Gopal Iyer, PhD

Gopal Iyer, PhD

Assistant Professor

Department of Human Oncology

I am an assistant professor in the Department of Human Oncology. My lab focuses on the molecular mechanisms of lung and pancreatic cancer development and its progression towards metastasis. Towards this effort, we apply experimental biophysical, optical imaging and computational approaches to analyze genes that are dysregulated in these cancers. We decipher the role of these gene mutations identified through genome sequencing efforts through external perturbations in cell lines, tissue slices and mouse models, closely mimicking therapeutic interventions. This approach could eventually lead to tailored treatments of patients and increase survival outcomes.

My laboratory fosters a multidisciplinary environment that bridges physics, chemistry and biology with the aim of training undergraduate, graduate and medical students towards aligning their interests in challenging questions in cancer. We constantly interact with biomedical engineers, medical and radiation oncologists and pathology experts to further our understanding of the clinical manifestations of the various stages of cancer. With our combined efforts, I hope to improve patients’ quality of life.

Education

Postdoc, University of California, Los Angeles & Stanford University, Biophysics (2011)

PhD, University of California, Berkeley & M.S. University of Baroda, India, Microbiology (2004)

MS, M.S. University of Baroda, India, Biotechnology (1990)

BS, M.S. University of Baroda, India, Chemistry, Botany (1988)

Academic Appointments

Assistant Professor, Human Oncology (2016)

Selected Honors and Awards

Rockefeller Foundation Fellowship (1997-2000)

Council of Scientific Industrial Research Scholarship (CSIR-INDIA) (1997-2000)

Department of Biotechnology Scholarship from Government of India for M.Sc Program (1988-1990)

Boards, Advisory Committees and Professional Organizations

ASTRO

Manuscript reviewer, ACS Nano

Manuscript reviewer, Chemistry

Manuscript reviewer, Optics Express

Manuscript reviewer, Nature Methods

Manuscript reviewer, Molecular Imaging

Manuscript reviewer, Journal of American Chemical Society

Manuscript reviewer, Angewandte Chemie

Manuscript reviewer, ACS Nanoletters

Manuscript reviewer, ACS Bioconjugate Chemistry

Manuscript reviewer, Small

Manuscript reviewer, Nanomedicine

Research Focus

Epigenetics and Phophorylation Processes, Acquired and Intrinsic Resistance to Cancer Therapy

The overarching goal of the Iyer Lab is to decipher the dynamics of signaling bias regulated by epigenetic and phosphorylation processes.

We use a head and neck cancer acquired resistance model and CRISPR models in pancreas and head and neck cancer that have knock-ins and knockouts of various oncogenic genes to decipher the dynamics of signaling bias regulated by epigenetic and phosphorylation processes. We are investigating perturbations of drug, radio and chemo-resistance. I was trained as a biochemist and molecular biologist in graduate school and switched to optical physics, microfluidics and chemistry in my postdoctoral career. My interdisciplinary skills are currently being used in my research to unravel the spatiotemporal kinetics of signaling modules during acquired and intrinsic resistance and apply quantitative methods to unravel the global genomic changes associated with these processes.

Androgen Receptor Signaling in Lung

Androgen receptor (AR) expression is necessary for early lung development. However, the presence of AR protein in non-small lung cancer adenocarcinoma (NSCLC) is intriguing. Recent cancer genome data have revealed the presence of mutations in AR in a subset (8 percent) of NSCLC patients that have unique mutation profiles compared to known mutations in lung cancer. In my lab, we explore the basic mechanism of AR transactivation in KRAS mutated and wild type NSCLC backgrounds. Using gene-editing cell lines, we are currently editing the AR mutations found from patient genome data and exploring its role towards the formation of lung adenocarcinoma by using optical imaging and mice models.

Genomics of Lung-Brain Metastasis

A combination of genetic and cellular events leads to tumor metastasis—the formation of secondary tumors at distant organs from the site of primary tumor. In order to alleviate the morbidity and mortality of this metastatic state, my lab has embarked on genomic approaches to sequence lung cancer patients in collaboration with oncologists and pathologists. We have performed transcriptome sequencing of more than 70 matched normal, primary lung tumor and brain metastatic patients. Extensive bioinformatics analyses revealed a unique set of brain specific genes that have potentially originated from the primary lung tumor site. Currently, we are exploring various approaches to validate these genes for their metastatic potential by fluorescent and bioluminescent tagging in vitro and in vivo models.

Targeting Bromodomain and extra-terminal (BET) Proteins in Solid Tumors

Bromodomains (BRDs) are evolutionarily conserved protein domains that interact with acetylated histones and set off a chain of events that includes recruiting transcription factors and other chromatin binding proteins to initiate RNA transcription. Using a pan-cancer approach, my lab is interested in exploring whether the inhibition of BRDs can lead to tumor regression either as a monotherapy or in combination with other drugs and chemo-radiation perturbations. The rationale of this approach is to block transcription of oncogenes like MYC and other onco-proteins that do not have inhibitors, leading to a block in cancer progression. Using nanomolar affinity inhibitors to BRDs, we unraveled a novel set of transcription factors that regulated differentially in several pancreatic and head and neck cancer lines leading to a remarkable suppression in cell proliferation and increased apoptosis. These findings have led us to explore if perturbation of BRDs across solid tumors is a conserved mechanism or has tissue specificity with respect to its transcriptional network. Using a systematic approach of RNA-sequencing, optical imaging and mice models, we are focused on addressing the relationship between BRD-BET inhibition with the mutational landscape of these solid tumors and whether we can tailor these inhibitors towards solid tumors that have specific mutations to create a more personalized treatment approach.

