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Large Molecule Therapeutics

Improved Antitumor Activity of the Fluoropyrimidine Polymer CF10 in Preclinical Colorectal Cancer Models through Distinct Mechanistic and Pharmacologic Properties

William H. Gmeiner, Anthony Dominijanni, Alex O. Haber, Lais P. Ghiraldeli, David L. Caudell, Ralph D’Agostino Jr, Boris C. Pasche, Thomas L. Smith, Zhiyong Deng, Sezgin Kiren, Chinnadurai Mani, Komaraiah Palle and Jonathan R. Brody
DOI: 10.1158/1535-7163.MCT-20-0516 Published March 2021

Abstract

Chemotherapy regimens that include 5-fluorouracil (5-FU) are central to colorectal cancer treatment; however, risk/benefit concerns limit 5-FU’s use, necessitating development of improved fluoropyrimidine (FP) drugs. In our study, we evaluated a second-generation nanoscale FP polymer, CF10, for improved antitumor activity. CF10 was more potent than the prototype FP polymer F10 and much more potent than 5-FU in multiple colorectal cancer cell lines including HCT-116, LS174T, SW480, and T84D. CF10 displayed improved stability to exonuclease degradation relative to F10 and reduced susceptibility to thymidine antagonism due to extension of the polymer with arabinosyl cytidine. In colorectal cancer cells, CF10 strongly inhibited thymidylate synthase (TS), induced Top1 cleavage complex formation and caused replication stress, while similar concentrations of 5-FU were ineffective. CF10 was well tolerated in vivo and invoked a reduced inflammatory response relative to 5-FU. Blood chemistry parameters in CF10-treated mice were within normal limits. In vivo, CF10 displayed antitumor activity in several colorectal cancer flank tumor models including HCT-116, HT-29, and CT-26. CF10’s antitumor activity was associated with increased plasma levels of FP deoxynucleotide metabolites relative to 5-FU. CF10 significantly reduced tumor growth and improved survival (84.5 days vs. 32 days; P < 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer model that spontaneously metastasized to liver. Improved survival in the orthotopic model correlated with localization of a fluorescent CF10 conjugate to tumor. Together, our preclinical data support an early-phase clinical trial of CF10 for treatment of colorectal cancer.

Introduction

Colorectal cancer is a leading cause of cancer-related mortality (1). 5-fluorouracil (5-FU)–based regimens were first shown to provide a survival benefit for postoperative adjuvant chemotherapy in 1988 (2), and have been used since to reduce risk for disease recurrence and improve survival. The antitumor activity of fluoropyrimidines (FP) results primarily from thymidylate synthase (TS) inhibition (3) by the deoxynucleotide FdUMP. However, 5-FU is inefficiently metabolized to FdUMP, and generates relatively higher levels of ribonucleotide metabolites (e.g., FUTP; ref. 4) that cause systemic toxicities as evidenced by reversal of gastrointestinal (GI) tract (5) and bone marrow toxicities (6) with uridine. 5-FU–related toxicities may be lethal (7), particularly in patients with cancer with deficiencies in pyrimidine catabolism due to naturally occurring polymorphisms in DPYD (8) and other genes. Risk/benefit considerations limit the applicability of 5-FU–based regimens for patients with stage II colon cancer (9), and dose reduction due to toxicity considerations limit 5-FU efficacy for patients with advanced disease.

Significant research effort continues to focus on enhancing the antineoplastic activity of FPs. Over the past decade, we demonstrated that a prototype FP polymer, F10 (10), displayed improved antitumor activity relative to 5-FU in multiple preclinical models (11–13) through a unique mechanism involving both TS inhibition and Top1 poisoning (10, 11). Optimal activity for FP polymers requires uptake of the intact polymer by malignant cells (12), followed by intracellular FdUMP release (14). However, premature F10 degradation likely limits antitumor activity. The second-generation FP polymer, CF10, includes chemical modifications to both reduce exonucleolytic degradation and enhance activity. In CF10, arabinosyl cytidine (AraC), a nucleoside analog with potent anticancer activity (15), and polyethylene glycol (PEG5; ref. 16), extend the F10 polymer at the 3′- and 5′-termini. AraC is a chain-terminating nucleotide analog relatively resistant to exonuclease removal, but removed from DNA via a Tdp1-mediated mechanism (17, 18). We predicted that enzymes that degraded ssDNA would not recognize AraC and, as a result, CF10 would display improved nuclease stability yet eventual AraC release would improve CF10 cytotoxicity to colorectal cancer cells relative to F10.

In this study, we demonstrated improved potency for CF10 relative to F10 and 5-FU in multiple colorectal cancer cell lines that correlated with reduced thymidine antagonism and improved stability to nuclease degradation, consistent with AraC directly contributing to CF10’s cytotoxicity and limiting its nuclease degradation. Mechanistically, both F10 (10) and AraC (19) induce Top1-mediated DNA damage and our studies showed CF10-induced greater Top1 cleavage complex (Top1cc) than F10. CF10 displayed reduced systemic toxicity relative to 5-FU and its antitumor activity was established in multiple colorectal cancer flank tumor models in mice, which correlated with increased and sustained FdU and FdUMP levels relative to 5-FU. CF10 displayed improved antitumor activity relative to 5-FU in an orthotopic tumor model and that correlated with its improved tumor localization relative to a free dye. Our results are consistent with a distinct cytotoxic mechanism for CF10 relative to F10 and 5-FU in colorectal cancer cells and distinct pharmacology that may increase FP exposure in GI malignancies resulting in strong antitumor activity. Our findings are consistent with CF10 having potential to improve outcomes for patients with colorectal cancer through its distinct mechanistic and pharmacologic properties.

