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. Author manuscript; available in PMC: 2022 Apr 16.
Published in final edited form as: Mol Cancer Res. 2021 Feb 16;19(4):565–572. doi: 10.1158/1541-7786.MCR-20-0985

AraC-FdUMP[10] (CF10) is a next generation fluoropyrimidine with potent antitumor activity in PDAC and synergy with PARG inhibition

Alex O Haber 1,2, Aditi Jain 1,2, Chinnadurai Mani 7, Avinoam Nevler 1,2, Lebaron C Agostini 1,2, Talia Golan 5,6, Komaraiah Palle 7, Charles J Yeo 1,2, William H Gmeiner 3,, Jonathan R Brody 4,
PMCID: PMC9013283  NIHMSID: NIHMS1675438  PMID: 33593942

Abstract

CF10 is a 2nd generation polymeric fluoropyrimidine (FP) that targets both thymidylate synthase (TS), the target of 5-fluorouracil (5-FU), and DNA topoisomerase 1 (Top1), the target of irinotecan, two drugs that are key components of FOLFIRNOX, a standard of care regimen for pancreatic ductal adenocarcinoma (PDAC). We demonstrated that F10 and CF10 are potent inhibitors of PDAC cell survival (in multiple cell lines including patient derived lines) with IC50s in the nanomolar range and are nearly 1000-fold more potent than 5-FU. The increased potency of CF10 relative to 5-FU correlated with enhanced TS inhibition and strong Top1 cleavage complex formation. Further, CF10 displayed single agent activity in PDAC murine xenografts without inducing weight-loss. Through a focused drug synergy screen, we identified that combining CF10 with targeting the DNA repair enzyme, Poly (ADP-ribose) Glycohydrolase (PARG), induces substantial DNA damage and apoptosis. This work moves CF10 closer to a clinical trial for the treatment of PDAC.

Graphical Abstract

graphic file with name nihms-1675438-f0004.jpg

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the U.S., with a 5-year survival rate of 10% [1]. While thousands of PDAC genomes have been whole-exome sequenced and core pathways have been identified as targets [2, 3], our best current options to treat PDAC, besides surgical resection, remain with cytotoxic chemotherapies. Clinical phase III trials in the metastatic setting show improved survival with a combination of cytotoxic agents called FOLFIRINOX (leucovorin, 5-fluorouracil/5-FU, irinotecan, oxaliplatin) which has become a standard-of-care option, particularly in healthy, high performance status patients with metastatic PDAC [46]. Still, improvement of overall survival is limited to just a few months, all patients responding to treatment eventually succumb to the disease, and many experience detrimental side effects such as neutropenia and extreme fatigue, indicating an unmet need for novel therapeutic strategies.

While 5-FU is an integral component of FOLFIRINOX, the anti-cancer activity of this regimen is likely sub-optimal because 5-FU is inefficiently metabolized to FdUMP [7, 8], the thymidylate synthase (TS) inhibitory metabolite that is responsible for 5-FU’s anti-cancer activity. To overcome the limitations of 5-FU we are developing polymeric FPs (F10, CF10) that we anticipate being more efficiently converted to the TS-inhibitory metabolite FdUMP by virtue of their higher FP content per molecule of 5-FU. Another expected benefit of these molecules is that they may generate relatively lower levels of ribonucleotide metabolites that are responsible for 5-FU’s systemic toxicities that can become serious and life-threatening in some patients. FdUMP[10] (F10) dually targets TS and DNA topoisomerase 1 (Top1) [912], which are well-established targets in PDAC by virtue of their targeting by 5-FU and irinotecan in FOLFIRINOX. Additionally, F10 achieves strong anti-tumor activity while causing fewer systemic toxicities than 5-FU in multiple preclinical cancer models [11, 1315].

In this study, we investigate a promising 2nd generation polymeric FP AraC-FdUMP[10] (CF10), as a potential new treatment for PDAC. CF10 includes a non-native nucleotide (AraC) at the 3’-terminus which we believe will enhance core F10 stability by differential recognition by exonucleases [16] and anti-cancer activity thru independent mechanisms of action. CF10 also includes PEG5 at the 5’-terminus, a modification known to promote binding to plasma proteins and increase circulation of molecules in-vivo [17]. We demonstrate therapeutic advantages for CF10 in PDAC models relative to both 5-FU and the prototype FP polymer F10. Additionally, we found that CF10 could be synergistically combined with a small molecule inhibitor of the DNA repair factor Poly (ADP) Glycohydrolase, PARG, suggesting the potential of novel combination therapy regimens that synergistically target DNA repair.

