The polymeric fluoropyrimidine CF10 overcomes limitations of 5-FU in pancreatic ductal adenocarcinoma cells through increased replication stress

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease soon to become the second leading cause of cancer deaths in the US. Beside surgery, current therapies have narrow clinical benefits with systemic toxicities. FOLFIRINOX is the current standard of care, one component of which is 5- Fluorouracil (5-FU), which causes serious gastrointestinal and hematopoietic toxicities and is vulnerable to resistance mechanisms. Recently, we have developed polymeric fluoropyrimidines (F10, CF10) which unlike 5-FU, are, in principle, completely converted to the thymidylate synthase inhibitory metabolite FdUMP, without generating appreciable levels of ribonucleotides that cause systemic toxicities while displaying much stronger anti-cancer activity. Here, we confirm the potency of CF10 and investigate enhancement of its efficacy through combination with inhibitors in vitro targeting replication stress, a hallmark of PDAC cells. CF10 is 308-times more potent as a single agent than 5-FU and was effective in the nM range in primary patient derived models. Further, we find that activity of CF10, but not 5-FU, is enhanced through combination with inhibitors of ATR and Wee1 that regulate the S and G2 DNA damage checkpoints and can be reversed by addition of dNTPs indicative of CF10 acting, at least in part, through inducing replication stress. Our results indicate CF10 has the potential to supersede the established benefit of 5-FU in PDAC treatment and indicate novel combination approaches that should be validated in vivo and may be beneficial in established regimens that include 5-FU.

Keywords: DNA damage; PDAC; fluoropyrimidine; pancreatic cancer; replication stress.

Figure 1.

(a) Structure of CF10 with PEG6 (blue) and AraC (yellow) modifications. (b) Differential metabolism of CF10 and 5-FU resulting in increased DNA damage with CF10 treatment and vulnerability to inhibitors of DNA repair. (c) Activation of the intra-S-phase checkpoint thru the ATR/Chk1 pathway and phosphorylation of Wee1. Our studies showed that inhibition of ATR or Wee1 enhances cytotoxicity of CF10, but not 5-FU, to PDAC cells. The combination of CF10+ATRi/Wee1 inhibition may overcome 5-FU resistance for improved treatment of PDAC.

Figure 2.

Dose-response viability curves for CF10, F10 and 5-FU in five conventional PDAC (BXPC-3, Capan1, Capan2, HPAF11, HS766T) and two primary patient-derived (7171-T and 4853-T) cell lines showing single agent efficacy. CF10 and F10 are displayed in nM while 5-FU is displayed in µm. IC50 values were calculated and reported in Table 1.

Figure 3.

(a) Representative Western blot detecting phosphorylation of ATR at threonine 1989 in MIA PaCa-2 cells after treatment with CF10, 5-FU, or GEM. Protein was collected at 5 time points between 8 and 48 hours, with each drug administered at IC50 concentration. Intensity of pATR (top) was normalized to a loading control (β-actin, bottom) and shown as fold increase as compared to vehicle treated (Veh.) cells. (b) Plotted relative pATR expression of three biological replicates in MIA PaCa-2 cells at 48 hours of CF10 (white), 5-FU (blue), or GEM (red) treatment. p values were calculated using an ordinary one-way ANOVA with significance defined as p < .05. *, p < .05; **, p < .01; ***, p < .001; ns, not significant.

Figure 4.

Viability effect of Wee1i or ATRi with single agent compounds in four PDAC cell lines. (a) The Wee1 inhibitor AZD1775 (adavosertib, green) showed increased single agent efficacy as compared to 5-FU (magenta), while treatment with both drugs were not additive (black). (b) ATRi (blue) showed increased single agent efficacy as compared to 5-FU (magenta), while treatment with both drugs did not show an additive effect (black).

Figure 5.

Wee1i and ATRi showed single agent compound efficacy in in four PDAC cell lines, and cytotoxicity is enhanced in combination with GEM. (a) The Wee1i (green) showed single agent efficacy comparable to or better than GEM (red), while concurrent treatment with both drugs displayed enhanced efficacy in all cell lines tested (black). (b) The ATRi (blue) showed single agent efficacy in all cell lines tested as well as enhanced cytotoxicity when administered concurrently to GEM (black).

Figure 6.

CF10, but not 5-FU, is enhanced by Wee1i and ATRi in four PDAC cell lines. (a, b) the Wee1i (green) and the ATRi (blue) showed enhanced cytotoxicity in all tested cell lines when administered concurrently to CF10 (black, single agent orange). (c, d) Conversely, enhanced effect is not seen in three out of four cell lines, and only modestly in the CFPAC1 cell line, when the Wee1i (green) and ATRi (blue) were administered either concurrently (black) to 5-FU (single agent, magenta).

Figure 7.

Adding of deoxynucleotides rescues viability in cells treated with CF10 or CF10+Wee1i/ATRi. (a) Four PDAC cell lines were treated with CF10 (orange) or CF10 and 10 µm dNTPs (black). (b) The same cell lines were tested with CF10 + Wee1i (green) or CF10 + Wee1i and 10 µm dNTPs (black). (c) The same cell lines were tested with CF10 + ATRi (blue) or CF10 + ATRi and 10 µm dNTPs (black).