Andrei Volgin

Targeting Src Family Kinases Inhibits Growth and Lymph Node Metastases of Prostate Cancer in an Orthotopic Nude Mouse Model

Aberrant expression and/or activity of members of the Src family of nonreceptor protein tyrosine kinases (SFK) are commonly observed in progressive stages of human tumors. In prostate cancer, two SFKs (Src and Lyn) have been specifically implicated in tumor growth and progression. However, there are no data in preclinical models demonstrating potential efficacy of Src inhibitors against prostate cancer growth and/or metastasis. In this study, we used the small molecule SFK/Abl kinase inhibitor dasatinib, currently in clinical trials for solid tumors, to examine in vitro and in vivo effects of inhibiting SFKs in prostate tumor cells. In vitro, dasatinib inhibits both Src and Lyn activity, resulting in decreased cellular proliferation, migration, and invasion. In orthotopic nude mouse models, dasatinib treatment effectively inhibits expression of activated SFKs, resulting in inhibition of both tumor growth and development of lymph node metastases in both androgen-sensitive and androgen-resistant tumors. In primary tumors, SFK inhibition leads to decreased cellular proliferation (determined by immunohistochemistry for proliferating cell nuclear antigen). In vitro, small interfering RNA (siRNA)-mediated inhibition of Lyn affects cellular proliferation; siRNA inhibition of Src affects primarily cellular migration. Therefore, we conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer and that Src and Lyn activities affect different cellular functions required for prostate tumor growth and progression.

Molecular imaging of active mutant L858R EGF receptor (EGFR) kinase-expressing nonsmall cell lung carcinomas using PET/CT

The importance of the EGF receptor (EGFR) signaling pathway in the development and progression of nonsmall cell lung carcinomas (NSCLC) is widely recognized. Gene sequencing studies revealed that a majority of tumors responding to EGFR kinase inhibitors harbor activating mutations in the EGFR kinase domain. This underscores the need for novel biomarkers and diagnostic imaging approaches to identify patients who may benefit from particular therapeutic agents and approaches with improved efficacy and safety profiles. To this goal, we developed 4-[(3-iodophenyl)amino]-7-{2-[2-{2-(2-[2-{2-([18F]fluoroethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}-ethoxy]-quinazoline-6-yl-acrylamide ([18F]F-PEG6-IPQA), a radiotracer with increased selectivity and irreversible binding to the active mutant L858R EGFR kinase. We show that PET with [18F]F-PEG6-IPQA in tumor-bearing mice discriminates H3255 NSCLC xenografts expressing L858R mutant EGFR from H441 and PC14 xenografts expressing EGFR or H1975 xenografts with L858R/T790M dual mutation in EGFR kinase domain, which confers resistance to EGFR inhibitors (i.e., gefitinib). The T790M mutation precludes the [18F]F-PEG6-IPQA from irreversible binding to EGFR. These results suggest that PET with [18F]F-PEG6-IPQA could be used for the selection of NSCLC patients for individualized therapy with small molecular inhibitors of EGFR kinase that are currently used in the clinic and have a similar structure (i.e., iressa, gefitinib, and erlotinib).

The selective hypoxia inducible factor-1 inhibitor PX-478 provides in vivo radiosensitization through tumor stromal effects.

Hypoxia inducible factor-1 (HIF-1) promotes tumor cell adaptation to microenvironmental stress. HIF-1 is up-regulated in irradiated tumors and serves as a promising target for radiosensitization. We initially confirmed that the orally bioavailable HIF-1 inhibitor PX-478 reduces HIF-1 protein levels and signaling in vitro in a dose-dependent manner and provides direct radiosensitization of hypoxic cancer cells in clonogenic survival assays using C6 glioma, HN5 and UMSCCa10 squamous cells, and Panc-1 pancreatic adenocarcinoma cell lines. However, PX-478 yields striking in vivo tumor sensitization to single-dose irradiation, which cannot be explained by incremental improvement in direct tumor cell killing. We show that PX-478 prevents postradiation HIF-1 signaling and abrogates downstream stromal adaptation in C6 and HN5 reporter xenografts as measured by serial ultrasound, vascular magnetic resonance imaging, and hypoxia response element–specific micro–positron emission tomography imaging. The primacy of indirect PX-478 in vivo effects was corroborated by our findings that (a) either concurrent or early postradiation sequencing of PX-478 provides roughly equivalent sensitization and (b) constitutive vascular endothelial growth factor expression maintains refractory tumor vessel function and progression following combined radiation and PX-478. These results confirm that disruption of postradiation adaptive HIF-1 signaling by PX-478 imparts increased therapeutic efficacy through blockade of HIF-1–dependent reconstitution of tumor stromal function. Successful translation of targeted HIF-1 radiosensitization to the clinical setting will require specific consideration of tumor microenvironmental effects and mechanisms. [Mol Cancer Ther 2009;8(4):947–58]