Materials and Methods

Cell lines and reagents

HCT116, LS174T, SW480, and T84 colorectal cancer cells and HIEC-6 intestinal cells were from ATCC and were cultured using recommended media, validated by short tandem repeat analysis, and regularly confirmed negative for Mycoplasma. CF10 and F10 (ST Pharm Co.) were validated by high-resolution mass spectrometry (Supplementary Fig. S1), and were dissolved in 0.9% sterile saline (Sigma). CF10 and F10 concentrations were determined from A260 UV absorbance using extinction coefficient for ssDNA. Stability of CF10 and F10 to exonuclease digestion with snake venom phosphodiesterase (SVPD) was determined as described previously (20). Clinical-grade 5-FU (50 mg/mL) was purchased from the Baptist Hospital clinical pharmacy. All drugs were filtered using a 0.22-μm filter before injection.

Cell viability and synergy analysis

Cell viability was determined using CellTiter-Glo (Promega) according to the manufacturer’s instructions. To determine drug interactions, cells were treated simultaneously with both agents (e.g., F10+thymidine) in a 5 × 9 matrix of concentrations chosen to encompass biologically relevant doses, as indicated in the Figures. Combination synergy was determined by Bliss independence analyses via the Combenefit program (21). The difference between the Bliss expectation and the observed growth inhibition of the combination is the Delta.Bliss, and these values were subject to median analysis using Graphpad PRISM.

Clonogenic assay

A total of 1,000–5,000 cells per well were plated in 6-well plates and treated with serial dilutions of drugs for 10 days with media changed at day 5. Cells were washed with PBS, fixed with 80% methanol, and stained with 0.05% Crystal Violet (Thermo Fisher Scientific). Imaging of colonies was done with the Bio-Rad Chemidoc MP on the Coomassie Blue setting with quantification via the ImageJ (NIH, Bethesda, MD) plugin Colony Area (22), and analyzed using Graphpad PRISM.

TS activity

TS catalytic assays were performed using well-established procedures (23) with freshly prepared 5,10 methylene tetrahydrofolate in 0.5 mol/L NaOH (Schircks Laboratories), 10 μmol/L dUMP, 200,000 dpm of 3H-dUMP (Moravek Biochemicals), 100 μmol/L 2-mercaptoethanol, and 25 mmol/L KH2PO4, pH 7.4. Reactions were quantified by scintillation counting.

DNA fiber assay and high-throughput alkaline comet assay

Cells were treated with either CF10, 5-FU, or DMSO for 16 hours and analyzed for replication dynamics using DNA fiber assay, as described previously (24). Experiments were performed in triplicate and mean values were presented with statistical significance. For Comet assays, colorectal cancer cells were treated with CF10, 5-FU, or DMSO for 24 hours. After treatment, cells were trypsinized and analyzed for percent of DNA tail (DNA damage) using 96-well comet chip system as described by the manufacturer (Trevigen).

Immunofluorescence

Immunofluorescence (IF) detection of pH2AX was performed in drug-treated colorectal cancer cells that were fixed and permeabilized using 100% cold methanol and then processed as described previously (24). Top1cc were detected using a primary antibody specific for the cleavage complex (Millipore MABE 1084 and gift of S. Kaufmann) using procedures similar to those described previously (25). Top1cc foci were captured using a Nikon A1R confocal microscope and positive cells were counted via ImageJ with >50 foci considered positive. Top1cc were also detected with the same antibody using a RADAR assay using methods described previously (26).

Western blotting

Proteins were isolated and their differential expressions were analyzed by Western blot analysis as described previously (24). Briefly, cells were lysed in ice-cold cytoskeletal buffer freshly supplemented with protease and phosphatase inhibitors (Roche). Concentration of the proteins were quantified using Bradford assay (Bio-Rad), and samples were normalized for equal loading. Samples were then heated at 100°C for 10 minutes in the presence of 6× Laemmli buffer (Boston BioProducts). Proteins were resolved by SDS-PAGE and their expression was analyzed by immunoblotting using specific antibodies.

Colorectal tumor models

Mice were housed and subjected to experiments in accordance with the protocols approved by the Institutional Animal Care and Use Committee at Wake Forest School of Medicine (Winston-Salem, NC). For flank tumor formation, colorectal cancer cells (3 × 106 cells) suspended in Matrigel:PBS were injected into the flanks of BALB/c nude mice (for HCT-116 and HT-29) or BALB/c mice (for CT-26; n = 3). Tumor growth and mice health were monitored daily. For orthotopic tumor formation, protocol was followed per Tseng and colleagues previous study (27). First, flank tumors were formed by subcutaneously injecting HCT116-luc cells and monitored until the tumor reached a volume large enough for transplantation, mice were euthanized, and tumor was removed and divided into 2- to 3-mm pieces. A laparotomy was performed in recipient mice to expose the cecum. A piece of tumor tissue was then attached to the outside of the cecum, the cecum was then returned to the cavity and the abdominal wall closed. Tumor volume and cell progression were monitored using an IVIS Lumina LT Series III and evaluated total photon flux. Pierce d-Luciferin (Thermo Fisher Scientific) was injected intraperitoneally and mice were transferred to the IVIS stage and imaged under 2% isoflurane anesthesia. All images were analyzed using Living Image software using an identical region of interest for each mouse. Two independent studies were performed with identical procedures except in the second study 5-FU (n = 9) was dosed 1×/week at 30 mg/kg (28) rather than 2×/week every other week at 70 mg/kg (29) because serious GI-tract toxicity was detected in the first study. CF10 was dosed 2×/week every week at 300 mg/kg (n = 8 in study 1; n = 2 in study 2). All drugs or vehicle (0.9% saline; n = 8 in study 1; n = 6 in study 2) were administered by intraperitoneal injection in 200-μL volume. Mice were removed from the study upon weight loss >20% of initial body weight or when deemed moribund due to tumor burden by veterinary staff.

