Mohammed Farhoud

Mice expressing activated PI3K rapidly develop advanced colon cancer

Aberrations in the phosphoinositide 3-kinase (PI3K) signaling pathway play a key role in the pathogenesis of numerous cancers by altering cellular growth, metabolism, proliferation, and apoptosis. Mutations in the catalytic domain of PI3K that generate a dominantly active kinase are commonly found in human colorectal cancers and have been thought to drive tumor progression but not initiation. However, the effects of constitutively activated PI3K upon the intestinal mucosa have not been previously studied in animal models. Here, we show that the expression of a dominantly active form of the PI3K protein in the mouse intestine results in hyperplasia and advanced neoplasia. Mice expressing constitutively active PI3K in the epithelial cells of the distal small bowel and colon rapidly developed invasive adenocarcinomas in the colon that spread into the mesentery and adjacent organs. The histologic characteristics of these tumors were strikingly similar to invasive mucinous colon cancers in humans. Interestingly, these tumors formed without a benign polypoid intermediary, consistent with the lack of aberrant WNT signaling observed. Together, our findings indicate a noncanonical mechanism of colon tumor initiation that is mediated through activation of PI3K. This unique model has the potential to further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification.

Alkylphosphocholine Analogs for Broad-Spectrum Cancer Imaging and Therapy

Tumor-specific alkylphosphocholine analogs were evaluated as imaging and therapy agents in patients and in animal models of human cancer. A Broad View of Cancer Many consider targeted or molecular imaging to be the optimal way to image cancer. Weichert and colleagues feel differently: Uptake of certain small molecules by all cancer cells can give a broad view of cancer, and perhaps also treat it. These small molecules are alkylphosphocholine (APC) analogs, which are taken up preferentially by cancer cells—as compared to, for example, fibroblasts—via plasma membranes and transported into the cells by lipid rafts. The authors tested the uptake of radiolabeled APC analogs in vitro and in vivo in animals in 57 different spontaneous and transgenic tumors, of both human and rodent origin. Because of the well-established efficacy of radiotherapy, the authors demonstrated that the APC analogs could be used to not only visualize tumors but also kill them. Translating this to cancer patients, Weichert et al. showed preliminary preferential uptake of a radiolabeled APC analog in brain tumors. These broadly applicable imaging and therapeutic APC-based agents have been tested in dozens of different human cancers, and preliminarily in people, and are now well poised for further translation to clinical trials. Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron emission tomography (PET) imaging (124I) or molecular radiotherapeutic (131I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. 131I-CLR1404 also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with 124I-CLR1404 or 131I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable personalized dual modality cancer therapy of using molecular 124I-CLR1404 tumor imaging for planning 131I-CLR1404 therapy.

Fluorescent cancer-selective alkylphosphocholine analogs for intraoperative glioma detection

BACKGROUND 5-Aminolevulinic acid (5-ALA)-induced tumor fluorescence aids brain tumor resections but is not approved for routine use in the United States. We developed and describe testing of 2 novel fluorescent, cancer-selective alkylphosphocholine analogs, CLR1501 (green) and CLR1502 (near infrared), in a proof-of-principle study for fluorescence-guided glioma surgery. OBJECTIVE To demonstrate that CLR1501 and CLR1502 are cancer cell-selective fluorescence agents in glioblastoma models and to compare tumor-to-normal brain (T:N) fluorescence ratios with 5-ALA. METHODS CLR1501, CLR1502, and 5-ALA were administered to mice with magnetic resonance imaging-verified orthotopic U251 glioblastoma multiforme- and glioblastoma stem cell-derived xenografts. Harvested brains were imaged with confocal microscopy (CLR1501), the IVIS Spectrum imaging system (CLR1501, CLR1502, and 5-ALA), or the Fluobeam near-infrared fluorescence imaging system (CLR1502). Imaging and quantitative analysis of T:N fluorescence ratios were performed. RESULTS Excitation/emission peaks are 500/517 nm for CLR1501 and 760/778 nm for CLR1502. The observed T:N ratio for CLR1502 (9.28±1.08) was significantly higher (P<.01) than for CLR1501 (3.51±0.44 on confocal imaging; 7.23±1.63 on IVIS imaging) and 5-ALA (4.81±0.92). Near-infrared Fluobeam CLR1502 imaging in a mouse xenograft model demonstrated high- contrast tumor visualization compatible with surgical applications. CONCLUSION CLR1501 (green) and CLR1502 (near infrared) are novel tumor-selective fluorescent agents for discriminating tumor from normal brain. CLR1501 exhibits a tumor-to-brain fluorescence ratio similar to that of 5-ALA, whereas CLR1502 has a superior tumor-to-brain fluorescence ratio. This study demonstrates the potential use of CLR1501 and CLR1502 in fluorescence-guided tumor surgery.

mTOR inhibition elicits a dramatic response in PI3K-dependent colon cancers.

