IGROV-1 Tumor Research Articles

Abstract 1550: Elucidating the role of ADAMTS9AS2 lncRNA in ovarian cancer

Abstract Introduction: Long non-coding RNAs (lncRNAs) are important regulators of gene expression and are involved in cancer progression and metastasis. Although studies have shown that aberrant expression of lncRNAs mediates disease progression in ovarian cancer (OC), the role of lncRNA ADAMTS9-AS2 has not been well studied. Objective: To determine the role of ADAMTS9-AS2 on OC pathogenesis. Methods: We examined ADAMTS9-AS2 expression in human OC cells and tumor tissues using in situ hybridization (ISH). ADAMTS9-AS2 was overexpressed in A2780cp20 and IGROV1cp20 cells and evaluated its effects on cell proliferation, viability and apoptosis. To evaluate the role of ADAMTS9-AS2 in tumor suppression, nude mice were injected with ADAMTS9-AS2 over expressing cells. Reverse phase protein array (RPPA) was performed to identify the downstream regulators. Luciferase reporter assay was performed to confirm the interaction of targeted protein with ADAMTS9-AS2. In addition, epigenetic regulation of ADAMTS9-AS2 was studied using 5-Azacytidine. Results: A2780cp20 and IGROV1cp20 cells expressed lower amounts of ADAMTS9-AS2, while SKOV3 and PEO4 cells showed higher expression levels. ISH analysis showed dense nuclear staining of ADAMTS9-AS2 in normal fallopian tube sections, whereas OC sections showed reduced expression. Kaplan-Meier survival analysis showed that patients with high expression of ADAMTS9-AS2 had significantly better overall survival (p=0.0079) and progression-free survival (p=0.00018) than those with low ADAMTS9-AS2 expression. Overexpression of ADAMTS9-AS2 in A2780cp20 cells resulted in decreased proliferation (p<0.01) and increased apoptosis (p<0.001). ADAMTS9-AS2 overexpressing A2780cp20 and IGROV1cp20 tumor bearing mice showed significant reduction in tumor growth (p=0.002; A2780cp20, p=0.01; IGROV1cp20) when compared to control A2780cp20 and IGROV1 tumor bearing mice. RPPA analysis showed that ANNEXIN A1 was the most upregulated protein upon ADAMTS9-AS2 overexpression. We confirmed that ANNEXIN A1 expression is regulated by ADAMTS9-AS2 by silencing ADAMTS9-AS2. Further, luciferase reporter assay results confirmed the interaction between ADAMTS9-AS2 and ANNEXIN A1 promoter and silencing ADAMTS9-AS2 with siRNA abolished this effect. Consistently with our previous in vitro findings, we observed a significant increase of ANNEXIN A1 expression in the ADAMTS9-AS2 overexpressing mice, compared with the A2780cp20 control. Further, azacytidine treated A2780cp20 cells showed an increase in ADAMTS9-AS2 expression. Conclusion: ADAMTS9-AS2 acts as a tumor suppressor, by enhancing the expression of ANNEXIN A1, and thereby regulating tumor growth. Thus, ADAMTS9-AS2-ANNEXIN A1 axis may represent a novel therapeutic target in OC. Citation Format: Emine Bayraktar, Guy Katz, Dahai Jiang, Cristian Rodriguez-Aguayo, Lingegowda S. Mangala, Santosh K. Dasari, Elaine Stur, Cristina Ivan, Gabriel Lopez-Berestein, Anil K. Sood. Elucidating the role of ADAMTS9AS2 lncRNA in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1550.

Abstract NT-090: PRECLINICAL ACTIVITY AND SAFETY OF STRO-002, A NOVEL ADC TARGETING FOLATE RECEPTOR ALPHA FOR OVARIAN AND ENDOMETRIAL CANCER

Abstract OBJECTIVES: Folate receptor alpha (FolRα) is a cell-surface glycoprotein, highly expressed in ovarian and endometrial adenocarcinoma, and thus a promising target for cancer therapy using antibody drug conjugates (ADCs). Most ADCs currently in development are generated by random attachment of the cytotoxic payload to the antibody and result in a heterogeneous mixture, comprised of many different forms that are likely to vary in stability and activity, and therefore may be suboptimal therapeutic agents. We have employed an E. coli cell-free expression system (XpressCFTM) and site-specific conjugation technology, to generate STRO-002, a novel homogenous FolRα-targeting ADC. STRO-002 was optimized by selection of the antibody, drug-linker, conjugation site and drug-antibody ratio (DAR) that conferred the best pharmacological properties. We have conducted preclinical studies to evaluate the stability of STRO-002 and characterize the pharmacological properties of the cytotoxic metabolite SC209. In vitro cytotoxicity assays and in vivo efficacy studies were conducted to evaluate the activity of STRO-002 in multiple ovarian cancer cell lines and xenografts. IND enabling toxicology studies were conducted to determine the safety profiles for STRO-002 and its metabolite SC209 in cynomolgous monkeys and rats, respectively. RESULTS: Based on optimization studies, the anti-FolRα human IgG1 antibody (H01/SP8166) conjugated to a proprietary cleavable drug-linker (SC239) was selected for the lead ADC STRO-002. SC239 contains a tubulin-targeting 3-aminophenyl hemiasterlin warhead, SC209, which has potent cytotoxic activity. Based on most favorable anti-tumor activity, positions 180 and 404 on each heavy chain were selected for conjugation of SC239 to SP8166 to yield an ADC with DAR of ~ 4. The drug-linkage in STRO-002 is highly stable and the released warhead, SC209, is a very weak substrate for cellular drug-resistance efflux pumps and is cleared rapidly from plasma. STRO-002 has potent but highly specific cytotoxic activity (0.1-3 nM) on multiple FolRα-positive ovarian cancer cell lines in vitro and anti-tumor efficacy in ovarian xenograft models. STRO-002 exhibits dose-dependent tumor growth inhibition in Igrov-1 tumor xenografts at a single dose and complete regression is achieved in Igrov-1 and OVCAR-3 tumors with a single dose at 10 and 5 mg/kg, respectively. In addition, administration of STRO-002 in combination with carboplatin confers added benefit in efficacy in Igrov-1 tumors. Toxicology studies show favorable safety profiles for STRO-002 and SC209. The main toxicity finding in monkeys dosed up to 9 mg/kg consists of reversible hematopoietic/lymphoid tissue toxicity, which is considered antigen-independent and is consistent with the anti-proliferative effects of SC209 observed in single-dose toxicology studies in rats. No evidence of ocular toxicity due to SC209 were observed in either species. CONCLUSIONS: STRO-002 is a highly specific FolRα targeting ADC with minimal drug moiety release in circulation and the potential for an improved safety and activity profile, and a reduced risk of tumor drug resistance. Our data supports the advancement of STRO-002 to the clinic as a potential treatment of FolRα expressing malignancies such as ovarian cancer. Citation Format: Cristina Abrahams, Stellanie Krimm, Xiaofan Li, Sihong Zhou, Jeffrey Hanson, Mary Rose Masikat, Krishna Bajjuri, Tyler Heibeck, Dharti Kothari, Abigail Yu, Robert Henningsen, Cuong Tran, Gang Yin, James Zawada, Julie Hang, Maureen Bruhns, Willy Solis, Alexander Steiner, Adam Galan, Toni Kline, Ryan Stafford, Alice Yam, Venita I. De Almeida, Mark Lupher, Jr., Trevor Hallam. PRECLINICAL ACTIVITY AND SAFETY OF STRO-002, A NOVEL ADC TARGETING FOLATE RECEPTOR ALPHA FOR OVARIAN AND ENDOMETRIAL CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-090.

