Susan E. Pratt

The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites

5′-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5′-fluoro-2′-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5′-fluoro-2′-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were ∼9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 μmol/L [3H]5-FdUMP, [3H]5-FUMP, [3H]dUMP, and not [3H]5-FUdR, or [3H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a Km of 1.1 mmol/L and Vmax of 439 pmol/min/mg protein. Uptake of 250 μmol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3′,5′-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 μmol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.

Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

Purpose: The oral prodrug of gemcitabine LY2334737 is cleaved systemically to gemcitabine; the mechanism responsible for hydrolysis is unknown. LY2334737 cytotoxicity was tested in the NCI-60 panel; mining of microarray expression data identified carboxylesterase (CES) as a top hydrolase candidate. Studies examined whether CES is responsible for hydrolysis and whether cellular CES expression confers prodrug sensitivity. Experimental Design: Human recombinant CES isozymes were assayed for LY2334737 hydrolysis. Stable CES-overexpressing HCT-116 transfectants and a SK-OV-3 knockdown were prepared. Cell lines were tested for drug sensitivity and CES expression by quantitative real time-PCR (qRT-PCR), Western blotting, and immunohistochemical staining. Bystander cytotoxicity studies were conducted with GFP-tagged PC-3 cells as the reporter cell line. Therapeutic response of the HCT-116 transfectants was evaluated in xenografts. Results: Of 3 human CES isozymes tested, only CES2 hydrolyzed LY2334737. Five cell lines that express CES2 responded to LY2334737 treatment. LY2334737 was less cytotoxic to a SK-OV-3 CES2 knockdown than parental cells. The drug response of CES2-transfected HCT-116 cells correlated with CES2 expression level. Bystander studies showed statistically greater PC-3–GFP growth inhibition by LY2334737 when cells were cocultured with CES2 and not mock transfectants. Oral treatment of xenograft models with 3.2 mg/kg LY2334737 once a day for 21 days showed greater tumor growth inhibition of CES2 transfectant than the mock transfectant (P ≤ 0.001). Conclusions: CES2 is responsible for the slow hydrolysis of LY2334737. Because intact prodrug circulates at high plasma levels after oral LY2334737 administration, improved response rates may be observed by tailoring LY2334737 treatment to patients with CES2 tumor expression. Clin Cancer Res; 19(5); 1159–68. ©2012 AACR.

Characterization of LY2228820 Dimesylate, a Potent and Selective Inhibitor of p38 MAPK with Antitumor Activity

p38α mitogen-activated protein kinase (MAPK) is activated in cancer cells in response to environmental factors, oncogenic stress, radiation, and chemotherapy. p38α MAPK phosphorylates a number of substrates, including MAPKAP-K2 (MK2), and regulates the production of cytokines in the tumor microenvironment, such as TNF-α, interleukin-1β (IL-1β), IL-6, and CXCL8 (IL-8). p38α MAPK is highly expressed in human cancers and may play a role in tumor growth, invasion, metastasis, and drug resistance. LY2228820 dimesylate (hereafter LY2228820), a trisubstituted imidazole derivative, is a potent and selective, ATP-competitive inhibitor of the α- and β-isoforms of p38 MAPK in vitro (IC50 = 5.3 and 3.2 nmol/L, respectively). In cell-based assays, LY2228820 potently and selectively inhibited phosphorylation of MK2 (Thr334) in anisomycin-stimulated HeLa cells (at 9.8 nmol/L by Western blot analysis) and anisomycin-induced mouse RAW264.7 macrophages (IC50 = 35.3 nmol/L) with no changes in phosphorylation of p38α MAPK, JNK, ERK1/2, c-Jun, ATF2, or c-Myc ≤ 10 μmol/L. LY2228820 also reduced TNF-α secretion by lipopolysaccharide/IFN-γ–stimulated macrophages (IC50 = 6.3 nmol/L). In mice transplanted with B16-F10 melanoma, tumor phospho-MK2 (p-MK2) was inhibited by LY2228820 in a dose-dependent manner [threshold effective dose (TED)70 = 11.2 mg/kg]. Significant target inhibition (>40% reduction in p-MK2) was maintained for 4 to 8 hours following a single 10 mg/kg oral dose. LY2228820 produced significant tumor growth delay in multiple in vivo cancer models (melanoma, non–small cell lung cancer, ovarian, glioma, myeloma, breast). In summary, LY2228820 is a p38 MAPK inhibitor, which has been optimized for potency, selectivity, drug-like properties (such as oral bioavailability), and efficacy in animal models of human cancer. Mol Cancer Ther; 13(2); 364–74. ©2013 AACR.