Materials and Methods

Cell Line Culture Conditions

All cells lines were purchased from American Type Culture Collection (ATCC). They were authenticated by short tandem repeat profiling at least twice per year and confirmed negative for mycoplasma contamination at least once per month. Cells were cultured according to ATCC specifications with the addition of prophylactic plasmocin (InvivoGen). Patient derived cell lines (PDX) were obtained from Dr. Talia Golan and were cultured as previously described [18, 19]. For all experiments cell line passage number was kept below 20.

Chemical Compounds

F10 was synthesized and characterized as previously described [20, 21]. CF10 was synthesized and provided by Dr. William Gmeiner [22] and both F10 and CF10 were prepared in saline. PDDX-04 was provided by Dr. Joseph Salvino (The Wistar Institute, Philadelphia, PA), and was prepared in DMSO. A list of other commercially available compounds used in the study is supplied in Supplementary Table S1.

5-day Picogreen Survival Assay and Bliss Additivity Analyses Assay

1000–3000 cells/well were plated in 96-well plates. 5-day drug treatment of cells was performed in sextuplet the following day. Pico green assay was carried out as previously described [19, 23]. For Bliss additivity analyses, survival percentages were calculated and then input into the Combenefit program as previously described [19, 23, 24].

10-day Colony Growth Inhibition Assay

1000–5000 cells/well were plated in 6 well plates and drug treatments were carried out the following day. After 5 days, cell culture media was changed, and new drug was added for another 5 days. Visualization of colonies was performed as previously described [19, 23] and colony area coverage was quantified using the Colony Area plugin for ImageJ (RRID:SCR_003070) [25].

In-vivo xenograft experiment and dosing

6-week old female aythmic nude mice (Envigo) were injected subcutaneously with 5×106 MIA-PaCa 2 cells suspended in a 1:1 mixture of Matrigrel (Corning) and 1x PBS on both left and right flanks. Tumors progressed to 100mm3 before mice were randomized into treatment groups. Mice were then treated either with F10 (200 mg/kg), CF10 (200 mg/kg), or vehicle control (saline) 3 times/week, alongside tumor volume measurement (Lx(WxW)/2) and mice weight recording. After completion of dosing (3 weeks for F10 and 5 weeks for CF10) mice were euthanized and tumors were extracted for further analyses. All animal studies and handling were performed and approved according to Institutional Animal Care and Use Committee (IACUC) guidelines.

Immunohistochemistry (IHC) for Ki67 and cleaved caspase-3 from formalin fixed-paraffin embedded tumor tissue

For Ki67 IHC staining formalin fixed-paraffin embedded tumor tissues, antigen retrieval was performed on the Roche Ventana Discovery ULTRA staining platform using Discovery CCI (Roche, Switzerland) for 36 minutes. Primary immunostaining was performed using a predilute Ki-67(Roche 790–4286) and incubated at 41°C for 32 minutes. Secondary immunostaining used a Horseradish Peroxidase (HRP) multimer cocktail (Roche) and immune complexes were visualized using the ultraView Universal DAB (diaminobenzidine tetrahydrochloride) Detection Kit (Roche). Slides were then washed with a Tris based reaction buffer (Roche) and counter-stained with Hematoxylin II (Roche cat) for 4 minutes. For cleaved-caspase-3 IHC staining, a similar protocol was followed using a Caspase-3-specific antibody (Biocare, PP229AA). For both IHC stains, analyses of positive tumor tissue from 5 randomly selected fields per tumor were done via ImageJ thresholding analysis.

Western blotting for thymidylate synthase classic complex (TS CC), total cell PARylation, yH2AX and cleaved caspase 3

7.5×105-1×106 cells were plated in 10 cm dishes and cells were treated with drugs the following day for indicated timepoints. Cell lysis and western blotting were performed as previously described [19, 23]. Antibodies used are supplied in Supplementary Table S2.

Immunofluorescence imaging of stalled Top1 complexes

Imagining of stalled Top1 complexes was performed using a specific antibody (MABE1084, Sigma-Millipore) as previously described [26, 27]. Slides were imaged with a Nikon A1R confocal microscope with a 60x oil objective. Quantification of Top1 foci was done via ImageJ by counting at least 100 cells and cells were declared positive if they had >50 foci per nuclei.

