Nagavarakishore Pillarsetty

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cells sensitivity to Hsp90 inhibition.

Monitoring the Efficacy of Adoptively Transferred Prostate Cancer–Targeted Human T Lymphocytes with PET and Bioluminescence Imaging

Noninvasive imaging technologies have the potential to enhance the monitoring and improvement of adoptive therapy with tumor-targeted T lymphocytes. We established an imaging methodology for the assessment of spatial and temporal distributions of adoptively transferred genetically modified human T cells in vivo for treatment monitoring and prediction of tumor response in a systemic prostate cancer model. Methods: RM1 murine prostate carcinoma tumors transduced with human prostate-specific membrane antigen (hPSMA) and a Renilla luciferase reporter gene were established in SCID/beige mice. Human T lymphocytes were transduced with chimeric antigen receptors (CAR) specific for either hPSMA or human carcinoembryonic antigen (hCEA) and with a fusion reporter gene for herpes simplex virus type 1 thymidine kinase (HSV1tk) and green fluorescent protein, with or without click beetle red luciferase. The localization of adoptively transferred T cells in tumor-bearing mice was monitored with 2′-18F-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-ethyluracil (18F-FEAU) small-animal PET and bioluminescence imaging (BLI). Results: Cotransduction of CAR-expressing T cells with the reporter gene did not affect CAR-mediated cytotoxicity. BLI of Renilla and click beetle red luciferase expression enabled concurrent imaging of adoptively transferred T cells and systemic tumors in the same animal. hPSMA-specific T lymphocytes persisted longer than control hCEA-targeted T cells in lung hPSMA-positive tumors, as indicated by both PET and BLI. Precise quantification of T-cell distributions at tumor sites by PET revealed that delayed tumor progression was positively correlated with the levels of 18F-FEAU accumulation in tumor foci in treated animals. Conclusion: Quantitative noninvasive monitoring of genetically engineered human T lymphocytes by PET provides spatial and temporal information on T-cell trafficking and persistence. PET may be useful for predicting tumor response and for guiding adoptive T-cell therapy.

18F-Labeled-Bioorthogonal Liposomes for In Vivo Targeting

Liposomes are attractive vehicles for the controlled release of drugs and cytotoxins and have a long-standing history in medical research and clinical practice. In addition to established therapeutic indications, liposomes have several favorable properties for molecular imaging, including high stability and the ability to be labeled with radioisotopes, as well as paramagnetic and fluorescent contrast agents. However, long circulation times and difficulties in creating targeted liposomes have proven challenges for imaging. In this study, we have addressed these limitations using a recently developed strategy for bioorthogonal conjugation, the reaction between tetrazines and trans-cyclooctenes. By coating radiolabeled liposomes with trans-cyclooctene and pretargeting with a tetrazine coupled to a targeted peptide, we were able to selectively enhance the retention of liposomes and bind them to tumor tissue in live animals. The rapid reaction between tetrazines and trans-cyclooctenes allowed imaging to be performed with the short-lived PET tracer (18)F, yielding signal-to-background activity ratios of 7:1. The covalent, bioorthogonally driven tumor-targeting of liposomes by in vivo click chemistry is promising and should be explored for more selective and rapid delivery of radiodiagnostics and radiotherapeutics, two classes of drugs which particularly benefit from fast clearance, low nonspecific binding, and the associated reduced toxicity to kidneys and bone marrow.

