non-small cell lung cancer (NSCLC), pancreatic cancer, squamous cell cancer of the head and neck (SCCHN), and colorectal cancer. Recent years have witnessed significant advances in our understanding of dysregulated signal transduction in cancer cells resulting from changes in the expression and/or mutational status of key signaling molecules that modulate sensitivity to drugs targeting EGFR. Based on this knowledge, we have an exciting opportunity to maximize the benefit provided to cancer patients by EGFR inhibitors. In this review article, we describe molecular determinants of sensitivity or resistance to EGFR-targeted agents, with specific emphasis on EGFR tyrosine kinase inhibitors (TKIs). The impact of these findings on our ability to evaluate candidate predictive biomarkers and to design robust mechanism-based combination strategies is also discussed.

Abstract P4-12-08: Use of an aptamer library based next generation omics platform for the development of a novel trastuzumab predictive assay

Introduction: Previous attempts to use individual aptamers as diagnostic reagents have failed to consistently achieve performance comparable to antibodies. Here we report a novel systems biology approach using poly-ligand aptamer libraries to identify responders and non-responders to traztuzumab-based regimens in metastatic breast cancer. Methods: To overcome the fundamental limitation of the individual aptamer binding affinities, large libraries (106 species) were created so that potentially thousands of aptamers could bind to each of a multitude of targets related to the whole cellular changes in response to trastuzumab therapy. A set of breast cancer patients, which received trastuzumab mono- or combined therapy for at least 7 months were classified as “Responders” (R); cases with particular regimen discontinued in the period not exceeding 5 months were classified as “Non-Responders”(NR). A library of 2x1012 unique 90-mer ssDNA oligodeoxynucleotides (ssODN) was trained on FFPE tissue of both R and NR patients. Partitioning of aptamer libraries was done by microdissection of the tumor tissue, after incubation of aptamer library with the entire tissue section, to drive selection pressure toward cancer cells. A total of 10 cases of R and NR, 6 Her2+ cases each, were used to train separate aptamer libraries, with 1 positive and 2 counter selection cases per enrichment. Enriched libraries were screened on 20 R and 20 NR cases (11 Her2+ cases each) by adopting modified immunohistochemistry protocol. Each library was used as an independent reagent (similar to an antibody in IHC) across all 40 cases to evaluate the efficacy of the aptamer library to distinguish differences between the R and NR groups. Staining (DAB chromogen) profiles were scored from 0 to 3+ (nuclear and cytoplasmic staining) by a pathologist without any knowledge of the clinical outcomes. Initial validation was done by t-test using raw histological scores. Four libraries showed significant p-values between groups of responders and non-responders, a classification algorithm was constructed and evaluated using area under the receiver-operator characteristic curve (AUC). The datasets of two best-performing libraries were combined into one model using logistic regression to further improved the classifier performance. Results: Of seventeen trained libraries, eight were evaluated and four showed significant correlation to clinical benefit with a minimum accuracy of 75% for each library when evaluated independently. Furthermore, two libraries showed exceptional performance (ROC curve AUC of 0.86 and 0.77). Combination of the profiling data from these two libraries using logistic regression resulted in an AUC of 0.985. A prospective validation of aptamer histochemical theranostic testing has been initiated. Summary: Enriched aptamer libraries appear to distinguish trastuzumab responsiveness in metastatic breast cancer. This technology could be used as an additional technique beyond FISH testing to determine sensitivity to anti-HER2 agents. The demonstrated platform is applicable to virtually any disease where the safe and effective use of corresponding drug is yet to be improved. Citation Format: Spetzler D, Domenyuk V, Santhanam R, Wei X, Stark A, Wang J, Gatalica Z, Miglarese M, Vidal G, Schwartzberg LS. Use of an aptamer library based next generation omics platform for the development of a novel trastuzumab predictive assay [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-08.

Abstract 2754: A novel liquid biopsy method for development of aptamer libraries that bind blood plasma exosomes from breast cancer patients

