Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jul 5;12(1):11346.
doi: 10.1038/s41598-022-14788-5.

Targeted therapy of pyrrolo[2,3-d]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment

Adrianne Wallace-Povirk  1 , Lisa Rubinsak  1 , Agnes Malysa  1 , Sijana H Dzinic  1   2 , Manasa Ravindra  3 , Mathew Schneider  1 , James Glassbrook  4 , Carrie O'Connor  1 , Zhanjun Hou  1   2 , Seongho Kim  1   2 , Jessica Back  1   2 , Lisa Polin  1   2 , Robert T Morris  1   2 , Aleem Gangjee  5 , Heather Gibson  6   7   8 , Larry H Matherly  9   10   11
Affiliations

Targeted therapy of pyrrolo[2,3-d]pyrimidine antifolates in a syngeneic mouse model of high grade serous ovarian cancer and the impact on the tumor microenvironment

Adrianne Wallace-Povirk et al. Sci Rep. .

Abstract

Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies that target unique vulnerabilities in the tumor microenvironment (TME) of EOC have largely been unrealized. One strategy to achieve selective drug delivery for EOC therapy involves use of targeted antifolates via their uptake by folate receptor (FR) proteins, resulting in inhibition of essential one-carbon (C1) metabolic pathways. FRα is highly expressed in EOCs, along with the proton-coupled folate transporter (PCFT); FRβ is expressed on activated macrophages, a major infiltrating immune population in EOC. Thus, there is great potential for targeting both the tumor and the TME with agents delivered via selective transport by FRs and PCFT. In this report, we investigated the therapeutic potential of a novel cytosolic C1 6-substituted pyrrolo[2,3-d]pyrimidine inhibitor AGF94, with selectivity for uptake by FRs and PCFT and inhibition of de novo purine nucleotide biosynthesis, against a syngeneic model of ovarian cancer (BR-Luc) which recapitulates high-grade serous ovarian cancer in patients. In vitro activity of AGF94 was extended in vivo against orthotopic BR-Luc tumors. With late-stage subcutaneous BR-Luc xenografts, AGF94 treatment resulted in substantial anti-tumor efficacy, accompanied by significantly decreased M2-like FRβ-expressing macrophages and increased CD3+ T cells, whereas CD4+ and CD8+ T cells were unaffected. Our studies demonstrate potent anti-tumor efficacy of AGF94 in the therapy of EOC in the context of an intact immune system, and provide a framework for targeting the immunosuppressive TME as an essential component of therapy.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Structures of 6-substituted pyrrolo[2,3-d]pyrmidine inhibitors of de novo purine nucleotide biosynthesis. Structures are shown for AGF94, AGF278, and AGF283, selective substrates for FRs and PCFT over RFC, and inhibitors of the folate-dependent purine biosynthetic enzyme GARFTase,.
Figure 2
Figure 2
Expression of GARFTase and AICARFTase transcripts in primary EOC patient samples and BR-5 and BR-Luc cells. Transcript levels for GARFTase (A) and AICARFTase (B) were measured using cDNAs from primary specimens including normal ovary (n = 8) and HGSOC (n = 39) (OriGene) and results were compared to those for EOC cell lines including IGROV1, SKOV3, A2780 and A2780 E-80. Transcript levels were normalized to β-actin transcripts. Statistical analyses were performed between normal samples/tissues and tumor samples/tissues using the Wilcoxon rank-sum test. (C,D) Transcript levels of cytosolic C1 metabolic targets (GARFTase and AICARFTase) were determined in the BR-5 and BR-Luc syngeneic mouse models of HGSOC by real-time RT-PCR. Transcripts were normalized to β-actin transcripts and results are shown relative to levels in mouse liver (assigned a value of 1). Results are presented as mean values ± standard errors from at least three experiments. The p values are as follows: ****p < 0.0001. See Supplementary Table S1 for patient characteristics and pathology.
Figure 3
Figure 3
Folate transporter expression and function in BR-5 and BR-Luc syngeneic HGSOC mouse models. Transcript levels for PCFT, RFC and FRα were measured by real-time RT-PCR for BR-5 (A) and BR-Luc (B) cells. Transcripts were normalized to β-actin transcripts and levels are shown relative to those in mouse liver (assigned a value of 1). (C) Total surface FRα was measured by titration with [3H]folic acid at 0 °C with and without unlabeled 10 µmol/L non-radioactive folic acid. (D) PCFT uptake was assayed using [3H]MTX (0.5 µM) at pH 5.5 at 37 °C in the absence and presence of 10 µM non-radiolabeled AGF94, as previously described. Results are presented as mean values (± standard errors) from at least three experiments.
Figure 4
Figure 4