  • Development and characterization of patient-derived xenografts from non-small cell lung cancer brain metastases Scientific reports
    Baschnagel AM, Kaushik S, Durmaz A, Goldstein S, Ong IM, Abel L, Clark PA, Gurel Z, Leal T, Buehler D, Iyer G, Scott JG, Kimple RJ
    2021 Jan 28;11(1):2520. doi: 10.1038/s41598-021-81832-1.
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      Non-small cell lung cancer (NSCLC) brain metastasis cell lines and in vivo models are not widely accessible. Herein we report on a direct-from patient-derived xenograft (PDX) model system of NSCLC brain metastases with genomic annotation useful for translational and mechanistic studies. Both heterotopic and orthotopic intracranial xenografts were established and RNA and DNA sequencing was performed on patient and matching tumors. Morphologically, strong retention of cytoarchitectural features was observed between original patient tumors and PDXs. Transcriptome and mutation analysis revealed high correlation between matched patient and PDX samples with more than more than 95% of variants detected being retained in the matched PDXs. PDXs demonstrated response to radiation, response to selumetinib in tumors harboring KRAS G12C mutations and response to savolitinib in a tumor with MET exon 14 skipping mutation. Savolitinib also demonstrated in vivo radiation enhancement in our MET exon 14 mutated PDX. Early passage cell strains showed high consistency between patient and PDX tumors. Together, these data describe a robust human xenograft model system for investigating NSCLC brain metastases. These PDXs and cell lines show strong phenotypic and molecular correlation with the original patient tumors and provide a valuable resource for testing preclinical therapeutics.

      PMID:33510214 | PMC:PMC7843608 | DOI:10.1038/s41598-021-81832-1


      View details for PubMedID 33510214
  • Geographical heterogeneity in the American Joint committee on Cancer oral cancer staging and prognostic implications Oral oncology
    Subramaniam N, Clark JR, Goldstein D, Almeida Jd, Abdalaty HA, Balasubramanian D, Thankappan K, Iyer S, Batstone M, Iyer NG, Smee RI, Chandrasekhar NH, Pillai V, Shetty V, Rangappa V, Veness M, Low TH
    2021 Feb;113:105122. doi: 10.1016/j.oraloncology.2020.105122. Epub 2020 Dec 21.
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      OBJECTIVES: The AJCC 8th edition (AJCC 8) has introduced depth of invasion (DOI) and extranodal extension (ENE) into staging for oral squamous cell carcinoma (OSCC). Although validations have been performed on institutional datasets have shown a good performance, particularly in early OSCC, there have been no studies on diverse patient populations that determine the impact on prognostic heterogeneity.

      MATERIALS AND METHODS: Retrospective analysis of 4710 patients with oral squamous cell carcinoma (OSCC) treated with surgery +/- adjuvant therapy in 8 institutions in Australia, North America and Asia. With overall survival (OS) as endpoint, the prognostic performance of AJCC 7th and 8th editions were compared using Akaike Information Criterion (AIC), Bayesian Information Criteria (BIC), Harrell's concordance index (C-index).

      RESULTS: When comparing AJCC 8 to AJCC 7, the heterogeneity in prediction of OS increased for T-category and N-category while remaining unchanged for TNM staging, suggesting AJCC 8 increased complexity with no improvement in predictive value. There were significant differences in median DOI and incidence of ENE between geographical regions, resulting in dissimilar rates of stage-migration when adopting AJCC 8.

      CONCLUSION: In an attempt to improve prognostic performance, AJCC 8 introduced more variables; however heterogeneity in these results in significant geographical differences in model discrimination and performance. Caution should be applied as this may result in inaccurate and unreliable prognostic predictions that may impact treatment recommendations.