Tumor localization, blood plasma levels, and blood chemistry

Tumor localization studies used Balb/c nude mice with orthotopic HCT-116-luc tumors. A modified CF10 (XF10A) was synthesized in which PEG5 at the 5′-terminus was replaced with a modified PEG5 containing a terminal alkyne. The terminal alkyne of XF10A was coupled to a fluorescent dye (Cy5.5-azide; Lumiprobe) via azide:alkyne Cu2+-catalyzed Click chemistry. The fluorescent CF10 analog (FL_CF10) was characterized by liquid chromatography with diode array detection at 680 and 260 nm and by LTQ Orbitrap XL mass spectrometry analysis. A total of 1 nmol of either the Cy 5.5 CF10 conjugate or the free dye were injected via tail vein (n = 3/group). Twenty-four hours after injection, mice underwent fluorescence imaging to detect Cy5.5 after which mice were euthanized and liver, tumor, and other organs were imaged ex vivo. Images were analyzed using Living Image software. To determine blood plasma levels of 5-FU, CF10, FdU, FdUMP, and other FP metabolites, BALB/c nude mice were injected with CF10 (300 mg/kg) or 5-FU (70 mg/kg) and groups of n = 3 at each time point (t = 0, 30 minutes, 1 hour, and 2 hours after treatment) mice underwent cardiac puncture under deep isoflurane anesthesia to collect blood samples. Plasma from the collected blood was analyzed by the mass spectrometry and pharmacokinetics shared resource at Wake Forest School of Medicine (Winston-Salem, NC) to quantify plasma levels of FPs and FP metabolites. Blood chemistry was analyzed in groups of n = 5 C57bl/6 mice with no treatment, 5-FU (70 mg/kg) or CF10 (300 mg/kg) 2×/week. Twenty-four hours after the second treatment, blood was collected by cardiac puncture under deep isoflurane anesthesia. A portion of the blood was used for complete blood count and plasma from a second portion was used for a complete metabolic panel. Blood testing was performed by IDEXX.

Statistical analysis

All statistical analyses were conducted using SAS 9.4 or GraphPad Prism. For comparing the tumor growth rates and changes in flux, a mixed model approach was used. Flux values were adjusted for the baseline by dividing observed flux by baseline flux from IVIS imaging. The model included terms for group, time, and group-by-time interactions as fixed effects. The animals were included as random effects to account for the within-animal correlation. Differences in survival times among treatment groups were examined using a Kaplan–Meier analysis and the survival curves were tested for differences using log-rank tests.

Histology analysis

Tissues from select mice from all treatment groups were removed from animals after undergoing euthanasia by CO2 asphyxiation and cervical dislocation. In some instances, mice were injected with luciferin approximately 20 minutes prior to euthanasia to permit IVIS imaging of extracted tissue. Tumors were weighed after removal and in instances where there was suspicion of possible metastases to liver, lung, or other organs tissue from these organs was removed and if luciferin was administered prior to euthanasia, these tissues were imaged using the IVIS system. All tissues were fixed in 10% neutral buffered formalin for 24 hours followed by transfer to 70% ethanol. Following routine processing in xylene, tissues were sectioned at 4 μm and stained with hematoxylin and eosin and evaluated by light microscopy by a veterinary pathologist.

Results

CF10 displays enhanced potency to colorectal cancer cells

First, we evaluated whether CF10 (Fig. 1A; Supplementary Fig. S1) displayed improved stability to exonuclease digestion relative to F10. AraC is not readily recognized by the proofreading activities of DNA polymerases and synthetic DNA extended with AraC may display reduced susceptibility to exonuclease degradation. We treated CF10 and F10 with SVPD for 0–16 hours, and evaluated the relative stabilities of the two FP polymers by gel electrophoresis and mass spectrometry. SVPD is a model nuclease with predominantly 3′-O-exonuclease activity (20). CF10 remained predominantly intact 1 hour after treatment (Fig. 1B) while F10 was degraded to shorter multimers (Fig. 1C). Furthermore, some full-length CF10 remained detectable 16 hours after treatment (Supplementary Fig. S2).

Figure 1.
Figure 1.

Chemical modification to CF10 increases stability to exonuclease degradation and improves potency toward colorectal cancer cells. A, Scheme depicting CF10's cytotoxic mechanism. AraC extension of the FP polymer reduces nuclease digestion but eventual release of FdUMP inhibits TS and incorporation of FdUTP and AraCTP into DNA causes Top1cc and replication stress. Snake venom phosphodiesterase hydrolysis of CF10 (B) and F10 (C) 1 hour posttreatment. Summary of clonogenic assays evaluating CF10, F10, and 5-FU in HCT-116 (D) and LS174T (E) cells.