Algorithmic guided screening of drug combinations of arbitrary size for activity against cancer cells

The standard treatment for most advanced cancers is multidrug therapy. Unfortunately, combinations in the clinic often do not perform as predicted. Therefore, to complement identifying rational drug combinations based on biological assumptions, we hypothesized that a functional screen of drug combinations, without limits on combination sizes, will aid the identification of effective drug cocktails. Given the myriad possible cocktails and inspired by examples of search algorithms in diverse fields outside of medicine, we developed a novel, efficient search strategy called Medicinal Algorithmic Combinatorial Screen (MACS). Such algorithms work by enriching for the fitness of cocktails, as defined by specific attributes through successive generations. Because assessment of synergy was not feasible, we developed a novel alternative fitness function based on the level of inhibition and the number of drugs. Using a WST-1 assay on the A549 cell line, through MACS, we screened 72 combinations of arbitrary size formed from a 19-drug pool across four generations. Fenretinide, suberoylanilide hydroxamic acid, and bortezomib (FSB) was the fittest. FSB performed up to 4.18 SD above the mean of a random set of cocktails or “too well” to have been found by chance, supporting the utility of the MACS strategy. Validation studies showed FSB was inhibitory in all 7 other NSCLC cell lines tested. It was also synergistic in A549, the one cell line in which this was evaluated. These results suggest that when guided by MACS, screening larger drug combinations may be feasible as a first step in combination drug discovery in a relatively small number of experiments. [Mol Cancer Ther 2009;8(3):521–32]

Optimizing heat shock protein expression induced by prostate cancer laser therapy through predictive computational models

Thermal therapy efficacy can be diminished due to heat shock protein (HSP) induction in regions of a tumor where temperatures are insufficient to coagulate proteins. HSP expression enhances tumor cell viability and imparts resistance to chemotherapy and radiation treatments, which are generally employed in conjunction with hyperthermia. Therefore, an understanding of the thermally induced HSP expression within the targeted tumor must be incorporated into the treatment plan to optimize the thermal dose delivery and permit prediction of the overall tissue response. A treatment planning computational model capable of predicting the temperature, HSP27 and HSP70 expression, and damage fraction distributions associated with laser heating in healthy prostate tissue and tumors is presented. Measured thermally induced HSP27 and HSP70 expression kinetics and injury data for normal and cancerous prostate cells and prostate tumors are employed to create the first HSP expression predictive model and formulate an Arrhenius damage model. The correlation coefficients between measured and model predicted temperature, HSP27, and HSP70 were 0.98, 0.99, and 0.99, respectively, confirming the accuracy of the model. Utilization of the treatment planning model in the design of prostate cancer thermal therapies can enable optimization of the treatment outcome by controlling HSP expression and injury.

Fluorinated cannabinoid CB2 receptor ligands: synthesis and in vitro binding characteristics of 2-oxoquinoline derivatives.

Cannabinoid receptor 2 (CB2) plays an important role in human physiology and the pathophysiology of different diseases, including neuroinflammation, neurodegeneration, and cancer. Several classes of CB2 receptor ligands, including 2-oxoquinoline derivatives, have been previously reported. We report the synthesis and results of in vitro receptor binding of a focused library of new fluorinated 2-oxoquinoline CB2 ligands. Twelve compounds, 13-1618, 19, 21-24, 27, and 28 were synthesized in good yields in multiple steps. Human U87 glioma cells expressing either hCB1 (control) or hCB2 were generated via lentiviral transduction. In vitro competitive binding assay was performed using [(3)H]CP-55,940 in U87hCB1 and U87hCB2 cells. Inhibition constant (K(i)) values of compounds 13-16, 18, 19, 21-24, 27, and 28 for CB2 were >10,000, 2.8, 5.0, 2.4, 22, 0.8, 1.4, >10,000, 486, 58, 620, and 2400 nM, respectively, and those for CB1 were >10,000 nM. Preliminary in vitro results suggest that six of these compounds may be useful for therapy of neuropathic pain, neuroinflammatory diseases and immune disorders. In addition, compound 19, with its subnanomolar K(i) value, could be radiolabeled with (18)F and explored for PET imaging of CB2 expression.