      PMID:33352532 | DOI:10.1016/j.oraloncology.2020.105122


      View details for PubMedID 33352532
  • FGFR Inhibition Enhances Sensitivity to Radiation in Non-Small Cell Lung Cancer Molecular cancer therapeutics
    SenthilKumar G, Fisher MM, Skiba JH, Miller MC, Brennan SR, Kaushik S, Bradley ST, Longhurst CA, Buehler D, Nickel KP, Iyer G, Kimple RJ, Baschnagel AM
    2020 Jun;19(6):1255-1265. doi: 10.1158/1535-7163.MCT-19-0931. Epub 2020 May 5.
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      FGFRs are commonly altered in non-small cell lung cancer (NSCLC). FGFRs activate multiple pathways including RAS/RAF/MAPK, PI3K/AKT, and STAT, which may play a role in the cellular response to radiation. We investigated the effects of combining the selective FGFR 1-3 tyrosine kinase inhibitor AZD4547 with radiation in cell line and xenograft models of NSCLC. NSCLC cell lines were assessed with proliferation, clonogenic survival, apoptosis, autophagy, cell cycle, and DNA damage signaling and repair assays. In vivo xenografts and IHC were used to confirm in vitro results. NSCLC cell lines demonstrated varying degrees of FGFR protein and mRNA expression. In vitro clonogenic survival assays showed radiosensitization with AZD4547 in two NSCLC cell lines. In these two cell lines, an increase in apoptosis and autophagy was observed with combined radiation and AZD4547. The addition of AZD4547 to radiation did not significantly affect γH2AX foci formation. Enhanced xenograft tumor growth delay was observed with the combination of radiation and AZD4547 compared with radiation or drug alone. IHC results revealed inhibition of pMAPK and pS6 and demonstrated an increase in apoptosis in the radiation plus AZD4547 group. This study demonstrates that FGFR inhibition by AZD4547 enhances the response of radiation in FGFR-expressing NSCLC in vitro and in vivo model systems. These results support further investigation of combining FGFR inhibition with radiation as a clinical therapeutic strategy.

      PMID:32371583 | PMC:PMC7272291 | DOI:10.1158/1535-7163.MCT-19-0931


      View details for PubMedID 32371583
  • Fibroblast Growth Factor Receptors as Targets for Radiosensitization in Head and Neck Squamous Cell Carcinomas International journal of radiation oncology, biology, physics
    Fisher MM, SenthilKumar G, Hu R, Goldstein S, Ong IM, Miller MC, Brennan SR, Kaushik S, Abel L, Nickel KP, Iyer G, Harari PM, Kimple RJ, Baschnagel AM
    2020 Jul 15;107(4):793-803. doi: 10.1016/j.ijrobp.2020.03.040. Epub 2020 Apr 13.
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      PURPOSE: We examined the capacity of the pan-fibroblast growth factor receptor (FGFR) inhibitor AZD4547 to augment radiation response across a panel of head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts.

      METHODS AND MATERIALS: FGFR1, FGFR2, and FGFR3 RNA in situ hybridization expression was assessed in a cohort of HNSCC patient samples, cell lines, and patient-derived xenografts (PDXs). In vitro effects of AZD4547 and radiation on cell survival, FGFR signaling, apoptosis, autophagy, cell cycle, and DNA damage repair were evaluated. Reverse phase protein array was used to identify differentially phosphorylated proteins in cells treated with AZD4547. In vivo tumor responses were evaluated in cell lines and PDX models.

      RESULTS: FGFR1, FGFR2, and FGFR3 RNA in situ hybridization were expressed in 41%, 81%, and 89% of 107 oropharynx patient samples. Sensitivity to AZD4547 did not directly correlate with FGFR protein or RNA expression. In sensitive cell lines, AZD4547 inhibited p-MAPK in a time-dependent manner. Significant radiosensitization with AZD4547 was observed in cell lines that were sensitive to AZD4547. The mechanism underlying these effects appears to be multifactorial, involving inhibition of the MTOR pathway and subsequent enhancement of autophagy and activation of apoptotic pathways. Significant tumor growth delay was observed when AZD4547 was combined with radiation compared with radiation or drug alone in an FGFR-expressing HNSCC cell line xenograft and PDX.

      CONCLUSIONS: These findings suggest that AZD4547 can augment the response of radiation in FGFR-expressing HNSCC in vivo model systems. FGFR1 and FGFR2 may prove worthy targets for radiosensitization in HNSCC clinical investigations.