We next evaluated whether CF10 was more effective than 5-FU and alternative FPs to colorectal cancer cells. CF10 was more potent than F10 and much more potent than 5-FU at reducing the clonogenic potential of HCT-116 (Fig. 1D), LS174T (Fig. 1E), T84, and SW480 colorectal cells (Supplementary Fig. S3). The potency advantage for CF10 relative to 5-FU far exceeded the 10-fold increased FP content as shown in Supplementary Table S1, which includes a summary of IC50 values based on the clonogenic assay results. A CF10 sample was also provided to the NCI developmental therapeutics program for testing in the 60-cell line screen. The NCI data confirmed CF10 (NSC 787877) displayed enhanced growth inhibitory properties relative to F10 (NSC S697912) toward all colorectal cancer cell lines in which both agents were tested. CF10 also was more potent than several FP drugs used clinically for colorectal cancer treatment including FdU (NSC 27640), 5-FU (NSC 19893), and trifluorothymidine (NSC 75520). A summary of the mean GI50 values (concentration required to inhibit growth by 50%) for CF10, F10, FdU, 5-FU, and trifluorothymidine from the NCI 60 cell line screen are included in Supplementary Table S2. CF10 was potent in all colorectal cancer cells regardless of the microsatellite instability/microsatellite stable status or KRAS mutation status, which are important factors determining whether patients with colorectal cancer are treated with FP drugs (30).

CF10 TS/Top1 dual targeting with contribution from AraC

TS is the established target of FP drugs and we previously showed FP polymers were cytotoxic through dual targeting of TS and Top1 (11). To assess TS inhibition by CF10, we detected TS ternary complex by Western blot analysis, together with the corresponding depletion of unbound TS (Fig. 2A). In HCT-116 cells, CF10 concentrations that inhibited colorectal cancer clonogenic survival resulted in TS detection almost exclusively as the ternary complex (Fig. 2A; Supplementary Fig. S4). F10, which has the same FP content as CF10, displayed similar TS ternary complex formation. In contrast, 5-FU, also at 10 nmol/L, did not induce detectable TS ternary complex formation (31), although at much higher 5-FU concentrations (10,000 nmol/L) TS ternary complex formation was evident (Fig. 2A). Similar results were obtained in CF10-treated and 5-FU–treated LS174T cells (Supplementary Fig. S4). We also evaluated changes in TS catalytic activity using a 3H-release assay (Fig. 2B). CF10 reduced TS catalytic activity at much lower concentrations than 5-FU, and induced a more sustained decrease in TS catalytic activity. Results are consistent with potent TS inhibition being important for CF10’s cytotoxicity to colorectal cancer cells. To gain further insight into the importance of TS inhibition for CF10 cytotoxicity, we cotreated colorectal cancer cells with CF10 ± thymidine, and evaluated drug interaction by Bliss synergy analysis. Identical studies were performed with F10 ± thymidine. Thymidine strongly antagonized F10 in both HCT-116 (Fig. 2C) and LS-174T cells (Supplementary Fig. S5) with antagonism ≥ 30 for 14/45 (HCT-116; median, −23 ± 3.6) and 22/45 (LS-174T; median, −26 ± 2.0) of F10+thymidine combinations tested. However, thymidine antagonism was reduced for CF10 relative to F10 with antagonism ≥ 30 for only 5/45 (HCT-116; median, −11 ± 2.5) (Fig. 2D) and 17/45 (LS-174T; median, −25 ± 1.7) CF10 + thymidine combinations tested. Decreased thymidine antagonism for CF10 was particularly evident in HCT-116 cells that were relatively more sensitive to CF10 compared with F10 (Fig. 1D and E). Our results are consistent with CF10 activating a thymidine-independent cell death mechanism, the extent of which is cell line dependent.

Figure 2.
Figure 2.

CF10 strongly inhibits TS but is less sensitive to thymidine antagonism than F10. A, Western blot analysis detecting TS ternary complex (TS CC) in HCT-116 cells 24 hours after treatment with the indicated treatment. B, TS activity assay showing reduced TS catalytic activity following CF10 treatment. Bliss synergy analysis showing deviation from expected activity based on strict additivity for the indicated concentrations of thymidine and F10 (C) and CF10 (D). E, IF data showing Top1cc in HCT-116 cells following the indicated treatments.

The enhanced cytotoxicity of CF10 relative to F10 could result from AraC directly contributing to CF10 cytotoxicity. We tested AraC in combination with F10 using Bliss synergy analysis. In HCT-116 cells, AraC was active as a single agent at concentrations that would be released from IC50 values of CF10. Furthermore, the F10+AraC combination was approximately additive with 21 of 45 of the concentration pairs evaluated displaying combination values of 0 ± 5 relative to the single agents (median, −7 ± 2.7; Supplementary Fig. S6). LS174T cells also were sensitive to AraC at concentrations that would be released from IC50 values of CF10. However, in LS174T cells, the AraC+F10 combination displayed uniform mild antagonism (Supplementary Fig. S6), a result consistent with CF10’s reduced potency advantage relative to F10 in LS174T cells (Fig. 1E). To gain further insight into AraC directly contributing to CF10 cytotoxicity, COMPARE analysis (http://dtp.nci.nih.gov; ref. 32) was conducted to identify mechanistically similar compounds. The compounds with the highest Pearson correlation coefficients (PCC), indicating close mechanistic similarities to CF10, are shown in Supplementary Table S3. CF10 was chosen as the seed and by definition has a Pearson correlation coefficient of 1. The compounds most closely correlated to CF10 included FdU and AraC ranked No. 1 and 3. However, the PCC values were only 0.642 and 0.596 indicating considerable mechanistic differences between CF10 and its constituent nucleosides. Topotecan ranked No. 5, consistent with mechanistic similarities between CF10 and Top1 poisons, as previously established for F10 (10, 11). AraC also induces Top1cc formation if stably misincorporated into DNA (19). To determine whether CF10 induced Top1cc formation in colorectal cancer cells, we performed IF studies with an mAb specific for Top1cc (25). CF10 and F10 concentrations that strongly inhibited colorectal cancer clonal survival also caused Top1cc (Fig. 2E; Supplementary Fig. S7) with data quantification indicating CF10 was relatively more effective than F10 (Supplementary Fig. S7). Collectively, our studies indicated that AraC may directly contribute to the enhanced cytotoxicity of CF10 relative to F10 in some colorectal cancer cells, although AraC’s effects on CF10 polymer stability may be more important, particularly in vivo (see below).