DNA fiber assay and High-Throughput Alkaline Comet Assay

Cells were treated with either DMSO or CF10 for 16 hours and analyzed for changes in replication dynamics via DNA fiber assay or DNA strand breaks via comet assay as described previously [28].

siRNA mediated knockdown of PARG

500,000–600,000 cells were plated in 10cm dishes and were transfected the following day. 10 nM of either siControl or siPARG siRNA were prepared with OPTI-MEM (Corning) and Lipofectamine RNAiMAX (ThermoFisher) per manufacturer’s instructions. After 48 hours of transfection, cells were split for downstream experiments and a separate pellet was taken for protein validation via western blot with a PARG antibody supplied in Supplementary Table S2.

Statistical Analyses

All statistical analyses were performed with the GraphPad Prism software (version 8.3.0, RRID:SCR_003070). For all in-vitro experiments 3 biological replicates were used for analysis and appropriate two-way ANOVA tests with Bonferroni correction were conducted with p<0.05 being considered as significant except where stated otherwise. For in-vivo xenograft experiments a standard t-test with Bonferroni correction on the final day of measurement was conducted via GraphPad Prism.

Additional experimental reagents are supplied in supplementary methods.

Results

CF10 has potent anti-tumor activity in PDAC cells

We first wanted to assess the in-vitro and in-vivo efficacy of CF10 and results from 5-day cell survival assays showed that CF10 had significantly lower IC50 values compared to F10 and 5-FU for inhibiting PDAC cell growth in-vitro (Figure 1A, Table 1, and Supplementary Figure 1A). We then conducted a 10-day colony growth inhibition assay to determine CF10’s impact on longer-term growth potential. (Supplementary Figure 1B). Quantification of colony area revealed that CF10 was significantly more potent than F10 and 5-FU at inhibiting colony growth in PANC1 (p< 0.01) and ASPC1 (p< 0.0001) cells, with a trend towards significance relative to F10 in MIA-PaCa 2 cells (Figure 1B).

Figure 1: CF10 has potent anti-tumor activity in PDAC cells.

Figure 1:

(A) Representative graphs of 5-day survival assay with quantification of relative cell number via picogreen in the indicated PDAC cell lines. (B) Colony area percentage quantification from 10-day colony growth inhibition assays in the indicated cell lines. (C) Graph of individual MIA-PaCA 2 flank-tumor volumes (n= 8 mice per arm) after 35 days of CF10 treatment (200mg/kg 3x/week) as compared to vehicle (saline) control with graph of changes in relative weight after initiation of CF10 treatment vs vehicle. (D) Representative images of Ki67 and cleaved caspase-3 IHC staining of tumor tissues from CF10- and vehicle-treated mice with quantification of IHC staining. For each in-vitro experiment, three independent experiments were conducted, and errors bars represent standard error of the means. Significance was tested via two-way ANOVA with Bonferroni correction for in-vitro assays and student t-test with Bonferroni correction for in-vivo assays denoted by *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001.

Table 1.

IC50 values with standard deviation for F10, CF10, and 5-FU across all PDAC cell lines examined. P-values indicate differences between CF10 IC50s compared to F10 and 5-FU.

IC50 (nM) MIA-PaCa 2 p-value PANC1 p-value ASPC1 p-value PDX139 p-value
CF10 7.06 ± 2.89 - 46.01 ± 1.19 - 85.30 ± 4.62 - 46.26 ± 3.01 -
F10 25.43 ± 4.00 0.003 345.10 ± 33.00 <0.0001 1203.00 ± 308.70 0.0033 226.30 ± 22.53 0.0002
5FU 6277.00 ± 426.00 <0.0001 12340.00 ± 1163.00 <0.0001 15820.00 ± 529.90 <0.0001 5451.00 ± 286.70 <0.0001