Combining Albumin-Binding Properties and Interaction with Pemetrexed to Improve the Tissue Distribution of Radiofolates.

Folic-acid-based radioconjugates have been developed for nuclear imaging of folate receptor (FR)-positive tumors; however, high renal uptake was unfavorable in view of a therapeutic application. Previously, it was shown that pre-injection of pemetrexed (PMX) increased the tumor-to-kidney ratio of radiofolates several-fold. In this study, PMX was combined with the currently best performing radiofolate ([177Lu]cm13), which is outfitted with an albumin-binding entity. Biodistribution studies were carried out in mice bearing KB or IGROV-1 tumor xenografts, both FR-positive tumor types. SPECT/CT was performed with control mice injected with [177Lu]folate only and with mice that received PMX in addition. Control mice showed high uptake of radioactivity in KB and IGROV-1 tumor xenografts, but retention in the kidneys was also high, resulting in tumor-to-kidney ratios of ~0.85 (4 h p.i.) and ~0.60 (24 h p.i.) or ~1.17 (4 h p.i.) and ~1.11 (24 h p.i.) respectively. Pre-injection of PMX improved the tumor-to-kidney ratio to values of ~1.13 (4 h p.i.) and ~0.92 (24 h p.i.) or ~1.79 (4 h p.i.) and ~1.59 (24 h p.i.), respectively, due to reduced uptake in the kidneys. It was found that a second injection of PMX—3 h or 7 h after administration of the radiofolate—improved the tumor-to-kidney ratio further to ~1.03 and ~0.99 or ~1.78 and ~1.62 at 24 h p.i. in KB and IGROV-1 tumor-bearing mice, respectively. SPECT/CT scans readily visualized the tumor xenografts, whereas accumulation of radioactivity in the kidneys was reduced in mice that received PMX. In this study, it was shown that PMX had a positive impact in terms of reducing the kidney uptake of albumin-binding radiofolates; hence, the administration of PMX resulted in ~1.3–1.7-fold higher tumor-to-kidney ratios. This is, however, a rather moderate effect in comparison to the previously shown effect of PMX on conventional radiofolates (without albumin binder), which led to 5–6-fold increased tumor-to-kidney ratios. An explanation for this result may be the different pharmacokinetic profiles of PMX and long-circulating radiofolates, respectively. Despite the promising potential of this concept, it is believed that a clinical translation would be challenging, particularly when PMX had to be injected more than once.

Tumor Targeting with Novel Pyridyl 6-Substituted Pyrrolo[2,3- d]Pyrimidine Antifolates via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of De Novo Purine Nucleotide Biosynthesis.

Tumor-targeted specificities of 6-substituted pyrrolo[2,3- d]pyrimidine analogues of 1, where the phenyl side-chain is replaced by 3',6' (5, 8), 2',5' (6, 9), and 2',6' (7, 10) pyridyls, were analyzed. Proliferation inhibition of isogenic Chinese hamster ovary (CHO) cells expressing folate receptors (FRs) α and β were in rank order, 6 > 9 > 5 > 7 > 8, with 10 showing no activity, and 6 > 9 > 5 > 8, with 10 and 7 being inactive, respectively. Antiproliferative effects toward FRα- and FRβ-expressing cells were reflected in competitive binding with [3H]folic acid. Only compound 6 was active against proton-coupled folate receptor (PCFT)-expressing CHO cells (∼4-fold more potent than 1) and inhibited [3H]methotrexate uptake by PCFT. In KB and IGROV1 tumor cells, 6 showed <1 nM IC50, ∼2-3-fold more potent than 1. Compound 6 inhibited glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis and showed potent in vivo efficacy toward subcutaneous IGROV1 tumor xenografts in SCID mice.

Improved Intraoperative Detection of Ovarian Cancer by Folate Receptor Alpha Targeted Dual-Modality Imaging.