      PMID:32298810 | PMC:PMC7321889 | DOI:10.1016/j.ijrobp.2020.03.040


      View details for PubMedID 32298810
  • <sup>177</sup>Lu-NM600 Targeted Radionuclide Therapy Extends Survival in Syngeneic Murine Models of Triple-Negative Breast Cancer Journal of nuclear medicine : official publication, Society of Nuclear Medicine
    Hernandez R, Grudzinski JJ, Aluicio-Sarduy E, Massey CF, Pinchuk AN, Bitton AN, Patel R, Zhang R, Rao AV, Iyer G, Engle JW, Weichert JP
    2020 Aug;61(8):1187-1194. doi: 10.2967/jnumed.119.236265. Epub 2019 Dec 20.
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      There is a clinically unmet need for effective treatments for triple-negative breast cancer (TNBC), as it remains the most aggressive subtype of breast cancer. Herein, we demonstrate a promising strategy using a tumor-targeting alkylphosphocholine (NM600) for targeted radionuclide therapy of TNBC. Methods: NM600 was radiolabeled with 86Y for PET imaging and 177Lu for targeted radionuclide therapy. 86Y-NM600 PET imaging was performed on female BALB/C mice bearing syngeneic 4T07 (nonmetastatic) and 4T1 (metastatic) TNBC tumor grafts (n = 3-5). Quantitative data derived from a PET-image region-of-interest analysis, which was corroborated by ex vivo biodistribution, were used to estimate the dosimetry of 177Lu-NM600 treatments. Weight measurement, complete blood counts, and histopathology analysis were performed to determine 177Lu-NM600 toxicity in naïve BALB/C mice administered 9.25 or 18.5 MBq. Groups of mice bearing 4T07 or 4T1 grafts (n = 5-6) received excipient or 9.25 or 18.5 MBq of 177Lu-NM600 as a single or fractionated schedule, and tumor growth and overall survival were monitored. Results: Excellent tumor targeting and rapid normal-tissue clearance of 86Y-NM600 were noted in both 4T07 and 4T1 murine models. Ex vivo biodistribution corroborated the accuracy of the PET data and validated 86Y-NM600 as a surrogate for 177Lu-NM600. 177Lu-NM600 dosimetry showed absorbed doses of 2.04 ± 0.32 and 1.68 ± 0.06 Gy/MBq to 4T07 and 4T1 tumors, respectively, which were larger than those delivered to liver (1.28 ± 0.09 Gy/MBq) and to bone marrow (0.31 ± 0.05 Gy/MBq). The 177Lu-NM600 injected activities used for treatment were well tolerated and resulted in significant tumor growth inhibition and prolonged overall survival in both tested TNBC models. A complete response was attained in 60% of treated mice bearing 4T07 grafts. Conclusion: Overall, our results suggest that 177Lu-NM600 targeted radionuclide therapy has potential for TNBC and merits further exploration in a clinical setting.

      PMID:31862799 | PMC:PMC7413241 | DOI:10.2967/jnumed.119.236265


      View details for PubMedID 31862799
  • Defining Reprogramming Checkpoints from Single-Cell Analyses of Induced Pluripotency Cell reports
    Tran KA, Pietrzak SJ, Zaidan NZ, Siahpirani AF, McCalla SG, Zhou AS, Iyer G, Roy S, Sridharan R
    2019 May 7;27(6):1726-1741.e5. doi: 10.1016/j.celrep.2019.04.056.
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      Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a combination of epigenomic modifiers and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress toward iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenomic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.

      PMID:31067459 | PMC:PMC6555151 | DOI:10.1016/j.celrep.2019.04.056


      View details for PubMedID 31067459
  • Enhanced Radiosensitivity in Solid Tumors using a Tumor-selective Alkyl Phospholipid Ether Analog Molecular cancer therapeutics
    Elsaid MY, Shahi A, Wang AR, Baiu DC, Li C, Werner LR, Singhal S, Hall LT, Weichert JP, Armstrong EA, Bednarz BP, Harari PM, Iyer G, Otto M
    2018 Nov;17(11):2320-2328. doi: 10.1158/1535-7163.MCT-17-0897. Epub 2018 Aug 14.
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      Antitumor alkyl phospholipid (APL) analogs comprise a group of structurally related molecules with remarkable tumor selectivity. Some of these compounds have shown radiosensitizing capabilities. CLR127 is a novel, clinical-grade antitumor APL ether analog, a subtype of synthetic APL broadly targeting cancer cells with limited uptake in normal tissues. The purpose of this study was to investigate the effect of CLR127 to modulate radiation response across several adult and pediatric cancer types in vitro as well as in murine xenograft models of human prostate adenocarcinoma, neuroblastoma, Ewing sarcoma, and rhabdomyosarcoma. In vitro, CLR127 demonstrated selective uptake in cancer cells compared to normal cells. In cancer cells, CLR127 treatment prior to radiation significantly decreased clonogenic survival in vitro, and led to increased radiation-induced double-stranded DNA (dsDNA) breakage compared with radiation alone, which was not observed in normal controls. In animal models, CLR127 effectively increased the antitumor response to fractionated radiotherapy and led to delayed tumor regrowth at potentially clinically achievable doses. In conclusion, our study highlights the ability of CLR127 to increase radiation response in several cancer types. Given almost universal uptake of CLR127 in malignant cells, future research should test whether the observed effects can be extended to other tumor types. Our data provide a strong rationale for clinical testing of CLR127 as a tumor-targeted radiosensitizing agent. Mol Cancer Ther; 17(11); 2320-8. ©2018 AACR.

      PMID:30108133 | PMC:PMC6215514 | DOI:10.1158/1535-7163.MCT-17-0897


      View details for PubMedID 30108133
  • Compartmentalization of HP1 Proteins in Pluripotency Acquisition and Maintenance Stem cell reports
    Zaidan NZ, Walker KJ, Brown JE, Schaffer LV, Scalf M, Shortreed MR, Iyer G, Smith LM, Sridharan R
    2018 Feb 13;10(2):627-641. doi: 10.1016/j.stemcr.2017.12.016.
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      The heterochromatin protein 1 (HP1) family is involved in various functions with maintenance of chromatin structure. During murine somatic cell reprogramming, we find that early depletion of HP1γ reduces the generation of induced pluripotent stem cells, while late depletion enhances the process, with a concomitant change from a centromeric to nucleoplasmic localization and elongation-associated histone H3.3 enrichment. Depletion of heterochromatin anchoring protein SENP7 increased reprogramming efficiency to a similar extent as HP1γ, indicating the importance of HP1γ release from chromatin for pluripotency acquisition. HP1γ interacted with OCT4 and DPPA4 in HP1α and HP1β knockouts and in H3K9 methylation depleted H3K9M embryonic stem cell (ESC) lines. HP1α and HP1γ complexes in ESCs differed in association with histones, the histone chaperone CAF1 complex, and specific components of chromatin-modifying complexes such as DPY30, implying distinct functional contributions. Taken together, our results reveal the complex contribution of the HP1 proteins to pluripotency.