Multimodality imaging of TGFβ signaling in breast cancer metastases

The skeleton is a preferred site for breast cancer metastasis. We have developed a multimodality imaging approach to monitor the transforming growth factor β (TGFβ) signaling pathway in bone metastases, sequentially over time in the same animal. As model systems, two MDA‐MB‐231 breast cancer cells lines with different metastatic tropisms, SCP2 and SCP3, were transduced with constitutive and TGFβ‐ inducible reporter genes and were tested in vitro and in living animals. The sites and expansion of metastases were visualized by bioluminescence imaging using a constitutive firefly luciferase reporter, while TGFP signaling in metastases was monitored by microPET imaging of HSV1‐TK/GFP expression with [18F]FEAU and by a more sensitive and cost‐effective bioluminescence reporter, based on nonsecreted Gaussia luciferase. Concurrent and sequential imaging of metastases in the same animals provided insight into the location and progression of metastases, and the timing and course of TGFP signaling. The anticipated and newly observed differences in the imaging of tumors from two related cell lines have demonstrated that TGFβ signal transduction pathway activity can be noninvasively imaged with high sensitivity and reproducibility, thereby providing the opportunity for an assessment of novel treatments that target TGFβ signaling.— Serganova, I.,Moroz, E., Vider, J., Gogiberidze, G., Moroz, M., Pillarsetty, N., Doubrovin, M., Minn, A., Thaler, H. T., Massague, J., Gelovani, J., Blasberg, R. Multimodality imaging of TGFβ signaling in breast cancer metastases. FASEB J. 23, 2662–2672 (2009)

Development of a minimal saponin vaccine adjuvant based on QS-21

Adjuvants are materials added to vaccines to enhance the immunological response to an antigen. QS-21 is a natural product adjuvant under investigation in numerous vaccine clinical trials, but its use is constrained by scarcity, toxicity, instability, and an enigmatic molecular mechanism of action. Herein, we describe the development of a minimal QS-21 analogue that decouples adjuvant activity from toxicity and provides a powerful platform for mechanistic investigations. We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required. Biodistribution studies revealed that active adjuvants were retained at the injection site and nearest draining lymph nodes preferentially compared to attenuated variants. Overall, these studies have yielded critical insights into saponin structure–function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.

The epichaperome is an integrated chaperome network that facilitates tumour survival

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes—dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically ‘rewired’ to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.

Increased KIT Inhibition Enhances Therapeutic Efficacy in Gastrointestinal Stromal Tumor

Purpose: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and a model of targeted molecular therapy. GIST depends on oncogenic KIT signaling and responds to the tyrosine kinase inhibitor imatinib. However, imatinib is rarely curative. We hypothesized that PLX3397, which inhibits KIT and colony-stimulating-factor-1 receptor (CSF1R), would be more efficacious than imatinib in GIST by also depleting tumor-associated macrophages, which are generally thought to support tumor growth. Experimental Design: We treated KitV558del/+ mice that develop GIST or mice with subcutaneous human GIST xenografts with imatinib or PLX3397 and analyzed tumor weight, cellular composition, histology, molecular signaling, and fibrosis. In vitro assays on human GIST cell lines were also performed. Results: PLX3397 was more effective than imatinib in reducing tumor weight and cellularity in both KitV558del/+ murine GIST and human GIST xenografts. The superiority of PLX3397 did not depend on depletion of tumor-associated macrophages, because adding CSF1R inhibition did not improve the effects of imatinib. Instead, PLX3397 was a more potent KIT inhibitor than imatinib in vitro. PLX3397 therapy also induced substantial intratumoral fibrosis, which impaired the subsequent delivery of small molecules. Conclusions: PLX3397 therapy has greater efficacy than imatinib in preclinical GIST models and warrants study in patients with GIST. The resultant intratumoral fibrosis may represent one of the barriers to achieving complete tumor eradication. Clin Cancer Res; 20(9); 2350–62. ©2014 AACR.

Sustained ERK inhibition maximizes responses of BrafV600E thyroid cancers to radioiodine