Complete resection of tumor lesions in advanced stage ovarian cancer patients is of utmost importance, since the extent of residual disease after surgery strongly affects survival. Intraoperative imaging may be useful to improve surgery in these patients. Farletuzumab is a humanized IgG1 antibody that specifically recognizes the folate receptor alpha (FRα). Labeled with a radiolabel and a fluorescent dye, farletuzumab may be used for the intraoperative detection of ovarian cancer lesions. The current aim is to demonstrate the feasibility of FRα-targeted dual-modality imaging using 111In-farletuzumab-IRDye800CW in an intraperitoneal ovarian cancer model. Biodistribution studies were performed 3 days after injection of 3, 10, 30, or 100 μg of 111In-farletuzumab-IRDye800CW in mice with subcutaneous IGROV-1 tumors (5 mice per group). In mice with intraperitoneal IGROV-1 tumors the nonspecific uptake of 111In-farletuzumab-IRDye800CW was determined by coinjecting an excess of unlabeled farletuzumab. MicroSPECT/CT and fluorescence imaging were performed 3 days after injection of 10 μg of 111In-farletuzumab-IRDye800CW. FRα expression in tumors was determined immunohistochemically. Optimal tumor-to-blood-ratios (3.4–3.7) were obtained at protein doses up to 30 μg. Multiple intra-abdominal tumor lesions were clearly visualized by microSPECT/CT, while uptake in normal tissues was limited. Fluorescence imaging was used to visualize and guide resection of superficial tumors. Coinjection of an excess of unlabeled farletuzumab significantly decreased tumor uptake of 111In-farletuzumab-IRDye800CW (69.4 ± 27.6 versus 18.3 ± 2.2% ID/g, p < 0.05). Immunohistochemical analyses demonstrated that the radioactive and fluorescent signal corresponded with FRα-expressing tumor lesions. FRα-targeted SPECT/fluorescence imaging using 111In-farletuzumab-IRDye800CW can be used to detect ovarian cancer in vivo and could be a valuable tool for enhanced intraoperative tumor visualization in patients with intraperitoneal metastases of ovarian cancer.

Abstract 3008: Preclinical characterization of AMG 900, a pan-aurora kinase inhibitor, alone and in combination with taxanes in ovarian cancer

Abstract BACKGROUND: Aurora kinases (AK) A and B play essential roles in multiple stages of mitosis and are frequently overexpressed in a subset of human cancers, including ovarian cancer (OC). AMG 900, a potent and highly selective small molecule inhibitor of AKs, showed promising single-agent activity in heavily pretreated patients with advanced OC in a Phase 1b clinical trial. In this study, we report the preclinical effects of AMG 900 in a panel of well-characterized human cancer cell lines representing clinically-relevant OC subtypes. METHODS: The anti-proliferative effects of AMG 900 were evaluated using a 5-day cell count assay. Cell lines were classified as sensitive to AMG 900 when lethality was &amp;gt; 15% at 10 nM. Molecular markers were profiled including TP53 mutation status, AURKA, CCNE1, MYC copy number, and p53, p21 and cyclin E1 protein by reverse phase protein array. Flow cytometry and imaging methods were used to evaluate the mechanism of action of AMG 900 alone and in combination with chemotherapy. The combination of AMG 900 plus docetaxel was evaluated in an IGROV-1 ovarian endometrioid carcinoma xenograft model. RESULTS: One third of the cell lines (11 of 35) were classified as sensitive to AMG 900 and showed enrichment for TP53 mutations and serous OC subtype. However, 10 of 24 resistant cell lines harbored TP53 mutations, indicating that TP53 mutational status alone was not sufficient for predicting AMG 900 sensitivity. Inhibition of AK activity by AMG 900 in OC cells resulted in aborted cell division leading to polyploidy and cell death (suggestive of aurora-B dominant phenotype). Re-plating of remnant cells after AMG 900 treatment showed an attenuation of cell regrowth, where TP53mut IGROV-1 cells showed minimal regrowth compared to TP53wt OVCAR-5 cells. AMG 900 inhibited proliferation at low nanomolar concentrations in the majority of OC cell lines and enhanced the effects of paclitaxel, carboplatin, and doxorubicin in IGROV-1 cells. In tumor-bearing mice, administration of AMG 900 at 7.5 mg/kg (PO) for two days per week or docetaxel at 10 mg/kg (IP) weekly for four cycles significantly inhibited the growth of IGROV-1 tumor xenografts (P &amp;lt; .0001 vs. vehicle alone). Notably, co-administration of AMG 900 with docetaxel enhanced efficacy and induced a delay in tumor regrowth compared to docetaxel alone. Single-agent-treated mice showed minimal body weight loss (BWL), whereas combination-treated mice showed moderate BWL (average &amp;lt; 10%) that was largely reversible (2 of 12 animals removed due to toxicity). CONCLUSIONS: AMG 900 alone or in combination with chemotherapy such as paclitaxel may be a promising clinical strategy to treat patients with ovarian cancer. Citation Format: Ondrej Kalous, Dylan Conklin, Kanthinh Manivong, William Wayne, Kelly Hanestad, Jude Canon, Robert Loberg, Gregory Friberg, Erick Gamelin, Florian D. Vogl, Gloria Juan, Angela Coxon, Dennis Slamon, Richard Finn, Marc Payton. Preclinical characterization of AMG 900, a pan-aurora kinase inhibitor, alone and in combination with taxanes in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3008.

6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Regioisomers as Targeted Antifolates for Folate Receptor α and the Proton-Coupled Folate Transporter in Human Tumors.