      PMID:29358085 | PMC:PMC5830946 | DOI:10.1016/j.stemcr.2017.12.016


      View details for PubMedID 29358085
  • Near-infrared peptide-coated quantum dots for small animal imaging Proceedings of SPIE--the International Society for Optical Engineering
    Iyer G, Li JJ, Pinaud F, Tsay JM, Bentolila LA, Michalet X, Weiss S
    2006 Jan;6096:60960B. doi: 10.1117/12.661777. Epub 2006 Mar 24.
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      We have synthesized high quality type-II CdTe/CdSe near infrared quantum dots using successive ion layer adsorption and reaction chemistry. Transmission electron microscopy reveals that CdTe/CdSe can be synthesized layer by layer yielding quantum dots of narrow size distribution. Excitation and photoluminescence spectra reveal discrete type-II transitions, which correspond to energy lower that type-I bandgap. We have used a peptide coating technique on type-II and commercial near infrared quantum dots for delivery in live animals and cultured cells.

      PMID:29151665 | PMC:PMC5693312 | DOI:10.1117/12.661777


      View details for PubMedID 29151665
  • Identification of stable housekeeping genes in response to ionizing radiation in cancer research Scientific reports
    Iyer G, Wang AR, Brennan SR, Bourgeois S, Armstrong E, Shah P, Harari PM
    2017 Mar 6;7:43763. doi: 10.1038/srep43763.
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      Housekeeping genes (HKGs) are essential for basic maintenance of a variety of cellular processes. They ideally maintain uniform expression independent of experimental conditions. However, the effects of ionizing radiation (IR) on HKG expression is unclear. Statistical algorithms, geNorm and Normfinder were used for estimating the stability of HKGs as raw quantification cycle (Cq) values were not a reliable factor for normalization. Head and neck, non-small lung and pancreas cells were exposed to 2, 4 and 6 Gy IR doses and expression of fourteen HKGs was measured at 5 min to 48 h post-irradiation within a given tissue. Paired and single cell line analyses under these experimental conditions identified TATA-Box Binding Protein (TBP) and Importin 8 (IPO8) to be stable in non-small cell lung cancer. In addition to these two genes, Ubiquitin C (UBC) in head and neck cancer and Transferrin receptor (TFRC) and β-Glucuronidase (GUSB) in pancreatic cancer were identified to be stable as well. In summary we present a resource for top ranked five stable HKGs and their transcriptional behavior in commonly used cancer model cell lines and suggest the use of multiple HKGs under radiation treatment conditions is a reliable metric for quantifying gene expression.

      PMID:28262749 | PMC:PMC5338320 | DOI:10.1038/srep43763


      View details for PubMedID 28262749
  • Erratum to: Insulin-like growth factor 1 receptor mediated tyrosine 845 phosphorylation of epidermal growth factor receptor in the presence of monoclonal antibody cetuximab BMC cancer
    Iyer G, Price J, Bourgeois S, Armstrong E, Huang S, Harari PM
    2016 Dec 1;16(1):928. doi: 10.1186/s12885-016-2958-x.
  • Insulin-like growth factor 1 receptor mediated tyrosine 845 phosphorylation of epidermal growth factor receptor in the presence of monoclonal antibody cetuximab BMC cancer
    Iyer G, Price J, Bourgeois S, Armstrong E, Huang S, Harari PM
    2016 Oct 6;16(1):773. doi: 10.1186/s12885-016-2796-x.
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      BACKGROUND: The epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and several other human cancers. Monoclonal antibodies, such as cetuximab that block EGFR signaling, have emerged as valuable molecular targeting agents in clinical cancer therapy. Prolonged exposure to cetuximab can result in cells acquiring resistance by a process that remains incompletely understood.

      METHODS: In this study, we analyzed the immediate early molecular response of cetuximab on physical interactions between EGFR and Insulin growth factor 1 like receptor (IGF-1R) in head and neck cancer cells that are resistant to cetuximab. Co-immunoprecipitation, small molecule inhibitors against phospho-Src and IGF-1R, quantitative western blot of EGFR and Src phosphorylation, cell proliferation assays were used to suggest the role of IGF-1R mediated phosphorylation of specific tyrosine Y845 on EGFR via increased heterodimerization of EGFR and IGF-1R in cetuximab resistant cells.