CF10 activates the DNA damage response

CF10 strongly inhibits TS (Fig. 2A) and also traps Top1cc at sites of FdU misincorporation into DNA (Fig. 2E), and may generate DNA damage by multiple mechanisms including collisions between advancing replication forks or active transcription complexes with trapped Top1cc (33). Trapped Top1cc at sites of FdU substitution may be particularly deleterious because repair proceeds under thymidine-depleted conditions resulting in FdUTP reincorporation into DNA during repair, potentially amplifying DNA damage through futile repair. We analyzed the time-dependent activation of the DNA damage response and demonstrated that 48 hours after treatment, CF10 increased monoubiquitination of FANCD2 and pChk1 (S317) levels relative to F10 and 5-FU (Fig. 3A; Supplementary Fig. S8), consistent with increased stalled replication forks, but with a delay potentially due to reduced nuclease susceptibility of CF10. DNA fiber analysis confirmed CF10-induced stalled replication forks (Supplementary Fig. S8). CF10 also increased pChk2 (T68), pH2AX, and Rad51 levels consistent with conversion of stalled forks to DNA double-strand breaks (DSB), which we validated by pH2AX IF (Fig. 3B and C) and COMET assays (Fig. 3D and E).

Figure 3.
Figure 3.

CF10 stimulates prolonged activation of the DNA damage response and induces formation of DNA DSB. A, Western blot analysis for markers of stalled replication forks (FANCD2, pChk1-S317) and DNA DSBs (pChk2-T68, Rad51, pH2AX) 24 or 48 hours with the indicated treatment in HCT-116 cells. B, IF imaging of pH2AX in HCT-116 cells following treatment with CF10 or 5-FU at 500 nmol/L. C, Quantification of IF data in B. D, Results of alkaline COMET assay following treatment with CF10 or 5-FU at 500 nmol/L. E, Quantification of data in D.

CF10 is well tolerated and active in vivo

Our previous studies demonstrated improved antitumor activity for F10 relative to 5-FU in multiple preclinical models (11–13), even with reduced systemic toxicities. To determine whether CF10 differed from 5-FU in selectivity for malignant versus nonmalignant cells, we compared CF10 and 5-FU effects in HIEC-6 intestinal cells and HCT-116 colorectal cancer cells. Consistent with our clonogenic data (Fig. 1; Supplementary Tables S1 and S2), CF10 was much more than 10-fold more potent than 5-FU to HCT-116 cells; however, CF10 (10−6 mol/L) reduced the viability of HIEC-6 cells relatively less than 5-FU (10−5 mol/L) at equivalent total FP concentrations (Fig. 4A). We next evaluated the effects of CF10 and 5-FU treatment in vivo to GI-tract tissue in BALB/c mice. Using a previously established dose for 5-FU (29), we observed a single treatment resulted in scattered neutrophils and expanded submucosa consistent with drug-induced inflammation. Conversely, a dose of CF10 that delivered relatively greater FP content had less damaging effects (Fig. 4B). Quantification of crypt and villi lengths in the small intestine revealed CF10 did not significantly decrease average villus length, but 5-FU did (Supplementary Fig. S9). To evaluate potential CF10 toxicity to liver and kidney, we analyzed blood chemistry. Values for alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine following CF10 treatment as dosed in efficacy studies (Fig. 5) were similar to control (Supplementary Table S4). While CF10 displayed minimal toxicity to normal tissues, it displayed significant antitumor activity at the doses tested in toxicity studies in three colorectal cancer flank xenograft models: HCT-116, HT-29, and CT-26 (Fig. 4C–F). In all three models, CF10 displayed significant antitumor activity and treatment resulted in no signs of systemic toxicities.

Figure 4.
Figure 4.

CF10 displays reduced toxicity to nonmalignant cells and tissue and antitumor activity in colorectal cancer flank tumor models. A, Effects of CF10 (10-6M), 5-FU (10-5M), and oxaliplatin (10-5M) in HIEC-6 intestinal cells and HCT-116 colorectal cancer cells with 72 hours treatment and viability determined using CellTiter-Glo (Promega). The doses of CF10 and 5-FU have equivalent FP content. B, Hematoxylin and eosin–stained sections from BALB/c mice 24 hours after treatment with (A and B)—vehicle—(4×), note normal colon and small intestine epithelium. C and D, CF10 300 mg/kg (20×). Minimal to scant nuclear debris in colonic epithelium (yellow arrow) and small intestine crypts have scattered to minimal nuclear debris (apoptosis). E and F, 5-FU 70 mg/kg (20×) colon with scattered neutrophils (yellow arrows) and expanded submucosa (green arrow; e.g., edema). Small intestine with increased crypt apoptosis and scattered neutrophils. C–E, Growth rates for HCT-116 (C), HT-29 (D), and CT-26 (E) flank tumors showing CF10 (300 mg/kg; 2×/week) significantly reduced tumor growth. F, CF10 increased survival in a CT-26 flank tumor model.