2-Amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidine antifolate thiophene regioisomers of AGF94 (4) with a thienoyl side chain and three-carbon bridge lengths [AGF150 (5) and AGF154 (7)] were synthesized as potential antitumor agents. These analogues inhibited proliferation of Chinese hamster ovary (CHO) sublines expressing folate receptors (FRs) α or β (IC50s < 1 nM) or the proton-coupled folate transporter (PCFT) (IC50 < 7 nM). Compounds 5 and 7 inhibited KB, IGROV1, and SKOV3 human tumor cells at subnanomolar concentrations, reflecting both FRα and PCFT uptake. AGF152 (6) and AGF163 (8), 2,4-diamino-5-substituted-furo[2,3-d]pyrimidine thiophene regioisomers, also inhibited growth of FR-expressing CHO and KB cells. All four analogues inhibited glycinamide ribonucleotide formyltransferase (GARFTase). Crystal structures of human GARFTase complexed with 5 and 7 were reported. In severe combined immunodeficient mice bearing SKOV3 tumors, 7 was efficacious. The selectivity of these compounds for PCFT and for FRα and β over the ubiquitously expressed reduced folate carrier is a paradigm for selective tumor targeting.

Abstract 3817: Suicidal autophagy induced by immunotherapy targeting folate receptor in ovarian cancer

Abstract Objective Cancer cells depend on folate metabolism for DNA replication, making folate receptor alpha (FRα) an important therapeutic target. Blocking FRα with a humanized anti-FRα monoclonal antibody, MORAB-003 offers a potent clinical opportunity; however, the mechanisms underlying its efficacy were not well understood. We investigated the functional mechanisms underlying MORAB-003 induced tumor inhibition in ovarian cancer. Methods Using FACS analysis and real-time PCR, FRα expression was determined in an array of human ovarian cancer cells. Next, we examined the effects of MORAB-003 monotherapy and combination with docetaxel in tumor growth using the xenograft mouse models. In vitro, we analyzed ovarian cancer cells with 3-dimensional (3-D) spheroid-like architecture. We next performed systems-based analysis including reverse-phase protein and genomic microarrays, and discovered an array of factors/genes involved in autophagy was upregulated by MORAB-003. Functional assays including FACS with AO staining, tandem mRFP/mGFP-LC3 fluorescence microscopy, transmission electron microscopy (TEM) and siRNAs knockdown were performed to further investigate MORAB-003-induced autophagy in tumor inhibition. Results IGROV1 and SKOV3ip1 cells express high-level FRα and A2780 cells express low-level FRα. In vivo, MORAB-003 monotherapy or a combination of MORAB-003 and docetaxel led to significantly decreased tumor growth in IGROV1 and SKOV3ip1 models, but not in A2780 model. Immunohistochemistry analysis on tumor sections indicated that MORAB-003 significantly reduced proliferative cancer cells, but had minimum effects on tumor cell apoptosis or angiogenesis. Network analysis on protein and gene expression profiles in IGROV1 and SKOV3ip1 tumors revealed that MORAB-003 significantly upregulated a number of factors/genes involved in autophagy, including LC3II, ATG3, ATG4B, S116-PEA-15 and BECN1. Furthermore, S116-PEA-15 and Beclin-1 were required for caspase-3-activated cell death in 3-D IGROV1 and SKOV3ip1 spheroids treated with MORAB-003. Results from autophagic flux assays indicated that MORAB-003 treatment led to an abundant accumulation of late-stage autophagic vacuoles. Blocking the autophagic vacuolization or fusion using bafilomycin A1 or hydroxychloroquine reversed the MORAB-003 induced reduction of cell viability in 3-D IGROV1 and SKOV3ip1 spheroids. Furthermore, queries from the Cancer Genome Atlas data (TCGA) revealed that copy-number gains of the FOLR1 gene significantly correlated with shorter disease-free survival in patients with the immunoreactive subtype of ovarian serous carcinoma and uterine carcinoma. Conclusion MORAB-003 inhibits tumor growth via a non-canonical autophagy associated with programmed cell death. This finding has substantial implications for clinical development of anti-FRα therapies, especially with regard to novel combinations and predictive biomarkers. Citation Format: Yun-Fei Wen, Anil K. Sood. Suicidal autophagy induced by immunotherapy targeting folate receptor in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3817. doi:10.1158/1538-7445.AM2015-3817

Abstract 1620: Synthesis and preclinical evaluation of novel 6-substituted pyrrolo[2,3-d]pyrimidines as targeted antifolates