We next investigated the in-vivo efficacy of CF10 in a murine flank xenograft study. Because neither CF10 nor F10 were previously tested in PDAC models, we initiated a pilot study with F10 to determine if it was efficacious at a dose similar to previous studies in other tumor types (200mg/kg 3x/week for 3 weeks) [14, 15, 29]. We found that F10 significantly reduced tumor volume (p< 0.05) compared to vehicle and was well tolerated, as F10-treated mice did not experience a decrease in body weight over the duration of the study (Supplementary Figure 1D). Quantification of Ki67 and cleaved caspase-3 (cCas3) immunohistochemistry (IHC) of F10-treated tumor tissue showed a significant reduction in Ki67 staining (p< 0.001) and increased cCas3 staining (p< 0.0001) (Supplementary Figure 1E) compared to vehicle, indicating that F10 treatment reduced proliferation and induced apoptosis in PDAC cells in-vivo. A similar in-vivo study with CF10 revealed significant reduction in tumor volume (p< 0.01) with no decrease in mouse body weights, indicating that CF10 was well tolerated and effective (Figure 1C and Supplementary Figure 1C). Ki67 and cCas3 IHC staining of CF10-treated tissues showed similar results as for F10 (p< 0.0001 for both Ki67 and cCas3, Figure 1D).

CF10 directly inhibits TS/Top1, disrupts replication fork dynamics, and is more DNA-directed than 5-FU

With the in-vitro and in-vivo efficacy of CF10 assessed, we next wanted to validate whether previously known mechanisms of action seen with F10 were active with CF10 in our PDAC models. Western blot for TS inhibitory covalent complex (TS CC) [3032] revealed that CF10 or F10 at 10nM for 24 hours in MIA-PaCa 2 cells strongly induced formation of the TS CC (Figure 2A). 5-FU at a similar dose of 10nM did not induce TS CC formation, although at 10uM, a concentration that greatly exceeded the 10-fold difference in FP content of CF10, 5-FU induced TS CC. These findings were replicated in PANC1 cells. Inhibition of Top1 in PDAC cells was assessed by Top1 cleavage complex (Top1cc) immunofluorescence as previously described [26]. Quantification of Top1cc positive foci in MIA-PaCa 2 and PANC1 cells revealed that 24h post-treatment both CF10 and F10 significantly increased the number of Top1cc-positive nuclear foci compared to the no treatment control (p< 0.001, Figure 2B) and to a level similar to the topotecan positive control [26, 33]. 5-FU at a similar dose to F10 and CF10 induced lower levels of Top1cc, suggesting less incorporation of 5-FU generated FdUMP into DNA. This finding prompted us to investigate whether 5-FU generated Top1ccs could be caused through an RNA- mediated mechanism through 5-FU conversion to FUMP as opposed to FdUMP. To assess this, we conducted a similar Top1cc immunofluorescence assay but supplemented MIA-PaCa2 or PANC1 cells with uridine to slow incorporation of FUMP into RNA and found that only 5-FU had a statistically significant decrease in Top1cc generation compared to all other conditions (Supplementary Figures 24). Taken together, these data suggest independent mechanisms of Top1cc inhibition between F10, CF10 and 5-FU that involve DNA-mediated effects with F10 and CF10 compared to RNA-mediated effects with 5FU.

Figure 2: CF10 directly inhibits TS/Top1, disrupts replication fork dynamics. and is more DNA-directed than 5FU.

Figure 2:

(A) Representative western blots detecting the TS Classic Complex (TS CC) after treatment in indicated PDAC cell lines with F10, CF10 and 5-FU at indicated concentrations for 24 hours. (B) Representative immunofluorescence images with an antibody specific for Top1 cleavage complexes (Top1cc) in indicated PDAC cell lines treated at indicated concentrations of F10, CF10, 5-FU for 24 hours and Topotecan for 1 hour with quantification graphs to the right. (C) Schematic depicting the dose and timing scheme for DNA fiber experiment, representative images from PANC1 cells used in DNA fiber analysis (yellow arrows indicate terminal replication forks), and graphs for quantification of fork velocity and percent terminal forks in PANC1 cells. (D) Quantifications of median bliss synergy scores in the indicated cell lines after co-treatment of F10, CF10 and 5-FU with thymidine and uridine. For each experiment, three independent experiments were conducted, and errors bars represent standard error of the means. Significance was tested via two-way ANOVA with Bonferroni correction for Top1cc immunofluorescence and student t-test with Bonferroni correction for DNA fiber and thymidine/uridine supplementation assays denoted by *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001.