Molecular–Genetic PET Imaging Using an HSV1-tk Mutant Reporter Gene with Enhanced Specificity to Acycloguanosine Nucleoside Analogs

Imaging 2 different molecular–genetic events in a single subject by PET is essential in a variety of in vivo applications. Using herpes simplex virus-1 thymidine kinase (HSV1-tk) mutants with narrower substrate specificities in combination with wild-type HSV1-tk (wtHSV1-tk) would enable differential imaging with corresponding radiotracers, namely 2′-deoxy-2′-18F-fluoro-5-ethyl-1-β-d-arabinofuranosyl-uracil (18F-FEAU) and the acycloguanosine derivative 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine (18F-FHBG). In this study, we evaluated wtHSV1-tk and the A168H mutant, which has been reported to exhibit enhanced acycloguanosine substrate catalytic activity and diminished pyrimidine phosphorylating activity, as PET reporter genes. Methods: Computational analysis was performed to assess the binding mode of FHBG and FEAU to wtHSV1-tk and the A168H variant. U87 cells were stably transduced with wtHSV1-tk or HSV1-tk(A168H) fused with green fluorescent protein and sorted to obtain equivalent transgene expression. In vitro uptake studies were performed to determine rates of substrate accumulation and retention. Nude mice bearing tumors expressing HSV1-tk variants were subsequently imaged using 18F-FHBG and 18F-FEAU. Results: Docking results indicate that binding of FHBG to the A168H variant is unaffected whereas the binding of FEAU is hindered because of a steric clash with the bulkier mutant residues. U87 cells expressing HSV1-tk(A168H) accumulated 18F-FHBG in in vitro uptake studies at a 3-fold higher rate than did cells expressing wtHSV1-tk without any detectable accumulation of 3H-FEAU. Furthermore, HSV1-tk(A168H) demonstrated no thymidine phosphorylation activity. In contrast, U87 cells expressing wtHSV1-tk preferentially accumulated 3H-FEAU at an 18-fold higher rate than they did 18F-FHBG. Tumors expressing wtHSV1-tk or HSV1-tk(A168H) were distinctly imaged with 18F-FEAU or 18F-FHBG, respectively. Hence, tumors expressing HSV1-tk(A168H) accumulated 8.4-fold more 18F-FHBG than did tumors expressing wtHSV1-tk. In addition, wtHSV1-tk tumors, compared with HSV1-tk(A168H)–expressing tumors (which retained baseline levels of the radiotracer), preferentially accumulated 18F-FEAU. Conclusion: The FEAU and FHBG substrate discrimination capacity of the wtHSV1-tk and HSV1-tk(A168H) reporter enzymes was validated in vivo by PET of mice with tumor xenografts established from U87 cells expressing these different reporters. Thus, HSV1-tk(A168H) may potentially be used as a second reporter gene in combination with wtHSV1-tk to achieve differential PET.

Synthesis and preliminary evaluation of [18F]-labeled 2-oxoquinoline derivatives for PET imaging of cannabinoid CB2 receptor

INTRODUCTION The cannabinoid receptor type 2 (CB(2)) is an important target for development of drugs and imaging agents for diseases, such as neuroinflammation, neurodegeneration and cancer. Recently, we reported synthesis and results of in vitro receptor binding of a focused library of fluorinated 2-oxoquinoline derivatives as CB(2) receptor ligands. Some of the compounds demonstrated to be good CB(2)-specific ligands with Ki values in the nanomolar to subnanomolar concentrations; therefore, we pursued the development of their (18)F-labeled analogues that should be useful for positron emission tomography (PET) imaging of CB(2) receptor expression. Here, we report the radiosynthesis of two (18)F-labeled 2-oxoquinoline derivatives and the preliminary in vitro and ex vivo evaluation of one compound as a CB(2)-specific radioligand. METHODS 4-[(18)F]fluorobenzyl amine [(18)F]-3 was prepared by radiofluorination of 4-cyano-N,N,N-trimethylanilinium triflate salt followed by reduction with LiAlH(4) and then coupled with acid chlorides 11 and 12 to afford [(18)F]-13 and [(18)F]-14. In vitro CB(2) receptor binding assay was performed using U87 cells transduced with CB(2) and CB(1) receptor. Ex vivo autoradiography was performed with [(18)F]-14 on spleen and on CB(2)- and CB(1)-expressing and wild-type U87 subcutaneous tumors grown in mice. RESULTS The radiochemical yields of [(18)F]-13 and [(18)F]-14 were 10%-15.0% with an average of 12% (n=10); radiochemical purity was >99% with specific activity 1200 mCi/μmol. The dissociation constant Kd for [(18)F]-14 was 3.4 nM. Ex vivo autoradiography showed accumulation of [(18)F]-14 in the CB(2)-expressing tumor. CONCLUSION Two new [(18)F]-labeled CB(2) ligands have been synthesized. Compound [(18)F]-14 appears to be a potential PET imaging agent for the assessment of CB(2) receptor expression; however, poor solubility restrain its use in vivo.