Abstract Reduced folates are essential cofactors for biosynthesis of purines and pyrimidines. Since humans do not synthesize folate, it is necessary to obtain these cofactors from dietary sources. In mammals, three specialized systems exist that mediate membrane transport of folates and antifolates across biological membranes. These include the reduced folate carrier (RFC), the primary route for the uptake of folates and antifolates in mammalian cells, folate receptors (FRs) α and β, and the proton-coupled folate transporter (PCFT). Whereas RFC is ubiquitously expressed, FRs and PCFT show a more narrow pattern of tissue expression. Toxicity of clinically used antifolates occurs in part, to their lack of selectivity for tumor cells over normal cells due to RFC transport. Antifolates with tumor-specific FR and/or PCFT drug uptake would circumvent major toxicities of currently used antifolates. Our analog AG17F had shown selective FR and PCFT transport over RFC with excellent cell inhibitory activity in KB tumor cell line (IC50 = 1.7 nM). On the basis of AG17F, AG140F with sulfur in place of carbon at C10, AG182F with oxygen in place of carbon at C10 and AG183F with nitrogen in place of carbon at C10 were designed and synthesized. This isosteric replacement of carbon with heteroatoms in the bridge provides compounds with different chain lengths, angles, conformations and extra hydrogen bond donors and/or acceptors compared to the parent carbon chain analogues. AG17F had shown inhibitory activity toward the growth of a CHO cell line (hRFC) expressing human RFC (IC50 = 304 nM), the three heteroatom analogues AG140F, AG182F and AG183F were inactive in the CHO cell line expressing RFC transport at concentrations up to 1000 nM, but were highly active in inhibiting CHO cell lines (RT16) expressing human FRα (IC50s of 0.62, 1.10, and 2.3 nM respectively) and inhibiting PCFT-expressing CHO cells (R2/PCFT4) (IC50s of 267, 57.3, and 87.4 nM respectively). AG140F, AG182F, and AG183F were potently inhibitory toward KB tumor cells (IC50s of 2, 0.32 and 0.34 nM respectively) and IGROV1 tumor cells (IC50s of 0.51, 0.58 and 0.48 nM respectively). AG140F, AG182F, and AG183F showed binding affinities to FRα in RT16 cells exceeding that for AG17F, and comparable to folic acid. With R2/PCFT4 cells, all compounds showed high affinities with binding (reflected in Ki values) in the order of AG17F&amp;gt;AG182F∼AG183F&amp;gt;AG140F. In KB cells, adenosine and 5-aminoimidazole-4-carboxamide protected from growth inhibition suggesting de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase) and the likely targets. By in situ assays in KB cells, GARFTase inhibition was confirmed at concentrations similar to those which inhibit cell proliferation. AG183F was further evaluated for against early stage human IGROV tumor xenograft in female SCID mice models and was found to be remarkably efficacious (T/C = 12%) without any acute or long term toxicities. Citation Format: Lalit Kumar Golani, Aleem Gangjee, Christina Cherian, Steven Orr, Shermaine Mitchell-Ryan, Lisa Polin, Adrianne Wallace, Larry H. Matherly. Synthesis and preclinical evaluation of novel 6-substituted pyrrolo[2,3-d]pyrimidines as targeted antifolates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1620. doi:10.1158/1538-7445.AM2014-1620

Abstract 673: A Monoclonal Antibody, MORAb-075, Targeting Claudin-3 &amp; Claudin-4 Inhibits human ovarian, pancreatic and prostate cancer progression in mice

Abstract Claudin (CLDN) is a family of integral membrane proteins. They are crucial for tight junction formation and functions. Studies have found CLDN3 and CLDN4 proteins are overexpressed in majority of primary ovarian, prostate, and pancreatic cancers. We have generated a rat-human chimeric monoclonal antibody, MORAb-075, which binds to CLDN3 and CLDN4 with high affinity in vitro. In order to evaluate its therapeutic potential in vivo, we have established a number of human cancer models where the tumors are grown orthotopically in nude mice. These tumors were confirmed positive for human CLDN3 or/and CLDN4 expressions via immunohistochemistry staining. The models include a CLDN3/4-expressing prostate cancer PC-3; a CLDN4-expressing pancreatic cancer, BxPc-3; a CLDN3-expressing ovarian cancer, IGROV-1. In these orthotopic tumor models, MORAb-075 demonstrated significant antitumor activities. When the mAb was administered intravenously at 25 mg/kg, twice weekly for 4 weeks, the tumor growth inhibition (TGI) of PC-3 tumors reached 50%; TGI of BxPC-3 tumors was nearly 60% and TGI of IGROV-1 tumors was greater than 75%. Our findings demonstrate the potency of MORAb-075, as a single agent, in inhibiting solid tumor growth in these models. These results suggest that mAb therapy targeting CLDN3 &amp; CLDN4 is a valuable option for therapeutic application in cancer therapy that may be improved in combination with standard of care chemotherapies for CLDN3 or CLDN4 positive cancers. Citation Format: Jian-Min Lin, allis soto, Christopher maddage, earl Albone, yuhong zhou, luigi grasso. A Monoclonal Antibody, MORAb-075, Targeting Claudin-3 &amp; Claudin-4 Inhibits human ovarian, pancreatic and prostate cancer progression in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2014-673

Future prospects for SPECT imaging using the radiolanthanide terbium-155 — production and preclinical evaluation in tumor-bearing mice

IntroductionWe assessed the suitability of the radiolanthanide 155Tb (t1/2=5.32days, Eγ=87keV (32%), 105keV (25%)) in combination with variable tumor targeted biomolecules using preclinical SPECT imaging. Methods155Tb was produced at ISOLDE (CERN, Geneva, Switzerland) by high-energy (~1.4GeV) proton irradiation of a tantalum target followed by ionization and on-line mass separation. 155Tb was separated from isobar and pseudo-isobar impurities by cation exchange chromatography. Four tumor targeting molecules – a somatostatin analog (DOTATATE), a minigastrin analog (MD), a folate derivative (cm09) and an anti-L1-CAM antibody (chCE7) – were radiolabeled with 155Tb. Imaging studies were performed in nude mice bearing AR42J, cholecystokinin-2 receptor expressing A431, KB, IGROV-1 and SKOV-3ip tumor xenografts using a dedicated small-animal SPECT/CT scanner. ResultsThe total yield of the two-step separation process of 155Tb was 86%. 155Tb was obtained in a physiological l-lactate solution suitable for direct labeling processes. The 155Tb-labeled tumor targeted biomolecules were obtained at a reasonable specific activity and high purity (>95%). 155Tb gave high quality, high resolution tomographic images. SPECT/CT experiments allowed excellent visualization of AR42J and CCK-2 receptor-expressing A431 tumors xenografts in mice after injection of 155Tb-DOTATATE and 155Tb-MD, respectively. The relatively long physical half-life of 155Tb matched in particular the biological half-lives of 155Tb-cm09 and 155Tb-DTPA-chCE7 allowing SPECT imaging of KB tumors, IGROV-1 and SKOV-3ip tumors even several days after administration. ConclusionsThe radiolanthanide 155Tb may be of particular interest for low-dose SPECT prior to therapy with a therapeutic match such as the β--emitting radiolanthanides 177Lu, 161Tb, 166Ho, and the pseudo-radiolanthanide 90Y.