Figure 5.
Figure 5.

CF10 treatment (300 mg/kg 2×/week for 8 weeks) inhibits tumor growth in an orthotopic model and provides a significant survival advantage relative to vehicle and 5-FU treatment (70 mg/kg 2×/week, every other week). A, Graph of mouse weights for control (blue), CF10 (red), and 5-FU (black) treatment groups. B, Graph of flux from HCT-116-luc orthotopic xenografts. Differences in the rate of change in flux among the groups were not significant during the first 3 weeks of treatment; however, the groups then diverged, and flux rate was significantly lower in CF10-treated mice (red) relative to vehicle (blue) or 5-FU (black)–treated mice. C, Kaplan–Meier plot showing a survival advantage for CF10 (red) relative to vehicle (blue) and 5-FU (black). Differences in survival were significant based on log-rank test (χ2 19.8, two degrees of freedom P < 0.0001). D, Representative IVIS images for mice after 5 weeks of the indicated treatment. E, Quantification of IVIS images from D.

We next evaluated CF10’s efficacy in an orthotopic human colorectal cancer tumor model using HCT-116-luc cells. Mice treated with CF10 (2×/week; 300 mg/kg) displayed stable weights and on average gained weight during a substantial portion of the study with treatment extending to 8 weeks (Fig. 5A). Mice treated with CF10 also remained active throughout the study with no signs of drug-related toxicity. Analysis of tumor growth rates showed the CF10 group had significantly reduced tumor growth than either 5-FU or vehicle (P = 0.014). A summary of tumor growth rates for the three treatment groups is included in Fig. 5B, and representative IVIS images and a summary of the week 5 flux values is presented in Fig. 5D and E. We examined whether differences in tumor growth among groups were more pronounced in the early (first 3 weeks) or later (3–6 week) time periods, but did not find evidence of this occurring. CF10 treatment also resulted in markedly prolonged survival in the HCT-116-luc orthotopic model (median, 84.5 days) relative to vehicle control (median, 33 days). When we examined survival times among groups, we found a highly significant difference based on the log-rank test (χ2 19.8, two degrees of freedom P < 0.0001)—with the CF10 animals having much longer survival (median, 84.5 days) than control or 5-FU (33 days and 32 days, respectively). Mice were removed from the study upon excessive weight loss or when deemed moribund due to tumor burden by Veterinary staff. A Kaplan–Meier presentation of the survival data is displayed in Fig. 5C. CF10 also trended toward improved survival relative to F10 (Supplementary Fig. S10). Our results demonstrate CF10 increases survival in an orthotopic model of HCT-116-luc CRC, whereas 5-FU had no significant effect on survival in this model. To determine whether the high tumor burden for mice in the 5-FU and vehicle treatment groups was associated with metastases, select mice that underwent euthanasia from morbidities associated with large tumor burden (i.e., lethargy, hunched posture) at week 5 were subjected to autopsy following euthanasia. Liver, lung, kidney, and other tissues were inspected for gross morphologic features consistent with metastases. Putative metastatic tissue was evident in two of four of 5-FU–treated mice and in three of five of the vehicle-treated group. One otherwise viable CF10-treated mouse was euthanized at week 8 and extensive analysis of liver tissue from autopsy revealed no signs of liver metastasis (Supplementary Fig. S11). The data indicate the HCT-116 orthotopic tumor model develops spontaneous metastases and are consistent with CF10 significantly reducing tumor burden and potentially reducing metastatic progression.

CF10 in vivo metabolism and targeting

To further investigate the pharmacologic basis for CF10’s improved antitumor activity relative to 5-FU in the orthotopic tumor model, we quantified FP metabolites from mouse plasma following intravenous CF10 or 5-FU injection (Fig. 6A), and evaluated CF10’s biodistribution using a fluorescent conjugate (Fig. 6B; Supplementary Fig. S12). Consistent with previous reports of 5-FU’s rapid plasma clearance, 5-FU plasma concentration rapidly decreased to <2% of initial values 30 minutes after treatment and was undetected at longer time points. Previous studies with phosphodiester oligonucleotides (ODN) of similar chemical composition as CF10 showed rapid plasma clearance via active transport into parenchymal and nonparenchymal liver tissue (34) followed by MRP2-dependent biliary secretion (35). We detected CF10 (Supplementary Fig. S13A) and shorter multimers (CF9, CF8, etc.; Supplementary Fig. S13B) immediately following injection, but not at later time points. However, FdUMP and FdU plasma concentrations were sustained over >1 hour following CF10 injection (Fig. 6), whereas these were barely detectable following 5-FU treatment (Fig. 6). To gain further insight into CF10’s biodistribution, we prepared a fluorescent conjugate of CF10 and performed IVIS imaging following intravenous injection in which HCT-116-luc orthotopic colorectal cancer tumors were established previously (Fig. 6B). Twenty-four hours after injection, FL_CF10 differed from the free dye in biodistribution (Fig. 6B). To further analyze CF10’s biodistribution, we euthanized the mice, rapidly extracted tissues, and performed ex vivo imaging of tumor and liver from all mice (Fig. 6B). Strong tumor localization was evident for FL_CF10 while the free dye localized primarily in liver. Data quantification demonstrated an increased tumor/liver ratio for FL_CF10 relative to free dye (Fig. 6B). Our results are consistent with CF10 localizing to orthotopic colon tumors following intravenous injection, which may occur in part through hepatobiliary clearance and contribute to CF10’s strong activity toward orthotopic colon tumors.