      RESULTS: Heterodimerization of EGFR with IGF-1R was increased in cetuximab resistant HNSCC cell line UMSCC6. Basal levels of phosphorylated EGFR Y845 showed significant increase in the presence of cetuximab. Surprisingly, this activated Y845 level was not inhibited in the presence of Src inhibitor PP1. Instead, inhibition of IGF-1R by picropodophyllin (PPP) reduced the EGFR Y845 levels. Taken together, these results suggest that heterodimerization of EGFR with IGF-1R can lead to increased activity of EGFR and may be an important platform for cetuximab mediated signaling in head and neck tumors that have become resistant to anti-EGFR therapy.

      CONCLUSIONS: EGFR-IGF-1R interaction has a functional consequence of phosphorylation of EGFR Y845 in cetuximab resistant HNSCC cells and dual targeting of EGFR and IGF-1R is a promising therapeutic strategy.

      PMID:27716204 | PMC:PMC5054590 | DOI:10.1186/s12885-016-2796-x


      View details for PubMedID 27716204
  • Microfluidic enrichment for the single cell analysis of circulating tumor cells Scientific reports
    Yeo T, Tan SJ, Lim CL, Lau PX, Chua YW, Krisna SS, Iyer G, Tan GS, Lim KH, Tan SW, Lim W, Lim CT
    2016 Feb 29;6:22076. doi: 10.1038/srep22076.
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      Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.

      PMID:26924553 | PMC:PMC4770429 | DOI:10.1038/srep22076


      View details for PubMedID 26924553
  • Small Molecule Inhibition of MDM2-p53 Interaction Augments Radiation Response in Human Tumors Molecular cancer therapeutics
    Werner LR, Huang S, Francis DM, Armstrong EA, Ma F, Li C, Iyer G, Canon J, Harari PM
    2015 Sep;14(9):1994-2003. doi: 10.1158/1535-7163.MCT-14-1056-T. Epub 2015 Jul 10.
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      MDM2-p53 interaction and downstream signaling affect cellular response to DNA damage. AMG 232 is a potent small molecule inhibitor that blocks the interaction of MDM2 and p53. We examined the capacity of AMG 232 to augment radiation response across a spectrum of human tumor cell lines and xenografts. AMG 232 effectively inhibited proliferation and enhanced radiosensitivity via inhibition of damage repair signaling. Combined AMG 232 and radiation treatment resulted in the accumulation of γH2AX-related DNA damage and induction of senescence with promotion of apoptotic and/or autophagic cell death. Several molecules involved in senescence, autophagy, and apoptosis were specifically modulated following the combined AMG 232/radiation treatment, including FoxM1, ULK-1, DRAM, and BAX. In vivo xenograft studies confirmed more potent antitumor and antiangiogenesis efficacy with combined AMG 232/radiation treatment than treatment with drug or radiation alone. Taken together, these data identify the capacity of AMG 232 to augment radiation response across a variety of tumor types harboring functional p53.

      PMID:26162687 | DOI:10.1158/1535-7163.MCT-14-1056-T


      View details for PubMedID 26162687
  • Single-step conjugation of antibodies to quantum dots for labeling cell surface receptors in mammalian cells Methods in molecular biology (Clifton, N.J.)
    Iyer G, Xu J, Weiss S
    2011;751:553-63. doi: 10.1007/978-1-61779-151-2_34.
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      Labeling of cell surface receptors in living cells can be achieved using antibody-conjugated semiconductor quantum dots (QDs). The inherent photostable property of QDs can be exploited for understanding the arrangement and distribution of receptors in the plasma membrane. We describe herein methods that allow conjugation of antibodies to QDs in a single step without the formation of side products. This protocol can be adapted universally for any type of QD structure with a coating of free amino groups.

      PMID:21674354 | DOI:10.1007/978-1-61779-151-2_34


      View details for PubMedID 21674354
  • Aromatic aldehyde and hydrazine activated peptide coated quantum dots for easy bioconjugation and live cell imaging Bioconjugate chemistry
    Iyer G, Pinaud F, Xu J, Ebenstein Y, Li J, Chang J, Dahan M, Weiss S
    2011 Jun 15;22(6):1006-11. doi: 10.1021/bc100593m. Epub 2011 May 16.
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      We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.

      PMID:21553893 | PMC:PMC3116077 | DOI:10.1021/bc100593m


      View details for PubMedID 21553893
  • Tracking single proteins in live cells using single-chain antibody fragment-fluorescent quantum dot affinity pair Methods in enzymology
    Iyer G, Michalet X, Chang Y, Weiss S
    2010;475:61-79. doi: 10.1016/S0076-6879(10)75003-5.
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      Quantum dots (QDs) are extremely bright fluorescent imaging probes that are particularly useful for tracking individual molecules in living cells. Here, we show how a two-component system composed of a high-affinity single-chain fragment antibody and its cognate hapten (fluorescein) can be utilized for tracking individual proteins in various cell types. The single-chain fragment antibody against fluorescein is genetically appended to the protein of interest, while the hapten fluorescein is attached to the end of the peptide that is used to coat the QDs. We describe (i) the method used to functionalize QDs with fluorescein peptides; (ii) the method used to control the stoichiometry of the hapten on the surface of the QD; and (iii) the technical details necessary to observe single molecules in living cells.