Abstract 3824: Design, synthesis and biological evaluation of 4-atom side chain pemetrexed (PMX) homolog AG127 as a potent inhibitor of tumors expressing folate receptors via 5-aminoimidazole-4-carboxamide ribonucleotide formyl transferase (AICARFTase) inhibition

Abstract Two major hurdles in cancer therapy are the inability of chemotherapeutic agents to selectively target tumor cells over normal tissues, and the development of tumor cell resistance. The multi-enzyme-targeted anticancer drug pemetrexed (PMX) suffers from dose-limiting toxicity, in part due to its membrane transport by the reduced folate carrier (RFC) which is ubiquitously expressed in normal and tumor cells. We recently described a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates. A highly active compound of this series (AG71) included a 4-carbon bridge and was selectively transported into cells by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) but not RFC. AG71 inhibited de novo purine biosynthesis at the level of glycinamide ribonucleotide formyltransferase (GARFTase). AG71 potentially inhibited proliferation of FR- and PCFT-expressing Chinese hamster ovary cells and of both KB and IGROV1 tumors in vitro; in SCID mice with subcutaneous KB tumors, AG71 was highly active against both early and advanced stage tumors. Based on the 5-substituted pyrrolo[2,3-d]pyrimidine scaffold of PMX and our results with the 6-substituted compound AG71, we synthesized 5-substituted pyrrolo[2,3-d]pyrimidine phenyl analogs with 1- to 6-carbon atoms in the bridge (AG124-AG129). Analogs were tested for anti-proliferative effects on KB human tumor cells and on Chinese hamster ovary (CHO) sublines engineered to individually express RFC (PC43-10), PCFT (R2/PCFT4), and FRα (RT-16). The 4-carbon atom chain analog (AG127) showed IC50 value of 1.9 nM, only slightly less than for AG71 (0.55 nM), in inhibiting proliferation of KB tumor cells in culture. In the CHO sublines, both AG127 and AG71 inhibited growth of RT-16 cells (IC50 = 3.1 nM and 1.8 nM respectively). AG71 was inactive toward PC43-10 cells and was inhibitory toward R2/PCFT4 cells (IC50 = 43.4 nM). Compared to RT-16 cells, AG127 showed reduced inhibitory activity toward PC43-10 cells (IC50 = 54 nM) and R2/PCFT4 cells (IC50 = 733 nM). PMX is a multi-targeted agent with its principle inhibitory effects on thymidylate synthase and secondary inhibitory effects on AICARFTase and GARFTase in de novo purine nucleotide biosynthetic. Inhibition of KB cell proliferation by AG127 was abolished by excess adenosine, but not thymidine or AICA, suggesting that AICARFTase was the principle intracellular target. Collectively, our results show that the side chain homologated analog of PMX, AG127, exhibits nanomolar inhibition of proliferation of KB tumor cells in culture. The unique and specific AICARFTase inhibition of AG127 makes it a promising option for cancer chemotherapy. 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 3824. doi:1538-7445.AM2012-3824

Abstract 4765: Synthesis and evaluation of nonclassical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates: Role of terminal amino acid moiety in membrane transport and antitumor activity.

Abstract Reduced folates are essential cofactors for biosynthesis of purines and pyrimidines. Since humans do not synthesize folate, it is necessary to obtain these cofactors from dietary sources. In mammals, three specialized systems exist that mediate membrane transport of folates and antifolates across biological membranes. These include the reduced folate carrier (RFC), the primary route for the uptake of folates and antifolates in mammalian cells, folate receptors (FRs) ≤ and β, and the proton-coupled folate transporter (PCFT). Whereas RFC is ubiquitously expressed, FRs and PCFT show a more narrow pattern of tissue expression. Toxicity of clinically used antifolates is due to their lack of selectivity for tumor cells over normal cells, since these drugs are transported by RFC. Antifolates with selective substrate activity for FRs and/or PCFT over RFC can potentially target tumors that express these non-RFC transporters relative to normal tissues. Thus, antifolates with tumor-specific FR and/or PCFT drug uptake would circumvent major toxicities of currently used antifolates. 6-Substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four carbon bridge length and with L-aspartic acid (AG170), aminomalonic acid (AG173) and α-glutamic acid (AG172) were designed and synthesized in place of the normal L-glutamic acid (AG118) of this series to evaluate the importance of L-glutamic acid for RFC, FRα, and PCFT transport and inhibition of cell proliferation. AG170 was slightly less active than AG118 in inhibiting growth of a CHO cell line (RT16) expressing human FRα (IC50s of 18.7 and 2.6 nM, respectively) but similarly active in inhibiting PCFT-expressing CHO cells (R2/PCFT4) (IC50s of 81.7 and 63.8, nM, respectively). AG173 and AG172 were inactive toward both RT16 and R2/PCFT4 cells (IC50&amp;gt;1000nM). All four analogues were inactive toward a CHO cell line (PC43-10) expressing human RFC but not FRα or PCFT. These analogues were further evaluated in KB and IGROV-1 human tumor cell lines in culture. Whereas AG118 was potently inhibitory toward KB and IGROV1 tumor cells (IC50s of 0.27 and 1.4 nM, respectively), AG170, AG173, and AG172 all were inert toward these tumor cells at concentrations up to 1000 nM. Collectively, these results establish an important role for the terminal amino acid in the anti-proliferative effects of this series of 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates. The disparate inhibitions of AG170 toward FRα-expressing CHO versus human tumor cells implies that there may be differences in metabolic pathways and/or target enzymes in CHO cell lines compared to human cancer cell lines that are responsible. 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 4765. doi:1538-7445.AM2012-4765

Integrated processing of contrast pulse sequencing ultrasound imaging for enhanced active contrast of hollow gas filled silica nanoshells and microshells.