Detection of pancreatic carcinomas by imaging lactose-binding protein expression in peritumoral pancreas using [18F] fluoroethyl-deoxylactose PET/CT

Background Early diagnosis of pancreatic carcinoma with highly sensitive diagnostic imaging methods could save lives of many thousands of patients, because early detection increases resectability and survival rates. Current non-invasive diagnostic imaging techniques have inadequate resolution and sensitivity for detection of small size (∼2–3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with β-O-D-galactopyranosyl-(1,4′)-2′-deoxy-2′-[18F]fluoroethyl-D-glucopyranose ([18F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [18F]FEDL is a novel radioligand of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which is overexpressed in peritumoral pancreatic acinar cells. Methodology/Principal Findings Dynamic PET/CT imaging demonstrated rapid accumulation of [18F]FEDL in peritumoral pancreatic tissue (4.04±2.06%ID/g), bi-exponential blood clearance with half-lives of 1.65±0.50 min and 14.14±3.60 min, and rapid elimination from other organs and tissues, predominantly by renal clearance. Using model-independent graphical analysis of dynamic PET data, the average distribution volume ratio (DVR) for [18F]FEDL in peritumoral pancreatic tissue was estimated as 3.57±0.60 and 0.94±0.72 in sham-operated control pancreas. Comparative analysis of quantitative autoradiographic images and densitometry of immunohistochemically stained and co-registered adjacent tissue sections demonstrated a strong linear correlation between the magnitude of [18F]FEDL binding and HIP/PAP expression in corresponding regions (r = 0.88). The in situ analysis demonstrated that at least a 2–4 fold apparent lesion size amplification was achieved for submillimeter tumors and to nearly half a murine pancreas for tumors larger than 3 mm. Conclusion/Significance We have demonstrated the feasibility of detection of early pancreatic tumors by non-invasive imaging with [18F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [18F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.

HIF-1–Dependent Stromal Adaptation to Ischemia Mediates In Vivo Tumor Radiation Resistance

Purpose: Hypoxia-inducible factor 1 (HIF-1) promotes cancer cell survival and tumor progression. The specific role played by HIF-1 and tumor–stromal interactions toward determining tumor resistance to radiation treatment remains undefined. We applied a multimodality preclinical imaging platform to mechanistically characterize tumor response to radiation, with a focus on HIF-1–dependent resistance pathways. Methods: C6 glioma and HN5 human squamous carcinoma cells were stably transfected with a dual HIF-1 signaling reporter construct (dxHRE-tk/eGFP-cmvRed2XPRT). Reporter cells were serially interrogated in vitro before and after irradiation as monolayer and multicellular spheroid cultures and as subcutaneous xenografts in nu/nu mice. Results: In vitro, single-dose irradiation of C6 and HN5 reporter cells modestly impacted HIF-1 signaling in normoxic monolayers and inhibited HIF-1 signaling in maturing spheroids. In contrast, irradiation of C6 or HN5 reporter xenografts with 8 Gy in vivo elicited marked upregulation of HIF-1 signaling and downstream proangiogenic signaling at 48 hours which preceded recovery of tumor growth. In situ ultrasound imaging and dynamic contrast-enhanced (DCE) MRI indicated that HIF-1 signaling followed acute disruption of stromal vascular function. High-resolution positron emission tomography and dual-contrast DCE-MRI of immobilized dorsal skin window tumors confirmed postradiotherapy HIF-1 signaling to spatiotemporally coincide with impaired stromal vascular function. Targeted disruption of HIF-1 signaling established this pathway to be a determinant of tumor radioresistance. Conclusions: Our results illustrate that tumor radioresistance is mediated by a capacity to compensate for stromal vascular disruption through HIF-1–dependent proangiogenic signaling and that clinically relevant vascular imaging techniques can spatially define mechanisms associated with tumor irradiation. Mol Cancer Res; 9(3); 259–70. ©2011 AACR.