Evaluation of an in vitro coculture model for the blood-brain barrier: comparison of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources.

SummaryCocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier. Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function. Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells. Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells. No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium. Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium. In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier. Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations. On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules.

Characterization and application of a vinblastine-selected Caco-2 cell line for evaluation of P-glycoprotein

SummaryThe role of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug-drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to upregulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.

Efficacy of Low-Dose Oral Metronomic Dosing of the Prodrug of Gemcitabine, LY2334737, in Human Tumor Xenografts

LY2334737, an oral prodrug of gemcitabine, is cleaved in vivo, releasing gemcitabine and valproic acid. Oral dosing of mice results in absorption of intact prodrug with slow systemic hydrolysis yielding higher plasma levels of LY2334737 than gemcitabine and prolonged gemcitabine exposure. Antitumor activity was evaluated in human colon and lung tumor xenograft models. The dose response for efficacy was examined using 3 metronomic schedules, once-a-day dosing for 14 doses, every other day for 7 doses, and once a day for 7 doses, 7 days rest, followed by an additional 7 days of once-a-day dosing. These schedules gave significant antitumor activity and were well tolerated. Oral gavage of 6 mg/kg LY2334737 daily for 21 days gave equivalent activity to i.v. 240 mg/kg gemcitabine. HCl administered once a week for 3 weeks to mice bearing a patient mesothelioma tumor PXF 1118 or a non–small cell lung cancer tumor LXFE 937. The LXFE 397 tumor possessed elevated expression of the equilibrative nucleoside transporter-1 (ENT1) important for gemcitabine uptake but not prodrug uptake and responded significantly better to treatment with LY2334737 than gemcitabine (P ≤ 0.001). In 3 colon xenografts, antitumor activity of LY2334737 plus a maximally tolerated dose of capecitabine, an oral prodrug of 5-fluorouracil, was significantly greater than either monotherapy. During treatment, the expression of carboxylesterase 2 (CES2) and concentrative nucleoside transporter-3 was induced in HCT-116 tumors; both are needed for the activity of the prodrugs. Thus, metronomic oral low-dose LY2334737 is efficacious, well tolerated, and easily combined with capecitabine for improved efficacy. Elevated CES2 or ENT1 expression may enhance LY2334737 tumor response. Mol Cancer Ther; 12(4); 481–90. ©2013 AACR.

Quantification of gemcitabine incorporation into human DNA by LC/MS/MS as a surrogate measure for target engagement.

In this study, we report a method for direct determination of gemcitabine incorporation into human DNA. Gemcitabine (dFdC), a structural analog of the nucleoside deoxycytidine (dC), derives its primary antitumor activity through interruption of DNA synthesis. Unlike other surrogate measures, DNA incorporation provides a mechanistic end point useful for dose optimization. DNA samples (ca. 25 microg) were hydrolyzed using a two-step enzymatic procedure to release dFdC which was subsequently quantified by LC-ESI-MS/MS using stable isotope labeled internal standards and selected reaction monitoring (SRM). dFdC was quantitated and reported relative to deoxyguanosine (dG) since dG is the complementary base for both dFdC and dC. The SRM channel for dG was detuned using collision energy as the attenuating parameter in order to accommodate the difference in relative abundance for these two analytes (>104) and enable simultaneous quantification from the same injection. The assay was shown to be independent of the amount of DNA analyzed. The method was validated for clinical use using a 3 day procedure assessing precision, accuracy, stability, selectivity, and robustness. The validated ranges for dFdC and dG were 5-7500 pg/mL and 0.1-150 microg/mL, respectively. Results are presented which confirm that the ratio of dFdC to dG in DNA isolated from tumor cells incubated with dFdC increases with increased exposure to the drug and that dFdC can also be quantified from DNA extracted from blood.