      PMID:20627153 | PMC:PMC3405731 | DOI:10.1016/S0076-6879(10)75003-5


      View details for PubMedID 20627153
  • Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI) Proceedings of the National Academy of Sciences of the United States of America
    Dertinger T, Colyer R, Iyer G, Weiss S, Enderlein J
    2009 Dec 29;106(52):22287-92. doi: 10.1073/pnas.0907866106. Epub 2009 Dec 14.
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      Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.

      PMID:20018714 | PMC:PMC2799731 | DOI:10.1073/pnas.0907866106


      View details for PubMedID 20018714
  • Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking Traffic (Copenhagen, Denmark)
    Pinaud F, Michalet X, Iyer G, Margeat E, Moore H, Weiss S
    2009 Jun;10(6):691-712. doi: 10.1111/j.1600-0854.2009.00902.x. Epub 2009 Mar 27.
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      Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

      PMID:19416475 | PMC:PMC2766537 | DOI:10.1111/j.1600-0854.2009.00902.x


      View details for PubMedID 19416475
  • High affinity scFv-hapten pair as a tool for quantum dot labeling and tracking of single proteins in live cells Nano letters
    Iyer G, Michalet X, Chang Y, Pinaud FF, Matyas SE, Payne G, Weiss S
    2008 Dec;8(12):4618-23. doi: 10.1021/nl8032284.
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      We describe a general approach to label cell surface proteins using quantum dots (QD) for single-molecule tracking. QDs coated with small-hapten modified peptides are targeted to cell surface fusion proteins containing the corresponding single-chain fragment antibody (scFv). The approach is illustrated with the small hapten fluorescein (FL) and a high-affinity anti-FL scFv fused to two different proteins in yeast and murine neuronal cell line N2a.

      PMID:19053789 | PMC:PMC3084663 | DOI:10.1021/nl8032284


      View details for PubMedID 19053789
  • Particle size, surface coating, and PEGylation influence the biodistribution of quantum dots in living mice Small (Weinheim an der Bergstrasse, Germany)
    Schipper ML, Iyer G, Koh AL, Cheng Z, Ebenstein Y, Aharoni A, Keren S, Bentolila LA, Li J, Rao J, Chen X, Banin U, Wu AM, Sinclair R, Weiss S, Gambhir SS
    2009 Jan;5(1):126-34. doi: 10.1002/smll.200800003.
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      This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

      PMID:19051182 | PMC:PMC3084659 | DOI:10.1002/smll.200800003


      View details for PubMedID 19051182
  • microPET-based biodistribution of quantum dots in living mice Journal of nuclear medicine : official publication, Society of Nuclear Medicine
    Schipper ML, Cheng Z, Lee S, Bentolila LA, Iyer G, Rao J, Chen X, Wu AM, Weiss S, Gambhir SS
    2007 Sep;48(9):1511-8. doi: 10.2967/jnumed.107.040071. Epub 2007 Aug 17.
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      This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice.

      METHODS: (64)Cu-Labeled 800- or 525-nm emission wavelength QD (21- or 12-nm diameter), with or without 2,000 MW (molecular weight) polyethylene glycol (PEG), were injected intravenously into mice (5.55 MBq/25 pmol QD) and studied using well counting or by serial microPET and region-of-interest analysis.

      RESULTS: Both methods show rapid uptake by the liver (27.4-38.9 %ID/g) (%ID/g is percentage injected dose per gram tissue) and spleen (8.0-12.4 %ID/g). Size has no influence on biodistribution within the range tested here. Pegylated QD have slightly slower uptake into liver and spleen (6 vs. 2 min) and show additional low-level bone uptake (6.5-6.9 %ID/g). No evidence of clearance from these organs was observed.

      CONCLUSION: Rapid reticuloendothelial system clearance of QD will require modification of QD for optimal utility in imaging living subjects. Formal quantitative biodistribution/imaging studies will be helpful in studying many types of nanoparticles, including quantum dots.

      PMID:17704240 | PMC:PMC4146342 | DOI:10.2967/jnumed.107.040071


      View details for PubMedID 17704240
  • Solubilization of quantum dots with a recombinant peptide from Escherichia coli Small (Weinheim an der Bergstrasse, Germany)
    Iyer G, Pinaud F, Tsay J, Weiss S
    2007 May;3(5):793-8. doi: 10.1002/smll.200600516.
  • Peptide coated quantum dots for biological applications IEEE transactions on nanobioscience
    Iyer G, Pinaud F, Tsay J, Li JJ, Bentolila LA, Michalet X, Weiss S
    2006 Dec;5(4):231-8. doi: 10.1109/tnb.2006.886563.
    • More

      Quantum dots (QDOTs) have been widely recognized by the scientific community and the biotechnology industry, as witnessed by the exponential growth of this field in the past several years. We describe the synthesis and characterization of visible and near infrared QDots--a critical step for engineering organic molecules like proteins and peptides for building nanocomposite materials with multifunctional properties suitable for biological applications.