Radioiodide (RAI) therapy of thyroid cancer exploits the relatively selective ability of thyroid cells to transport and accumulate iodide. Iodide uptake requires expression of critical genes that are involved in various steps of thyroid hormone biosynthesis. ERK signaling, which is markedly increased in thyroid cancer cells driven by oncogenic BRAF, represses the genetic program that enables iodide transport. Here, we determined that a critical threshold for inhibition of MAPK signaling is required to optimally restore expression of thyroid differentiation genes in thyroid cells and in mice with BrafV600E-induced thyroid cancer. Although the MEK inhibitor selumetinib transiently inhibited ERK signaling, which subsequently rebounded, the MEK inhibitor CKI suppressed ERK signaling in a sustained manner by preventing RAF reactivation. A small increase in ERK inhibition markedly increased the expression of thyroid differentiation genes, increased iodide accumulation in cancer cells, and thereby improved responses to RAI therapy. Only a short exposure to the drug was necessary to obtain a maximal response to RAI. These data suggest that potent inhibition of ERK signaling is required to adequately induce iodide uptake and indicate that this is a promising strategy for the treatment of BRAF-mutant thyroid cancer.

A New Acycloguanosine-Specific Supermutant of Herpes Simplex Virus Type 1 Thymidine Kinase Suitable for PET Imaging and Suicide Gene Therapy for Potential Use in Patients Treated with Pyrimidine-Based Cytotoxic Drugs

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene is widely used as a suicide gene in combination with ganciclovir (GCV) and as a nuclear imaging reporter gene with an appropriate reporter probe. Wild-type HSV1-tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogs, including clinically used antiviral drugs. PET of HSV1-tk reporter gene expression will be compromised in patients receiving nucleoside-based antiviral treatment. With the use of an acycloguanosine-specific mutant of the enzyme, PET of HSV1-tk reporter gene expression can be successfully performed with acycloguanosine-based radiotracers without interference from pyrimidine-based antiviral drugs. Methods: The levels of expression of wild-type HSV1-tk and HSV1-A167Ytk, HSV1-sr39tk, and HSV1-A167Ysr39tk mutants fused with green fluorescent protein (GFP) and transduced into U87 cells were normalized to the mean fluorescence of GFP measured by fluorescence-activated cell sorting. The levels of enzymatic activities of wild-type HSV1-tk and its mutants were compared by 2-h in vitro radiotracer uptake assays with 3H-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-ethyluracil (3H-FEAU), 3H-pencyclovir (3H-PCV), and 3H-GCV and by drug sensitivity assays. PET with 18F-FEAU and 18F-9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) was performed in mice with established subcutaneous tumors, expressing wild-type HSV1-tk and its mutants, followed by tissue sampling. Results: FEAU accumulation was not detected in HSV1-A167Ysr39tk–expressing cells and xenografts. Lack of conversion of pyrimidine derivatives by the HSV1-A167Ysr39tk supermutant was also confirmed by a drug sensitivity assay, in which the 50% inhibitory concentrations for thymine 1-β-d-arabinofuranoside and bromovinyldeoxyuridine were found to be similar to those in nontransduced cells. In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate 3H-GCV at levels similar to those of wild-type HSV1-tk and HSV1-A167Ytk but showed enhanced activity with 3H-PCV in vitro and with 18F-FHBG in vivo. Conclusion: We developed a new reporter gene, HSV1-A167Ysr39tk, which exhibits specificity and high phosphorylation activity for acycloguanosine derivatives. The resulting supermutant can be used for PET with 18F-FHBG and suicidal gene therapy protocols with GCV in patients treated with pyrimidine-based cytotoxic drugs.

Building Blocks for the Construction of Bioorthogonally Reactive Peptides via Solid-Phase Peptide Synthesis

The need for post-synthetic modifications and reactive prosthetic groups has long been a limiting factor in the synthesis and study of peptidic and peptidomimetic imaging agents. In this regard, the application of biologically and chemically orthogonal reactions to the design and development of novel radiotracers has the potential to have far-reaching implications in both the laboratory and the clinic. Herein, we report the synthesis and development of a series of modular and versatile building blocks for inverse electron-demand Diels–Alder copper-free click chemistry: tetrazine-functionalized artificial amino acids. Following the development of a novel peptide coupling protocol for peptide synthesis in the presence of tetrazines, we successfully demonstrated its effectiveness and applicability. This versatile methodology has the potential to have a transformational impact, opening the door for the rapid, facile, and modular synthesis of bioorthogonally reactive peptide probes.