Abstract B31: Potential for patient tailoring for prodrug of gemcitabine (LY2334737) by assessment carboxylesterase II expression in solid tumor biopsies

Carboxylesterase II (CES2), a serine ester hydrolase, is the major carboxylesterase responsible for the conversion of the prodrug of gemcitabine into its active form, gemcitabine (LY2334737), an anticancer chemotherapeutic. In humans, the ratio of plasma levels of prodrug to active form is 10:1. Therefore, increased availability of LY2334737 can result in cleavage of the prodrug into its active form at the tumor site and has potential for greater tumor cytotoxicity. Relatively high levels of expression of CES2 within non-neoplastic human tissue occur in the liver, kidney and gastrointestinal tract as visualized by immunohistochemistry (IHC). CES2 is also reported to be expressed in human cancers such as non-small cell lung carcinoma and colon adenocarcinomas. In this study, we analyzed CES2- transfected in vitro cell lines by real-time PCR, chromogenic IHC, and automated quantitative analysis (AQUA) of immunofluorescent in situ protein expression and showed that high levels of CES2 correlate with higher cytotoxicity. qRT-PCR analysis revealed that the mRNA levels of CES2 measured in formalin-fixed paraffin-embedded tumors correlate with protein levels. CES2 transcriptional profiling was performed to identify additional tumor types with high levels of expression of CES2. CES2 overexpression was detectable by IHC and/or AQUA analysis in human colon carcinoma, mesothelioma, non-small cell lung cancer, and tumors of the breast and ovary. CES2 IHC labeling in colonic adenocarcinomas was diffuse. However, labeling in non-neoplastic mucosa showed low levels in proliferating cells at the crypt base and increasingly higher expression toward the terminally differentiated cells at the tips—cells that would not progress through the cell cycle and should not be affected by antiproliferative agents. In conclusion, we have developed robust IHC and AQUA-based assays for differential expression of CES protein levels in a variety of archival human tumor types and have shown high correlation with CES2 transcript levels by qRT-PCR. These methodologies can identify tumors with high levels of CES2, a biomarker that may further be investigated in biomarker-driven clinical trials to identify patients who will more likely respond to LY2334737. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B31.

Abstract B36: Identification of carboxylesterase 2 as a hydrolase that cleaves the prodrug of gemcitabine LY2334737

Gemcitabine hydrochloride is a nucleoside anticancer agent registered for the treatment of pancreatic, NSCLC, breast, and ovarian cancers. A prodrug of gemcitabine (LY2334737) was prepared that is a gemcitabine analog with an amide-linked valproate. This prodrug is noncytotoxic and must be hydrolyzed to release gemcitabine. In man, the prodrug is orally absorbed intact and converted to gemcitabine systemically. This study was undertaken to identify the enzyme responsible for hydrolysis of the prodrug to gemcitabine. The NCI-60 cell line panel was screened for prodrug sensitivity and microarray data were used to evaluate the expression of hydrolase enzymes in responsive and unresponsive cell lines. Cell extracts and candidate enzymes were tested for LY hydrolysis using HPLC and cell lines were evaluated for enzyme expression using qRT-PCR and Western methods. Screening of the NCI-60 panel demonstrated that LY was not cytotoxic to the majority of cell lines, however sensitivity was observed in a few cell lines such as SK-OV-3 and COLO205. Other cell lines were also evaluated. Extracts of drug sensitive cell lines hydrolyzed LY to gemcitabine and its inactive metabolite 29,29-difluorodeoxyuridine (dFdU), indicating that LY hydrolysis was necessary for activity. Analysis of the expression of known hydrolases in the NCI-60 microarray data identified the serine ester hydrolase, carboxylesterase 1 (CES1) among the top candidates. In humans, there are 3 CESs and these are known to hydrolyze other prodrugs. Human recombinant protein of each CES was tested for LY cleavage. With long incubation periods, only CES2 hydrolyzed the drug. Loperamide is an inhibitor of CES1 and CES2 that is permeable to cells and noncytotoxic. At low micromolar concentrations this inhibitor only affects CES2, therefore cytotoxicity assays were conducted with SK-OV-3 in the presence and absence of 10 µM loperamide. The cytotoxicity of LY was decreased with loperamide CES2 inhibition without effect on gemcitabine cytotoxicity. Expression of CES2 was evaluated in drug-sensitive and -insensitive cell lines by qRT-PCR and Western analysis; LY sensitive lines expressed CES2 while insensitive lines did not. An immunohistochemical assay was developed that showed expression of CES2 to be heterogeneously expressed in these cell lines. Taken together, these data indicate that CES2 hydrolyzes the prodrug. Cancer cells that express CES2 may have an enhanced response to treatment with the prodrug LY2334737. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B36.