The phosphatidylinositide-3-kinase (PI3K) signaling pathway is critical for multiple cellular functions including metabolism, proliferation, angiogenesis, and apoptosis, and is the most commonly altered pathway in human cancers. Recently, we developed a novel mouse model of colon cancer in which tumors are initiated by a dominant active PI3K (FC PIK3ca*). The cancers in these mice are moderately differentiated invasive mucinous adenocarcinomas of the proximal colon that develop by 50 days of age. Interestingly, these cancers form without a benign intermediary or aberrant WNT signaling, indicating a non-canonical mechanism of tumorigenesis. Since these tumors are dependent upon the PI3K pathway, we investigated the potential for tumor response by the targeting of this pathway with rapamycin, an mTOR inhibitor. A cohort of FC PIK3ca* mice were treated with rapamycin at a dose of 6 mg/kg/day or placebo for 14 days. FDG dual hybrid PET/CT imaging demonstrated a dramatic tumor response in the rapamycin arm and this was confirmed on necropsy. The tumor tissue remaining after treatment with rapamycin demonstrated increased pERK1/2 or persistent phosphorylated ribosomal protein S6 (pS6), indicating potential resistance mechanisms. This unique model will further our understanding of human disease and facilitate the development of therapeutics through pharmacologic screening and biomarker identification.

Creating physical 3D stereolithograph models of brain and skull.

The human brain and skull are three dimensional (3D) anatomical structures with complex surfaces. However, medical images are often two dimensional (2D) and provide incomplete visualization of structural morphology. To overcome this loss in dimension, we developed and validated a freely available, semi-automated pathway to build 3D virtual reality (VR) and hand-held, stereolithograph models. To evaluate whether surface visualization in 3D was more informative than in 2D, undergraduate students (n = 50) used the Gillespie scale to rate 3D VR and physical models of both a living patient-volunteers brain and the skull of Phineas Gage, a historically famous railroad worker whose misfortune with a projectile tamping iron provided the first evidence of a structure-function relationship in brain. Using our processing pathway, we successfully fabricated human brain and skull replicas and validated that the stereolithograph model preserved the scale of the VR model. Based on the Gillespie ratings, students indicated that the biological utility and quality of visual information at the surface of VR and stereolithograph models were greater than the 2D images from which they were derived. The method we developed is useful to create VR and stereolithograph 3D models from medical images and can be used to model hard or soft tissue in living or preserved specimens. Compared to 2D images, VR and stereolithograph models provide an extra dimension that enhances both the quality of visual information and utility of surface visualization in neuroscience and medicine.

The effects of lobeline on α4β2* nicotinic acetylcholine receptor binding and uptake of [18F]nifene in rats

UNLABELLED Lobeline is a potential smoking cessation drug with affinity for the α4β2 nicotinic acetylcholine receptor and may inhibit the blood-brain barrier (BBB) amine transporter. The goal of this work was to use PET imaging to evaluate the effects of lobeline on the kinetic properties of [(18)F]nifene in the rat brain. METHODS Direct α4β2* competition of lobeline with [(18)F]nifene was evaluated using imaging experiments with both displacing and blocking doses of lobeline (1mg/kg, i.v.) given between two injections of [(18)F]nifene separated by 50min. Inhibition of the BBB amine transporter was examined using a separate imaging protocol with three injections of [(18)F]nifene, first at baseline, then following (-)nicotine blocking, and finally following lobeline blocking. RESULTS Rapid displacement of [(18)F]nifene was observed in the α4β2*-rich thalamus following lobeline administration, suggesting direct competition of the drug at α4β2* sites. Slight decreases in BBB transport of [(18)F]nifene were observed when the α4β2* system was first saturated with (-)nicotine and then given lobeline. This perturbation may be due to inhibition of the BBB amine transporter by lobeline or reductions in blood flow. Significant cerebellar displacement of [(18)F]nifene was found following the administration of both lobeline and (-)nicotine, indicating detectable specific binding in the rat cerebellum. CONCLUSION The competition of lobeline with [(18)F]nifene is largely dominated at the α4β2* binding site and only small perturbations in BBB transport of [(18)F]nifene are seen at the 1mg/kg dose. Similar experiments could be used to study other drugs as therapeutic agents for smoking cessation with PET.