Figure 6.
Figure 6.

CF10 results in sustained plasma concentrations of FP deoxynucleosides (FdUMP and FdU) and localizes to orthotopic colorectal cancer tumors. A, Plasma concentrations of 5-FU (black), FdU (blue), and FdUMP (red) determined by LC/MS following tail vein injection of 5-FU (left) and CF10 (right). B, A fluorescent conjugate of CF10 localizes to orthotopic colon tumors, in vivo. Mice with preestablished orthotopic HCT-116 tumors were injected with either FL_CF10 or free dye and underwent near-infrared imaging 24 hours after injection. Ex vivo imaging of tumor (top row) and liver (bottom row) from mice treated with FL_CF10 (top) or free dye (bottom).

Discussion

FP drugs remain central to colorectal cancer treatment (3) and 5-FU, the most widely used FP, is used to treat >2 million patients with cancer each year (36). Despite decades of optimizing its schedule and modulating activity by cotreatment with leucovorin and other agents (37, 38), the narrow therapeutic index of 5-FU remains a major obstacle to improving outcomes with FP drugs, particularly for patients with metastatic colorectal cancer (30, 39). In this study, we highlighted the improved activity of a novel second-generation FP polymer, CF10 relative to both 5-FU and F10, a prototype FP polymer with established anticancer activity in multiple preclinical models. We demonstrated that the chemical modifications that differentiate CF10 from F10, AraC extension and PEG5 conjugation (Fig. 1A; Supplementary Fig. S1), improved both CF10’s stability to enzymatic degradation (Fig. 1B and C) and its potency relative to FP drugs used to treat colorectal cancer, including 5-FU (Fig. 1D and E), FdU, and trifluorothymidine (Supplementary Tables S1 and S2). Importantly, CF10 displayed strong antitumor activity, which we also demonstrated in vivo with flank (Fig. 4) and orthotopic tumor xenografts (Fig. 5).

CF10 was chemically modified from the prototype FP polymer F10 by extension at the termini with AraC and PEG5. AraC is a chain-terminating nucleoside analog that is not readily removed by proofreading activities of DNA polymerases and we reasoned that extending the 3′-terminus of F10 would decrease susceptibility to enzymatic hydrolysis and potentially enhance antitumor activity because AraC is cytotoxic to malignant cells. Consistent with this goal, our findings established that CF10 is stabilized to exonuclease degradation (Fig. 1B and C) and more potent than F10 and much more potent than 5-FU in multiple colorectal cancer cell lines (Supplementary Tables S1 and S2). Our data also support a direct contribution of AraC release to CF10 cytotoxicity as concentrations of AraC that would be released from effective concentrations of CF10 displayed single-agent activity in colorectal cancer cells (Supplementary Fig. S6). Furthermore, the increased potency of CF10 relative to F10 correlates with decreased thymidine antagonism (Fig. 2C and D) consistent with a non-FP component to cytotoxicity. Hence, CF10’s cytotoxicity advantage relative to F10 results in part, from AraC both decreasing susceptibility to nuclease degradation and a direct cytotoxic component resulting from AraC release.

Mechanistically, CF10’s substantial potency advantage relative to 5-FU in colorectal cancer cells is associated with increased replication stress. Malignant cells experience high levels of replication stress (40), and drugs enhancing replication stress display strong anticancer activity (41, 42). We demonstrated that CF10, but not 5-FU, significantly reduced replication fork velocity and increased terminal replication forks (Supplementary Fig. S8), consistent with increased replication stress. The causes of replication stress are diverse and include deoxynucleotide depletion and imbalance that result from TS inhibition and Top1cc formation. CF10 strongly inhibits TS as evidenced by TS ternary complex formation (Fig. 2A). A source of CF10-induced replication stress in addition to thymidine depletion is Top1cc formation. Top1 is the sole target for topotecan, and Top1 is also poisoned by FdU (10) and AraC (19) misincorporation into DNA. In these studies, we demonstrated using IF that CF10 induced significantly greater Top1cc in colorectal cancer cells than F10 (Fig. 2E).

5-FU causes serious GI and hematologic toxicities in many patients, particularly patients deficient in pyrimidine catabolism due to polymorphisms in DPYD (8). In these studies, 5-FU was administered at a previously established MTD (70 mg/kg, 2×/week every other week; ref. 29), and even a single dose at this concentration resulted in an inflammatory response to the GI tract as evidenced by increased neutrophils and edema (Fig. 4B). Clinically, both the GI and hematologic toxicities of 5-FU are reversed with uridine consistent with an RNA-directed origin. FP polymers, in principle, are converted to deoxyribonucleotides, which we confirmed both by enzymatic hydrolysis ex vivo (Fig. 1B and C; Supplementary Fig. S2) and by analyzing plasma metabolite levels in vivo (Fig. 6A; Supplementary Fig. S13). Because of reduced RNA-mediated effects, CF10 causes less GI tract damage than equivalent 5-FU concentrations (Supplementary Fig. S9) enabling higher CF10 doses to be tolerated. Evaluation of blood chemistry at these higher doses did not reveal evidence for kidney or liver toxicity (Supplementary Table S4), and observational studies revealed these higher doses to be tolerated even with extensive, multiweek treatment.