Since both TS inhibition and Top1cc formation could affect replication fork progression [34], we next investigated CF10’s effects on DNA replication fork dynamics via DNA fiber assay. Analysis of DNA fiber length and structure via immunofluorescence in PANC1 cells revealed that CF10 significantly reduced fork velocity (p< 0.0001) and increased the number of terminal replication forks (p< 0.001, Figure 2C), indicating that CF10 induces replication stress in PDAC cells. These findings demonstrate CF10 is cytotoxic to PDAC cells via dual targeting of TS and Top1, which disrupts replication fork progression and causes replication stress. To further investigate the origin of CF10’s DNA-directed cytotoxic mechanism and its improved potency relative to 5-FU, we performed thymidine and uridine rescue experiments. Previous studies demonstrated F10-induced apoptosis was reversed by exogenous thymidine, but not uridine [35]. In contrast, 5-FU-induced apoptosis was reversed with uridine consistent with an RNA-directed mechanism [36, 37]. We employed Bliss additivity analysis [19, 24] to examine whether the combination of CF10 with thymidine affected cell viability. We observed that thymidine antagonized both CF10 and F10 cytotoxicity in MIA-PaCa 2 and ASPC1 cells (Figure 2D and Supplementary Figure 56). However, thymidine was a less potent antagonist of CF10, consistent with a non-TS-dependent cytotoxic mechanism that results from AraC release, a non-TS-dependent DNA damaging nucleoside analog. Consistent with a non-TS-directed cytotoxic mechanism, the 5-FU plus thymidine combination displayed relatively less antagonism relative to F10 or CF10. In contrast to our thymidine antagonism studies, Bliss additivity analysis revealed only mild antagonism when either CF10 and F10 were combined with uridine, but much stronger antagonism between 5-FU and uridine (p< 0.05, Figure 2D). We believe that these supplementation studies highlight the increased “DNA-directed” vs. “RNA-directed” effects of CF10 relative to 5-FU in PDAC cells, consistent with an improved therapeutic index.

CF10 and PDDX-04 synergy produces unresolved DNA damage which triggers apoptosis

Because combinations of drugs are historically more effective than single agents for PDAC treatment [5, 38], we next conducted a focused drug synergy screen with CF10 and various potential combinatorial partners using Bliss additivity analysis. Combinations of CF10 with standard-of-care PDAC agents such as 5-FU, irinotecan, oxaliplatin, or gemcitabine in either MIA-PaCa 2 or PANC1 cells did not yield robust synergy (data not shown). We then screened inhibitors of Poly (ADP) Ribose Polymerase 1 (PARP1) and Poly(ADP) Ribose Glycohydrolase (PARG), Olaparib and PDDX-04, respectively, two key enzymes involved in DNA damage repair [39, 40]. PARP1 is important for Top1cc repair and PARP inhibitors produce increased sensitivity to Top1 inhibitors [41, 42]. Further, PARP1 is important for the resolution of replication fork stress [40], which our DNA fiber data indicate CF10 produces. PARG opposes PARP1 function by removing PARylation, and we recently reported studies demonstrating that PARG inhibition sensitized PDAC cell lines to DNA damaging agents [19, 43, 44] We observed antagonism for the CF10 plus olapraib combination but intriguingly, the CF10 plus PDDX-04 combination displayed strong synergy in both MIA-PaCa 2 and PANC1 cells (Figure 3A and Supplementary Figure 7A). To gain further insight into the mechanism for this synergy, we determined separately the relative contributions of the F10 and AraC components. Bliss additivity analysis of all possible combinations of PDDX-04, F10, and AraC revealed extensive synergy for the PDDX-04 plus F10 combination relative to PDDX-04 plus AraC and mild antagonism between F10 plus AraC in both MIA-PaCa 2 and PANC1 cells (Figure 3A and Supplementary Figure 7AB), suggesting that the bulk of synergy between CF10 and PDDX-04 is indeed due to the FP component. We validated the observed synergy between CF10 and PDDX-04 via siRNA oligos targeted against PARG (siPARG). siPARG treatment sensitized MIA-PaCa 2 cells to both CF10 and F10, confirming the specificity of effects seen with the CF10 plus PDDX-4 combination (Table 2 and Supplementary Figure 8A).

Figure 3: CF10 and PDDX-04 synergy produces unresolved DNA damage which triggers apoptosis.