PET imaging of acetylcholinesterase inhibitor‐induced effects on α4β2 nicotinic acetylcholine receptor binding

Acetylcholinesterase inhibitors (AChEIs) are drugs that increase synaptic acetylcholine (ACh) concentrations and are under investigation as treatments for symptoms accompanying Alzheimers disease. The goal of this work was to use PET imaging to evaluate alterations of in vivo α4β2 nicotinic acetylcholine receptor (nAChR) binding induced by the AChEIs physostigmine (PHY) and galanthamine (GAL). The α4β2 nAChR‐specific radioligand [18F]nifene was used to examine the effects of 0.1–0.2 mg/kg PHY, 5 mg/kg GAL, and saline in three separate experiments all performed on each of two rat subjects. A 60‐min bolus‐infusion protocol was used with drug administered after 30 min. Data from the thalamus and cortex were analyzed with a graphical model accounting for neurotransmitter activation using the cerebellum as a reference region to test for transient competition with bound [18F]nifene. Significant [18F]nifene displacement was detected in both regions during one PHY and both GAL studies, while no significant competition was observed in both saline studies. This preliminary work indicates the viability of [18F]nifene in detecting increases in synaptic ACh induced by AChEIs. Synapse 67:882–886, 2013.

Positron emission tomography assessment of 8-OH-DPAT-mediated changes in an index of cerebral glucose metabolism in female marmosets

As part of a larger experiment investigating serotonergic regulation of female marmoset sexual behavior, this study was designed to (1) advance methods for PET imaging of common marmoset monkey brain, (2) measure normalized FDG uptake as an index of local cerebral metabolic rates for glucose, and (3) study changes induced in this index of cerebral glucose metabolism by chronic treatment of female marmosets with a serotonin 1A receptor (5-HT(1A)) agonist. We hypothesized that chronic treatment with the 5-HT(1A) agonist 8-OH-DPAT would alter the glucose metabolism index in dorsal raphe (DR), medial prefrontal cortex (mPFC), medial preoptic area of hypothalamus (mPOA), ventromedial nucleus of hypothalamus (VMH), and field CA1 of hippocampus. Eight adult ovariectomized female common marmosets (Callithrix jacchus) were studied with and without estradiol replacement. In a crossover design, each subject was treated daily with 8-OH-DPAT (0.1mg/kg SC daily) or saline. After 42-49 days of treatment, the glucose metabolism radiotracer FDG was administered to each female immediately prior to 30 min of interaction with her male pairmate, after which the subject was anesthetized and imaged by PET. Whole brain normalized PET images were analyzed with anatomically defined regions of interest (ROI). Whole brain voxelwise mapping was also used to explore treatment effects and correlations between alterations in the glucose metabolism index and pairmate interactions. The rank order of normalized FDG uptake was VMH/mPOA>DR>mPFC/CA1 in both conditions. 8-OH-DPAT did not induce alterations in the glucose metabolism index in ROIs. Voxelwise mapping showed a significant reduction in normalized FDG uptake in response to 8-OH-DPAT in a cluster in medial occipital cortex as well as a significant correlation between increased rejection of mount attempts and reduced normalized FDG uptake in an overlapping cluster. In conclusion, PET imaging has been used to measure FDG uptake relative to whole brain in marmoset monkeys. Voxelwise mapping shows that 8-OH-DPAT reduces this index of glucose metabolism in medial occipital cortex, consistent with alterations in female sexual behavior.

Abstract 4536: Combination external beam and internal radiation via 131I-CLR1404 in the treatment of head and neck squamous cell carcinoma xenografts.