Improved technologies capable of characterizing system-wide changes associated with complex diseases will be required to be able to detect millions of proteins and their isoforms as well as multi-molecular complexes. We present a method for developing aptamer libraries using blood plasma exosomes that provides unprecedented system-wide coverage of native exosomal complexes. To train a naive aptamer library toward cancer samples (positive selection), the library (~1013 biotinylated ssODN species) was incubated with plasma from individual cancer patients and aptamer-bound exosomes were isolated using polymer-based precipitation. Negative selection was performed by contacting the aptamer library with exosomes from donors without breast cancer and recovering unbound aptamers from the supernatant. In all, 12 libraries trained toward 12 individual breast cancer patients were used to probe additional samples. Exosome-bound aptamers were identified and quantified by Next Generation Sequencing (NGS) to build highly accurate signatures for cancer/healthy donor classification. Using these signatures, cancer patients’ binding profiles were easily distinguishable from controls. Interestingly, cancer-trained libraries did not distinguish any of the negative control samples from each other, indicating that the selection pressure for cancer was high and noise due to inherent inter-healthy donor heterogeneity was minimal. Full validation studies are ongoing. Aptamer libraries may ultimately be deployed as a minimally-invasive diagnostic adjunct in breast and other cancers. Citation Format: Valeriy Domenyuk, Symon Levenberg, Adam Stark, Mark Miglarese, David Spetzler. A novel liquid biopsy method for development of aptamer libraries that bind blood plasma exosomes from breast cancer patients [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 2754. doi:10.1158/1538-7445.AM2017-2754

Abstract 4437: Small non-coding RNA profiling from prostate cancer plasma by deep sequencing

Background: Prostate cancer (PCa) is the most common non-skin cancer among American men. MicroRNAs (miRNAs) are critical post-transcriptional regulators and involved in prostate cancer tumorigenesis. The aim of this study is to identify a PCa-specific expression profile of miRNAs from plasma to guide prostate cancer diagnosis and therapeutic treatment. Methods: Plasma was collected from 5 PCa patients and 5 normal men. Circulating RNA was extracted from 1) 200ul plasma or 2) the pellets of anti-Ago2 immunoprecipitations from 500ul plasma using the miRNeasy Serum/Plasma kit (Qiagen), with addition of glycogen as a carrier. Small RNA libraries were constructed using the NEBNext Multiplex Small RNA Prep Set for Illumina® (New England. BioLabs). The cDNA library fragments were purified by Blue Pippin (Sage Science) for extraction of 140-160 bp size fraction containing small RNA inserts. Equimolar amounts of cDNA library samples were pooled and were sequenced in a single flowcell on an Illumina HiSeq2500 with 50 cycle kit and rapid run model. Bioinformatics analysis: Adaptor was firstly removed from the raw reads, and the sequences were mapped to several small RNA databases by using bowtie1 with 1 mismatch. The multiple aligned reads were weighted to the mapped small RNAs based on their unique mapped reads counts. We then calculated the RPM (reads per million) as indicator of the expression levels of the small RNA. To get confident analysis results, we discarded the small RNAs whose averages of the raw reads counts in cancer and normal groups are smaller than 25 and only focus on the mature microRNAs. The moderate t-test is applied to find the differently expressed (DE) microRNAs between normal and cancer group. Results: Two major small RNA classes identified from total plasma are miRNA (47.7%) and yRNA (35.0%). The percentage of miRNA increased to 85.3% by Ago2-IP method. The compositions of categories of small RNAs in cancer and normal samples are similar. We identified 28 and 22 differential miRNAs (>2 fold change between cancer and normal group) by total plasma and Ago-2 IP methods respectively. Conclusions: We discovered a unique expression profile of miRNA detectable in the plasma from prostate cancer patients. Extracted RNA from the pellets of anti-Ago2 immunoprecipitations can enhance the detection of miRNA. Expand study to confirm these findings are needed. Citation Format: Xianghua Liu, Andrew Hunter, Qing Zhang, Quanyuan He, Annemarie Benton, Gerri Ortiz, Vaishali Pannu, Nick Xiao, Mark Miglarese, David Spetzler. Small non-coding RNA profiling from prostate cancer plasma by deep sequencing [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 4437. doi:10.1158/1538-7445.AM2017-4437

Abstract 2469: The B-cell lymphoma specific aptamer C10.36 binds a ribonucleoprotein complex on the cell surface of cancer cells