The ability to dose CF10 at higher levels than 5-FU without causing serious systemic toxicities likely contributes to CF10’s improved antitumor activity relative to 5-FU, but other factors are important. CF10 treatment resulted in increased FdU and FdUMP plasma levels relative to 5-FU (Fig. 6A), and these metabolites are important for antitumor activity. Furthermore, with CF10, we observed greater selectivity for malignant (i.e., HCT-116) relative to nonmalignant cells (i.e., HIEC-6) compared with 5-FU (Fig. 4A), which could result from increased reliance of malignant cells on de novo thymidine biosynthesis. Finally, a CF10 fluorescent conjugate displayed an altered biodistribution relative to the unconjugated dye and strong tumor uptake was evident for the conjugate (Fig. 6B). Phosphodiester ODNs of similar chemical composition as CF10 undergo receptor-mediated uptake in liver (34) followed by MRP2-mediated biliary excretion (35). Similar routing of CF10 could be responsible for the improved tumor localization of the fluorescent CF10 conjugate relative to the free dye. Furthermore, hepatobiliary routing of CF10 could result in increased FP exposure in the GI tract contributing to improved antitumor activity (Fig. 5). Unlike purine metabolites that are degraded to uric acid before absorption, pyrimidines remain intact, and are readily absorbed from the intestine (43). Interestingly, the HCT-116 orthotopic tumor model used displayed evidence of metastatic progression for the vehicle and 5-FU treatment groups (Supplementary Fig. S11A and S11B). However, while animal numbers were limited, our data suggest CF10 treatment may impede metastatic progression (Supplementary Fig. S11C), although further studies are needed to conclusively test this. CF10 also displayed a trend toward improved survival relative to F10 (Supplementary Fig. S10), consistent with its improved potency relative to F10 toward colorectal cancer cells in cell culture.

Overall, our findings underscore that CF10 is more potent than F10 or current FP drugs and that the improved activity of CF10 relative to F10 results from AraC’s effects contributing to both polymer stability and cytotoxicity. The AraC component of CF10 likely is responsible for reduced thymidine antagonism relative to F10 (Fig. 2C and D), as well as increased potency to colorectal cancer cells (Supplementary Tables S1 and S2). CF10 is well tolerated in vivo (Fig. 4B) and increased tolerability together with enhanced selectivity to malignant cells (Fig. 4A), improved conversion to deoxynucleotide FP metabolites (Fig. 6A), and altered biodistribution (Fig. 6B), all likely contribute to CF10’s improved potency relative to 5-FU toward orthotopic colorectal cancer tumors (Fig. 5). Our findings support advanced preclinical testing of CF10 and initiation of clinical studies in colorectal cancer and other aggressive tumor types.

Authors’ Disclosures

W.H. Gmeiner reports grants from NIH during the conduct of the study, other from DeepCreek Pharma, LLC outside the submitted work and a patent for PCT/US2019/033902 pending. R. D’Agostino Jr. reports grants from NCI during the conduct of the study. J.R. Brody reports personal fees and other from Perthera outside the submitted work. No disclosures were reported by the other authors.

Authors' Contributions

W.H. Gmeiner: Conceptualization, supervision, funding acquisition, investigation, writing–original draft, writing–review and editing. A. Dominijanni: Investigation, writing–review and editing. A.O. Haber: Investigation, writing–review and editing. L.P. Ghiraldeli: Investigation. D.L. Caudell: Investigation, writing–review and editing. R. D’Agostino: Formal analysis, writing–review and editing. B. C. Pasche: Writing–review and editing. T.L. Smith: Investigation, writing–review and editing. Z. Deng: Investigation. S. Kiren: Investigation. C. Mani: Investigation, writing–review and editing. K. Palle: Investigation, writing–review and editing. J.R. Brody: Supervision, funding acquisition, investigation, writing–review and editing.

Acknowledgments

This study was performed with grant support from 1R21 CA218933 (to W.H. Gmeiner), 1R01CA212600-01 (to J.R. Brody), R01 CA212600 (to K. Palle), Weitlauf Endowment Funds (K. Palle), and R01CA219187 (K. Palle). This work was also supported, in part, by a NCI of the NIH under a Cancer Center Support Grant P30CA012197 (WFBHCCC) and P30CA056036 SKCC Core Grant (Thomas Jefferson University, Philadelphia, PA) and P30 CA069533 (Druker, OHSU). The authors wish to acknowledge the support of the Wake Forest Baptist Comprehensive Cancer Center Biostatistics Shared Resource, Cell and Viral Vector Shared Resource, and the Proteomics and Metabolomics Shared Resource and the WFU mass Spectrometry facility. The authors are grateful for technical assistance from B. Kuhlman, T. Bor, M. Manandhar, M. Regan, C. Tracy, E. Routh, and S. Rideout.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

  • Mol Cancer Ther 2021;20:553–63

  • Received June 19, 2020.
  • Revision received October 26, 2020.
  • Accepted December 16, 2020.
  • Published first December 23, 2020.

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Improved Antitumor Activity of the Fluoropyrimidine Polymer CF10 in Preclinical Colorectal Cancer Models through Distinct Mechanistic and Pharmacologic Properties
William H. Gmeiner, Anthony Dominijanni, Alex O. Haber, Lais P. Ghiraldeli, David L. Caudell, Ralph D’Agostino Jr, Boris C. Pasche, Thomas L. Smith, Zhiyong Deng, Sezgin Kiren, Chinnadurai Mani, Komaraiah Palle and Jonathan R. Brody
Mol Cancer Ther March 1 2021 (20) (3) 553-563; DOI: 10.1158/1535-7163.MCT-20-0516
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