Figure 3:

(A) Representative Bliss synergy plots of indicated PDAC cell lines concurrently treated with serial dilutions of CF10 plus Olaparib, CF10 plus PDDX-04 and F10 plus PDDX-04. (B) Representative immunofluorescence images of MIA-PaCa 2 cells from an alkaline comet assay after treatment with CF10, PDDX-04 or their combination for 16 hours at the indicated concentrations and quantification of DNA tail moments in indicated PDAC cell lines. (C) Representative Western blot for PAR, gH2AX, and cleaved caspase-3 with appropriate loading controls in indicated PDAC cell lines treated with CF10, PDDX-04 or their combination for 48 hours with quantifications below. For each experiment, three independent experiments were conducted, and errors bars represent standard error of the means. Significance was tested via two-way ANOVA with Bonferroni correction and denoted by *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001.

Table 2.

IC50 values with standard deviation from 5-day survival assay of MIA-PaCa 2 cells treated with CF10 or F10 after 48h of siRNA knockdown of PARG compared to control scrambled siRNA. P-values indicate differences between siCon and siPARG cells.

MIA-PaCa 2 IC50 (nM) siCon siPARG p-value
CF10 24.54 ± 7.32 2.41 ± 0.33 0.0064
F10 90.14 ± 8.73 5.61 ± 0.70 <0.0001

Since F10 is a known inducer of DNA damage [9, 28, 29], we hypothesized that combining CF10 with an inhibitor of DNA repair (i.e. PARG) would mechanistically result in unresolved DNA damage followed by induction of cell death. Alkaline comet assay was performed with CF10 and/or PDDX-04 and quantification of DNA tail length revealed that the combination of CF10 and PDDX-04 caused significant increase in tail length as compared to either treatment alone in MIA-PaCa 2 (p< 0.01) and PANC1 cells (p< 0.001, Figure 3B). Western blot analysis at 48 hours also revealed increased total cell PARylation, γH2AX serine-139 phosphorylation, and cCas3 with the CF10 and PDDX-04 combination in MIA-PaCa 2 (p< 0.0001) and PANC1 cells (p< 0.05, Figure 3C), compared to either drug alone suggesting that breaks in DNA observed previously are unable to be repaired and ultimately trigger apoptosis. These data were validated by siPARG treatment when we observed that 48h post-CF10 treatment, siPARG-treated MIA-PaCa2 cells exhibit higher levels of total cell PARylation, γH2AX, and cCas3 compared to siControl cells (p< 0.01, Supplementary Figure 8B). Ultimately, we believe that these data support our hypothesis that the combination of CF10 and PDDX-04 induces substantial DNA damage that is not resolved in the context of pharmacologic or genetic PARG inhibition and eventually triggers apoptosis.

Discussion

Ultimately, this study represents the first investigations of the novel polymeric FP CF10 for the treatment of PDAC and we believe that our data moves CF10 closer to a clinical trial. The identification of the novel CF10 and PDDX-04 combination invites more study, beyond Chk1 and Wee1 inhibition [28, 44], involving the exploration of CF10 with other DNA repair inhibitors. One limitation of our study is the lack of bioavailability of PDDX-04 which prevents in-vivo validation of CF10-PDDX synergy, but future investigations will involve validation using CF10 combined with genetic models of PARG inhibition [19, 44] and next generation bioavailable PARG inhibitors. Future directions will include understanding why CF10 appears to preferentially synergize with inhibition of PARG but not PARP1 which could be due in part to the differential consequences of inhibiting these two DNA repair factors [40].

Supplementary Material

1

Implications:

CF10 is a promising polymeric fluoropyrimidine with dual mechanisms of action (i.e., TS and Top1 inhibition) for the treatment of PDAC and synergizes with targeting of DNA repair.

Acknowledgements

This work was supported by NIH-NCI R21CA218933 (W.H. Gmeiner), R01CA212600-01 (J.R. Brody), and in part by NIH U01CA224012 (R.C. Sears); P30CA069533 (B.J. Druker); and P30CA056036 (K.E. Knudsen). J. R. Brody is also supported by funding from the Pancreatic Cancer Cure Foundation, Pancreatic Cancer Action Network-AACR Research Acceleration Network Grant, Grant ID 15-90-25-BROD (J.R. Brody), and the Richard Alan Parry Pancreatic Cancer Research Fund.

Footnotes

Conflict of interest: W. Gmeiner is an inventor on a patent application for CF10 in colon cancer.

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