Aptamers have recently gained prominence for their diagnostic and therapeutic potential. The DNA aptamer C10.36 forms a G-quadruplex and has been shown to bind the Ramos Burkitt’s lymphoma cell line. However, its binding partner on the cell surface remains unknown. Here we report on the identification of the molecular target of C10.36, which suggests its application in the therapy of B-cell lymphoma and leukaemia. Aptamer-affinity purification, followed by LC-MS/MS revealed unique proteins pulled down with C10.36 associated with Ramos cells but not Jurkat, a T-cell lymphocyte cell line. The majority of the identified target molecules were found to be associated within ribonucleoprotein complexes, of which the abundant and consistent ones belong to the nucleolin complex including nucleolin (NCL) itself and its interacting partners, i.e. nucleophosmin (NPM1), heterogeneous nuclear ribonucleoprotein (HNRNP) family members such as HNRNP C1C2 and U, rRNA 29-O-methyltransferase fibrillarin (FBL), actin (ACTB), nucleolar RNA helicase 2 (DDX21), and proline- and glutamine-rich splicing factors (SFPQ). All proteins identified in the above ribonucleoprotein complex are aberrantly expressed on the surface of several disparate cancer cell types and have been shown to play an oncogenic role in cancer. Another G-rich anti-NCL aptamer, AS1411, has been shown to induce cell death in >100 cancer cell lines. Therefore, we tested the effect of C10.36 on viability of Ramos and other non-Hodgkin B-cell lymphoma (NHL) cancer cell lines. Our results indicate that C10.36 treatment causes specific cell death of certain lymphoma cell lines such as Ramos but not Jurkat cells. The present study identifies C10.36 as a novel anti-B cell lymphoma aptamer and underscores its potential for the development of a new targeted therapy to treat B-cell lymphomas. Citation Format: Sonal S. Tonapi, Janet E. Duncan, Matthew Rosenow, Melissa Richards, Teresa L. Tinder, Heather A. O9Neill, Mark R. Miglarese, David Spetzler, Michael Famulok, Gunter Mayer. The B-cell lymphoma specific aptamer C10.36 binds a ribonucleoprotein complex on the cell surface of cancer cells [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 2469. doi:10.1158/1538-7445.AM2017-2469

Abstract LB-135: Adaptive dynamic artificial poly-ligand targeting (ADAPT) enables plasma-based exosome profiling with potential diagnostic utility

Introduction: Single stranded DNA (ssDNA) libraries consisting of several trillion oligodeoxynucleotides (ODNs) can adopt a nearly infinite number of three-dimensional structures. These structures can potentially bind any biomolecule and can be screened for specificity toward important biomarkers by employing suitable enrichment schemes. Since no prior knowledge on the binding partner is required, massively parallel biomarker identification is possible even on complex matrices like biological fluids and across a wide range of biological conditions. Here we present Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT) as a platform for biomarker and target discovery. We employed ADAPT for the molecular profiling of exosome-associated proteins in small volume plasma samples from women with breast cancer and healthy donors. Results: Random ssDNA-libraries of 10 11 unique ODNs were subjected to a number of selection and counter-selection steps on pooled blood plasma of breast cancer and healthy women. Several positive and negative enrichment schemes were employed, and exosome isolation and ODNs library partitioning were performed by ultracentrifugation and/or PEG precipitation. After library enrichment reduction of complexity to 10 6 -10 7 ), ODN libraries were used to probe an independent set of individual plasma samples from women with or without breast cancer. Two thousand differentially-binding aptamers with significant p-values were re-synthesized and combined in equimolar amounts to create a profiling library (L2000). The L2000 library was used to probe plasma samples from 323 individuals (206 from breast cancer patients and 117 from healthy donors) in triplicate. Using Next Generation Sequencing, we quantitated bound ODN from each plasma sample. ANOVA revealed 350 aptamers with significant p-values in distinguishing plasma samples from cancer patients and healthy donors, far in excess of the number of ODNs that would have achieved statistical significance by random sampling of the 2000 ODNs. Generalized linear model showed an AUC in a ROC curve of 0.94 for the training set. Random forest modelling was used to assess classification performance and revealed an AUC of 0.73 (p Conclusions: We have demonstrated the feasibility of aptamer library enrichment directly on blood plasma and have identified a set of 2000 DNA aptamers that distinguish plasma from women with breast cancer from women without breast cancer. This liquid biopsy approach requires only 200 microliters of plasma and is amenable to high-throughput processing. By employing a number of statistical approaches including rigorous cross-validation, we consistently achieve ROC AUC values >0.6. Further optimization of the aptamer library and testing on additional samples is ongoing. Upon complete validation, an ADAPT TM - derived breast cancer test may serve as a vital diagnostic adjunct that can be easily incorporated into standard clinical practice. Citation Format: Valeriy Domenyuk, Zhenyu Zhong, Jie Wang, Adam Stark, Nianqing Xiao, Mark Miglarese, George Poste, Michael Famulok, Gunter Mayer, David Spetzler. Adaptive dynamic artificial poly-ligand targeting (ADAPT) enables plasma-based exosome profiling with potential diagnostic utility. [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 LB-135.