In recent years, there have been increasing developments in the field of contrast-enhanced ultrasound both in the creation of new contrast agents and in imaging modalities. These contrast agents have been employed to study tumor vasculature in order to improve cancer detection and diagnosis. An in vivo study is presented of ultrasound imaging of gas filled hollow silica microshells and nanoshells which have been delivered intraperitoneally to an IGROV-1 tumor bearing mouse. In contrast to microbubbles, this formulation of microshells provided strong ultrasound imaging signals by shell disruption and release of gas. Imaging of the microshells in an animal model was facilitated by novel image processing. Although the particle signal could be identified by eye under live imaging, high background obfuscated the particle signal in still images and near the borders of the tumor with live images. Image processing techniques were developed that employed the transient nature of the particle signal to selectively filter out the background signal. By applying image registration, high-pass, median, threshold, and motion filtering, a short video clip of the particle signal was compressed into a single image, thereby resolving the silica shells within the tumor. © 2012 American Vacuum Society.

Protein profiles in human ovarian cancer cell lines correspond to their metabolic activity and to metabolic profiles of respective tumor xenografts

Many solid tumors show a large variability in glycolytic activity and lactate accumulation, which has been correlated with different metastatic spread, radioresistance and patient survival. To investigate potential differences in protein profiles underlying these metabolic variances, the highly glycolytic human ovarian cancer cell line OC316 was investigated and compared with the less glycolytic line IGROV-1. Extracellular acidification and oxygen consumption were analyzed with an extracellular flux analyzer. Glycolysis-associated proteins, including specific membrane transporters, were quantified through in-cell western analyses. Metabolic properties of corresponding tumor xenografts were assessed via induced metabolic bioluminescence imaging. Extracellular flux analyses revealed elevated bioenergetics of OC316 cells. Hexokinase II, pyruvate kinase, pyruvate dehydrogenase E1 beta subunit and pyruvate dehydrogenase kinase 1, as well as the glucose transporter 1 and the monocarboxylate transporter 4, were overexpressed in these cells compared with IGROV-1. When generating tumor xenografts in SCID mice, cells maintained their glycolytic behavior, i.e. OC316 showed higher lactate concentrations than IGROV-1 tumors. In summary, a congruency between protein profiles and metabolic properties has been demonstrated in the human ovarian cancer lines investigated. Also, a perpetuation of glycolytic characteristics during the transition from in vitro to the in vivo situation has been documented. This model system could be useful for systematic studies on therapeutic intervention by manipulation of tumor glycolysis and associated pathways.

Synthesis, Biological, and Antitumor Activity of a Highly Potent 6-Substituted Pyrrolo[2,3-d]pyrimidine Thienoyl Antifolate Inhibitor with Proton-Coupled Folate Transporter and Folate Receptor Selectivity over the Reduced Folate Carrier That Inhibits β-Glycinamide Ribonucleotide Formyltransferase

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1-3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with l-glutamate diethyl ester, followed by saponification, afforded 1-3. Compound 3 selectively inhibited the proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1-, 2-, and 4-atom bridge lengths for the activity of this series.

Abstract 3250: Design, synthesis and biological evaluation of side chain pemetrexed (PMX) analogues as potent inhibitors of tumors expressing folate receptors via GARFTase inhibition

Abstract Two major hurdles in cancer therapy are the inability of chemotherapy agents to selectively target tumor cells over normal tissues, and the development of tumor cell resistance. The multi-enzyme-targeted anticancer drug pemetrexed (PMX) suffers from dose-limiting toxicity due to its membrane transport by the reduced folate carrier (RFC) which is ubiquitously expressed in normal and tumor cells. We recently reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates. The most active of this series (AG71) included a 4 carbon bridge and was selectively transported into cells by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) (but not RFC) whereupon it inhibited de novo purine biosynthesis at the level of glycinamide ribonucleotide formyltransferase (GARFTase). AG71 was highly active toward both KB and IGROV1 tumors; in SCID mice with KB tumors, AG71 was highly active against both early and advanced stage tumors. Based on the 5-substituted pyrrolo[2,3-d]pyrimidine structure of PMX and our results with the 6-substituted compound AG71, we synthesized 5-substituted pyrrolo[2,3-d]pyrimidine thienoyl analogs with 1 to 6 carbon atoms in the bridge (AG133-AG138) as hybrid molecules of PMX and AG71. The compounds were synthesized via a Heck coupling of the thiophene iodide with the alkene alcohols followed by α-bromonation and cyclization with the 2,4-diamino-6-oxo-pyrimidine to afford the 5-substituted pyrrolo[2,3-d]pyrimidine and elaborated to the L-glutamate. These analogs could be envisaged to afford the multi-targeted attributes of PMX, while preserving FRα and/or PCFT specificity of our 6-substituted analogs. Analogs were tested for anti-proliferative effects on KB human tumor cells and on Chinese hamster ovary (CHO) sublines engineered to individually express RFC (PC43-10), PCFT (R2/PCFT4), and FRα (RT-16). Analogs with 4 and 5-carbon atom chains (AG136 and AG137) showed IC50 values of 17.1 nM and 18.1 nM, respectively, against KB tumor cells in culture; other analogs of this series were 13- to &amp;gt;58-fold less active. In the CHO sublines, AG136 and AG137 were growth inhibitory in the order RT-16 = PC43-10 &amp;gt; R2/PCFT4 and RT-16 &amp;gt;&amp;gt; PC43-10 = R2/PCFT4, respectively. Thus, at least for AG136 unlike AG71, significant RFC activity is preserved. Whereas PMX is a multi-targeted agent with inhibitory effects on both de novo thymidylate and purine nucleotide biosynthetic pathways, the anti-proliferative effects of AG136 and AG137 were abolished by adenosine but not thymidine, establishing exclusive inhibition of purine biosynthesis. Our results suggest that 6-subtitution of the pyrrolo[2,3-d]pyrimidine scaffold is more effective in conferring selective transport of FRα over RFC, although inhibition of de novo purine biosynthesis is preserved in the 5-substituted compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3250. doi:10.1158/1538-7445.AM2011-3250