Abstract 317: Combination of a novel ERK1/2 inhibitor (LY3214996) with CDK4 and CDK6 inhibitor (abemaciclib) enhances antitumor efficacy in KRAS mutant non-small cell lung cancer (NSCLC)

ERK1/2, a key downstream effector of RAS mutations, is involved in the signaling network which drives cell proliferation, survival, metastasis and cancer resistance to drug treatment (including MEK and BRAF inhibitors). Lung cancer is a leading cause of cancer death worldwide. KRAS mutation present in up to 30% of NSCLC patients is associated with a poor prognosis and represents an unmet medical need. In KRAS mutant NSCLC, enhanced ERK activation cooperates with dysregulation of the cell cycle checkpoint (e.g., cyclin D, CDK4 and CDK6 complex), and contributes to tumor progression; thus, the simultaneous inhibition of ERK and the CDK4/6 pathway is hypothesized to augment tumor growth inhibition. LY3214996, a novel and highly selective small molecule inhibitor of ERK1 and ERK2, is currently in phase I clinical trial and has been shown to inhibit cell proliferation in RAS or BRAF mutant tumor cells in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4 and CDK6-selective inhibitor is currently in phase III studies for ER positive breast cancer and KRAS mutant NSCLC. In this study we explore the potential efficacy of combined inhibition of ERK1/2 and CDK4 and CDK6 in KRAS mutant NSCLC. The combination of LY3214996 and abemaciclib synergistically inhibited cell proliferation in 85% of KRAS mutant cells in an unbiased NSCLC panel. Combination treatment with LY3214996 and abemaciclib significantly decreased levels of phospho- p90RSK, phospho-Rb, phospho-S6 and Ki67; and synergistically inhibited cell proliferation and survival in KRAS mutant NSCLC cell lines including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). Subsequent in vivo studies showed that the combination treatment with LY3214996 and abemaciclib was well tolerated and led to more robust tumor growth inhibition or regression in all KRAS mutant NSCLC xenograft models (H2122, A549 and H441) compared with either single agent treatment (p≤0.002). Furthermore, in xenograft tumors the combination of LY3214996 and abemaciclib resulted in more significant reduction of phospho-p90RSK, phospho-Rb, phospho-S6 and Ki67 in H2122 tumors compared with either single agent. Overall, the combined inhibition of ERK1/2 and CDK4 and CDK6 was tolerated and enhanced antitumor efficacy in several KRAS mutant NSCLC preclinical models. These data support the feasibility of combining ERK inhibitor LY3214996 with CDK4 and CDK6 inhibitor abemaciclib as a promising strategy for the treatment of KRAS mutant NSCLC patients, and provides the rationale for the combination study in the on-going phase I LY3214996 clinic trial (NCT02857270). Citation Format: Wenjuan Wu, Shripad V. Bhagwat, Constance King, Susan Pratt, Xueqian Gong, Julie Stewart, Bonita Jones, Robert Flack, Richard Beckman, Beverly Falcon, Jason Manro, William T. McMillen, Ramon V. Tiu, Sheng-Bin Peng, Christoph Reinhard, Sajan Joseph, Sean Buchanan. Combination of a novel ERK1/2 inhibitor (LY3214996) with CDK4 and CDK6 inhibitor (abemaciclib) enhances antitumor efficacy in KRAS mutant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 317. doi:10.1158/1538-7445.AM2017-317