Radiotherapy (RT) is a central treatment modality for head and neck cancer (HNC). However, optimal outcome is commonly limited by the radiation tolerance of adjacent normal tissue structures. Radiation is sometimes valuable in the setting of locoregional tumor recurrence, however the risk of significant normal tissue toxicity is substantial with repeat irradiation and can have a profound adverse impact on patient quality of life. Although technical advances including intensity modulated radiotherapy (IMRT) can reduce normal tissue effects, the problem of repeat exposure of normal tissues to radiation is frequently limiting. The radiolabeled drug 124/131I-CLR1404 is a phospholipid ether analogue (PLE) that accumulates preferentially within malignant tumors and enables internal delivery of radiation. This allows a combination of external and internal radiotherapy delivery with a goal of improving treatment outcome without enhanced normal tissue toxicities. In this study, we characterized the ability of 131I-CLR1404 to augment RT response using xenografted mice harboring human squamous cell carcinomas (SCC). We determined tumor growth delay as well as tumor and normal tissue dose distribution of 131I-CLR-1404 via Monte Carlo simulation using PET data generated with 124I-CLR1404 imaging. Tumor growth delay was extended by 15.5 days using combined 3.7MBq 131I-CLR1404 and RT compared to RT or 131I-CLR1404 alone. In combination therapy, single dose administration of 7.4 MBq 131I-CLR1404 as well as split dosing with 2 x 3.7 MBq separated by one week showed the same additional growth delay of 27 days compared to single dose of 3.7 MBq. Monitoring drug distribution via PET/CT showed high tumor selectivity with a tumor-to-liver ratio of 6.2±0.65 and a tumor-to-muscle ratio of 6.7±0.21. These data illustrate the tumor selectivity of the CLR1404 PLE delivery platform as well as augmentation of tumor response with combined RT and 131I-CLR1404 treatment. De-intensification of external beam radiation dose, with attendant reduction in normal tissue toxicities so common in HNC patients represents a central objective to be tested in controlled clinical trials with the ultimate goal of improving treatment outcome and quality of life for HNC patients. Citation Format: Jarob Saker, Eric Armstrong, Bryan Bednarz, Mohammed Farhoud, Abigail Besemer, Jamey Weichert, Paul Harari. Combination external beam and internal radiation via 131I-CLR1404 in the treatment of head and neck squamous cell carcinoma xenografts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4536. doi:10.1158/1538-7445.AM2013-4536

Abstract 3831: 131I-CLR1404 and CLR1404: Broad spectrum, cancer-targeted molecular radio- and chemotherapeutic phospholipid ether analogs

The effectiveness of current approaches to cancer therapy is often limited by off-target toxicity or, if relatively selective, by lack of drug target expression in tumors. To address these shortcomings, structure-activity relationship studies were undertaken and identified a series of iodophenyl-containing phospholipid ether (PLE) analogs that selectively accumulate in cancer cells/cancer stem cells compared to normal cells/stem cells, in vitro and in vivo. Isosteric iodine substitution in CLR1404 affords either a diagnostic/imaging agent (e.g. using 124 I for cancer-selective PET imaging) or a molecular radiotherapeutic agent (e.g. using 131 I for cancer-selective cytotoxicity), both of which are in clinical development. We suggest the term “diapeutic” to describe such drugs which can be used in one form to identify and characterize patients who will benefit from a specific therapy and, in another form, to effect that therapy. Here we describe the broad-spectrum efficacy of the 131 I-labeled homolog of one such diapeutic compound (CLR1404) in human tumor xenograft models in mice including Caki-2 (renal; clear cell carcinoma), HCT-116 (colorectal carcinoma), Ovcar-3 (ovarian adenocarcinoma), MDA-MB-231 (triple negative mammary gland adenocarcinoma, MES-SA/Dx5 (uterine sarcoma), U87 (glioma), Mia Paca-2 (pancreatic carcinoma) and PC-3 (prostate carcinoma). In these models, a single i.v. injection of 131 I-CLR1404 (100 αCi, 3.8 μg/mouse) resulted in significant tumor growth suppression and extension of survival. A separate radiotoxicology study in normal rats followed for six months or until death after a single dose of 131 I-CLR1404 did not find significant radiation toxicity below 5 mCi suggesting that 131 I-CLR1404 may display an acceptable therapeutic index in man. At 100-fold higher mass dose (380 μg/mouse, i.v.) CLR1404, the non-radioactive, 127 I-containing homolog, was also highly efficacious in human tumor mouse xenograft models including MDA-MB-231 (triple negative breast cancer) and A549 (non-small cell lung cancer). CLR1404 inhibited proliferation of multiple human cancer cell lines in vitro with an IC 50 of ∼5 μM compared to ∼50 μM for growth suppression of normal fibroblasts. Similar concentrations inhibited the PI3K/Akt cell signaling pathway and stimulated pro-apoptotic caspases in human cancer cell lines but not in normal fibroblasts. Thus, 131 I-CLR1404 and CLR1404 each combine wide-ranging, cancer cell-selective targeting with cytotoxicity mechanisms known be broadly effective (intracellular radiation or PI3K/Akt inhibition, respectively). As such, both agents have the potential to provide effective, well-tolerated therapy across numerous cancer types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3831. doi:1538-7445.AM2012-3831