Abstract 1364: Design, synthesis and evaluation of potent antiproliferative and cytotoxic antifolates with selective FRs and PCFT transport over RFC and potent GARFTase inhibition

Abstract Lack of selectivity for folate transporters is one of the major causes of toxicity and chemotherapy failure with clinically used antifolates. Three folate transporters, the reduced folate carrier (RFC), folate receptors (FR) and the proton-coupled folate transporter (PCFT), account for most of the folate influx activity in mammalian cells. We recently reported potent inhibition of cell proliferation by a classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine with a thienoyl for benzoyl-substituted side chain and a 4-carbon bridge (AG71). AG71 was selective for transport by FRs and PCFT over RFC and inhibited glycinamideribonucleotide formyl transferase (GARFTase) in de novo purine biosynthesis. In SCID mice with KB tumors, AG71 was highly active against both early and advanced stage tumors. A series of five thienoyl regioisomers of AG71 with [1,3] (AG117 and AG118) and [1,2] (AG114, AG115, AG116) thienoyl substitutions by the 4-carbon bridge and the L-glutamate were synthesized. Analogs were evaluated for anti-proliferative activities in engineered Chinese hamster ovary (CHO) cells expressing RFC, FR, or PCFT to determine the optimum structures for selective transport by FRs and/or PCFT. Additional studies were performed in KB and IGROV1 tumor cells, using inhibition of cell proliferation or colony formation as metrics. Like AG71, none of the novel thienyol regioisomers were substrates for RFC. AG117 and AG118 were potently active toward FR- and PCFT-expressing CHO cells and toward KB and IGROV1 tumor cells, equivalent or exceeding levels for AG71. In colony-forming assays in KB cells, AG117 and AG118 were cytotoxic. AG114, AG115, and AG116 were substantially less active or completely inert. AG117 and AG118, like AG71, were high affinity ligands for FRα by competitive ligand binding with 3H-folic acid, and were potent substrates for PCFT by direct competition 3H-methotrexate transport. By protection assays, AG71 and AG117 preserved potent anti-proliferative activity even in the presence of 50 nM leucovorin; adenosine and 5-imidazole-4-carboxamide were completely protective, establishing the de novo purine biosynthetic pathway and GARFTase as the principal intracellular targets. GARFTase inhibition was confirmed by in situ GARFTase metabolic assays with 14C-glycine as a radiotracer. Our results identify 6-subsituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers of AG71 as potent anti-proliferative and cytotoxic antifolates, reflecting selective transport by PCFT and FR over RFC and inhibition of GARFTase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1364. doi:10.1158/1538-7445.AM2011-1364

A Low Molecular Weight Folate Receptor Targeted Contrast Agent for Magnetic Resonance Tumor Imaging

This study aims to develop a low molecular weight folate receptor (FR) contrast agent for MR tumor imaging. Gadolinium-tetraazacyclododecane tetraacetic acid (Gd.DOTA) was conjugated to folic acid to create Gd.DOTA.Folate. The efficacy of Gd.DOTA.Folate to bind FR was evaluated in vitro by inductively coupled mass spectrometry (ICP-MS) and in vivo by magnetic resonance imaging (MRI) tumor enhancement over 14 h, utilizing an overexpressing α-FR cell line (IGROV-1), compared to an α-FR-negative cell line (OVCAR-3). Gd.DOTA.Folate localization ex vivo was verified by laser ablation ICP-MS. ICP-MS confirmed Gd.DOTA.Folate uptake by IGROV-1 cells and competitive binding with free folic acid inhibited binding. IGROV-1 tumors showed an increase in R (1) at 2 h, which increased significantly over 14 h post-Gd.DOTA.Folate with clear enhancement on MR images. This was not observed in controls. These data support the use of FR-targeted small molecular weight MRI contrast agents for tumor imaging in vivo.

Abstract 4541: The design, synthesis and evaluation of selective FR α and β substrates with potent GARFTase inhibitory activity as antitumor agents

Abstract Glycinamide ribonucleotide formyl transferase (GARFTase) catalyzes the first of two formyl transfer steps in the de novo purine biosynthetic pathway. Potent inhibitors of GARFTase and de novo purine biosynthesis have shown promise as antitumor drugs. However, a lack of selectivity and toxicity has been a major hurdle for GARFTase inhibitors as cancer chemotherapeutic agents. We recently reported a series of thieno[2,3-d]pyrimidine classical antifolates that are specifically taken up by the folate receptor (FR) and inhibit FR expressing tumor cells at nanomolar IC50 values (IC50= 4.9 nM in human KB tumor cells, IC50= 5.9 nM in human IGROV1 tumor cells,). GARFTase was confirmed as the target. Notably, the thieno[2,3-d]pyrimidine antifolates are unique and distinct from all other classical antifolates evaluated, including pemetrexed (PMX), raltitrexed (RTX), and methotrexate (MTX) in that they are neither substrates for reduced folate carrier (RFC) nor for the proton-coupled folate transporter (PCFT). This characteristic results in selective targeting of FR-expressing cells, without toxicity to normal cells that express RFC. To further investigate the structural requirements of antifolates with respect to FR substrate activity, a series of four 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines with different aryl side chains were synthesized. This afforded potent antitumor agents with increased inhibitory activity against FRα expressing tumor cells (IC50= 2 nM in human KB tumor cells) 2.5- fold more potent than the lead benzoyl compound. In addition, these thieno[2,3-d]pyrimidines were specific for FRα and were not transported by RFC or PCFT and indicated that the nature of the bicyclic scaffold is an important structural determinant for transport specificity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4541.