      PMID:17181021 | PMC:PMC3074086 | DOI:10.1109/tnb.2006.886563


      View details for PubMedID 17181021
  • VIB-1 is required for expression of genes necessary for programmed cell death in Neurospora crassa Eukaryotic cell
    Dementhon K, Iyer G, Glass NL
    2006 Dec;5(12):2161-73. doi: 10.1128/EC.00253-06. Epub 2006 Sep 29.
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      Nonself recognition during somatic growth is an essential and ubiquitous phenomenon in both prokaryotic and eukaryotic species. In filamentous fungi, nonself recognition is also important during vegetative growth. Hyphal fusion between genetically dissimilar individuals results in rejection of heterokaryon formation and in programmed cell death of the fusion compartment. In filamentous fungi, such as Neurospora crassa, nonself recognition and heterokaryon incompatibility (HI) are regulated by genetic differences at het loci. In N. crassa, mutations at the vib-1 locus suppress nonself recognition and HI mediated by genetic differences at het-c/pin-c, mat, and un-24/het-6. vib-1 is a homolog of Saccharomyces cerevisiae NDT80, which is a transcriptional activator of genes during meiosis. For this study, we determined that vib-1 encodes a nuclear protein and showed that VIB-1 localization varies during asexual reproduction and during HI. vib-1 is required for the expression of genes involved in nonself recognition and HI, including pin-c, tol, and het-6; all of these genes encode proteins containing a HET domain. vib-1 is also required for the production of downstream effectors associated with HI, including the production of extracellular proteases upon carbon and nitrogen starvation. Our data support a model in which mechanisms associated with starvation and nonself recognition/HI are interconnected. VIB-1 is a major regulator of responses to nitrogen and carbon starvation and is essential for the expression of genes involved in nonself recognition and death in N. crassa.

      PMID:17012538 | PMC:PMC1694810 | DOI:10.1128/EC.00253-06


      View details for PubMedID 17012538
  • Advances in fluorescence imaging with quantum dot bio-probes Biomaterials
    Pinaud F, Michalet X, Bentolila LA, Tsay JM, Doose S, Li JJ, Iyer G, Weiss S
    2006 Mar;27(9):1679-87. doi: 10.1016/j.biomaterials.2005.11.018. Epub 2005 Nov 28.
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      After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed, the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo.

      PMID:16318871 | PMC:PMC3073483 | DOI:10.1016/j.biomaterials.2005.11.018


      View details for PubMedID 16318871
  • Purification and characterization of laccase from the rice blast fungus, Magnaporthe grisea FEMS microbiology letters
    Iyer G, Chattoo BB
    2003 Oct 10;227(1):121-6. doi: 10.1016/S0378-1097(03)00658-X.
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      A 70-kDa extracellular laccase was purified from the rice blast fungus Magnaporthe grisea using gel filtration and ion exchange chromatography The procedure provided 282-fold purification with a specific enzyme activity of 225.91 U mg(-1) and a yield of 11.92%. The enzyme oxidized a wide range of substrates. The highest level of oxidation was detected with syringaldazine as the substrate. Using syringaldazine as the substrate, the enzyme exhibited a pH optimum of 6 and temperature optimum of 30 degrees C, and its K(m) was 0.118 mM. The enzyme was strongly inhibited by Cu-chelating agents.

      PMID:14568157 | DOI:10.1016/S0378-1097(03)00658-X


      View details for PubMedID 14568157
  • Nonself recognition is mediated by HET-C heterocomplex formation during vegetative incompatibility The EMBO journal
    Sarkar S, Iyer G, Wu J, Glass NL
    2002 Sep 16;21(18):4841-50. doi: 10.1093/emboj/cdf479.
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      Nonself recognition during vegetative growth in filamentous fungi is mediated by heterokaryon incompatibility (het) loci. In Neurospora crassa, het-c is one of 11 het loci. Three allelic specificity groups, termed het-c(OR), het-c(PA) and het-c(GR), exist in natural populations. Heterokaryons or partial diploids that contain het-c alleles of alternative specificity show severe growth inhibition, repression of conidiation and hyphal compartmentation and death (HCD). Using epitope-tagged HET-C, we show that nonself recognition is mediated by the presence of a heterocomplex composed of polypeptides encoded by het-c alleles of alternative specificity. The HET-C heterocomplex localized to the plasma membrane (PM); PM-bound HET-C heterocomplexes occurred in all three het-c incompatible allelic interactions. Strains containing het-c constructs deleted for a predicted signal peptide sequence formed HET-C heterocomplexes in the cytoplasm and showed a growth arrest phenotype. Our finding is a step towards understanding nonself recognition mechanisms that operate during vegetative growth in filamentous fungi, and provides a model for investigating relationships between recognition mechanisms and cell death.

      PMID:12234924 | PMC:PMC126278 | DOI:10.1093/emboj/cdf479


      View details for PubMedID 12234924

Contact Information

Gopal Iyer, PhD

1111 Highland Avenue,
3133 WIMR
Madison, WI 53705
(608) 263-0662