AGF94 efficacy trial in the IP BR-Luc model. Overall survival (A) and tumor burden (C) are shown for IP BR-Luc tumors in FVB mice following treatment with AGF94 (32 mg/kg × 4 doses). (A) A Kaplan–Meier survival analysis was performed on 4 mice treated with AGF94 (32 mg/kg × 4 doses). For control mice, a median of 22 days was measured compared to a median of 33 days for the AGF94-treated mice. Statistical analysis was performed using the log-rank test. (B) The treatment scheme is shown. (C) Luminescent images are shown for a separate cohort (3 mice) over 3 min for control and AGF94-treated mice treated with 4 doses at 32 mg/kg and overlayed on top of an X-ray image.
Figure 5
Figure 5
AGF94 efficacy in a subcutaneous BR-Luc model. (A) Luminescent images were collected over 3 min and overlayed on top of an X-ray image. The image was obtained 7 days after the tumor was allografted and 1 day prior to treatment initiation. (B) The trial design schematic is shown. BR-Luc tumors were engrafted SC bilaterally; treatment with AGF94 began at 8 days when the tumors were palpable (~ 400 mg). Tumors were harvested, dissociated and flow cytometry was performed 24 h after 2, 3, and 4 doses of AGF94. (C) Results are plotted for the BR-Luc trial efficacy arm with AGF94 by individual mice; the median results for 6 mice are shown as broken lines. Female FVB mice were implanted bilaterally SC with BR-Luc tumors and AGF94 treatment was initiated on day 8 following tumor implantation. AGF94 was dosed as Q4dx4 at 32 mg/kg/IV injection. %T/C values were determined on day 11. (D) The table summarizes the results of the in vivo trial with SC BR-Luc xenografts treated with AGF94 for mice maintained on both the folate-deficient and standard folate replete diets.
Figure 6
Figure 6
Impact of AGF94 treatment on tumor infiltrating macrophages. To assess the impact on the immune populations in mice treated with AGF94, the immune populations from tumors and spleens were harvested after 2, 3, and 4 treatments. (A) Results are shown for the percentage of CD11b+ and F4/80+ macrophages. (B) Percentage of FRβ+, gated off CD11b+ and F4/80+ macrophages, are shown with 2, 3, or 4 doses of AGF94. (C) Percentage of Arg1+ FRβ+, gated off CD11b+ and F4/80+ macrophages, are shown following 2, 3 and 4 doses of AGF94. (D) Percentages of CD80+ FRβ+, gated off CD11b+ and F4/80+ macrophages, are shown following 2, 3, and 4 doses of AGF94. Results are shown for individual mice. Horizontal bars represent median values. Statistical comparisons were made between AGF94-treated and control groups. *p < 0.05.
Figure 7
Figure 7
Impact of AGF94 treatment on tumor infiltrating T cells. Percentages of CD3+ T cells (A) are shown from single cell suspensions of BRLuc tumor and infiltrating lymphocytes following 2, 3 and 4 doses of AGF94. The CD3+ population was gated off CD45+ cells. Percentage of CD4+ (B) and CD8+ (C) T cells are shown from single cell suspensions of tumor and infiltrating lymphocytes following 2, 3 and 4 doses of AGF94. The CD4+ and CD8+ populations were gated off CD45+ and CD3+ cells. Results are shown for individual mice. Horizontal bars represent median values. Statistical comparisons were made between AGF94-treated and control groups. *p < 0.05.
Figure 8
Figure 8
Immunofluorescence staining of FRβ-expressing tumor-associated macrophage population following treatment with AGF94. Slides were stained with FRβ (Genetex) with Alexa Fluor 488 secondary antibody and pan-Macrophage, F4/80, conjugated to APC-R700 (BD Biosciences). Images were acquired by confocal microscopy. Immunofluorescence staining is shown for representative tumor sections from control mice and mice treated with 4 doses of AGF94. The scale is 20 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Torre LA, et al. Ovarian cancer statistics, 2018. CA Cancer J. Clin. 2018;68:284–296. doi: 10.3322/caac.21456. - DOI - PMC - PubMed
    1. Yang C, et al. Folate-mediated one-carbon metabolism: A targeting strategy in cancer therapy. Drug Discov. Today. 2021;26:817–825. doi: 10.1016/j.drudis.2020.12.006. - DOI - PubMed
    1. Lheureux S, Gourley C, Vergote I, Oza AM. Epithelial ovarian cancer. Lancet. 2019;393:1240–1253. doi: 10.1016/S0140-6736(18)32552-2. - DOI - PubMed
    1. Jackman, A. L., Jansen, G. & Ng, M. in Targeted Drug Strategies for Cancer and Inflammation (eds Jackman, A. L. & Leamon, C. P.) 93–117 (Springer US, 2011).
    1. Moore KN, et al. Phase 1 dose-escalation study of mirvetuximab soravtansine (IMGN853), a folate receptor alpha-targeting antibody-drug conjugate, in patients with solid tumors. Cancer. 2017;123:3080–3087. doi: 10.1002/cncr.30736. - DOI - PMC - PubMed

Publication types