pISSN 1598-2998, eISSN 2005-9256 Cancer Res Treat. 2020;52(1):149-166 https://doi.org/10.4143/crt.2019.183 Original Article Open Access Therapeutic Co-targeting of WEE1 and ATM Downregulates PD-L1 Expression in Pancreatic Cancer Mei Hua Jin, MD, MS1 Purpose Ah-Rong Nam, MS1 Pancreatic cancer (PC) is one of the most lethal cancers worldwide, but there are currently no effective treatments. The DNA damage response (DDR) is under investigation for the Ji Eun Park, MS1 development of novel anti-cancer drugs. Since DNA repair pathway alterations have been MS1 Ju-Hee Bang, found frequently in PC, the purpose of this study was to test the DDR-targeting strategy in 1,2 Yung-Jue Bang, MD, PhD PC using WEE1 and ATM inhibitors. Do-Youn Oh, MD, PhD1,2 Materials and Methods We performed in vitro experiments using a total of ten human PC cell lines to evaluate anti- tumor effect of AZD1775 (WEE1 inhibitor) alone or combination with AZD0156 (ATM inhi- bitor). We established Capan-1–mouse model for in vivo experiments to confirm our findings. Results In our research, we found that WEE1 inhibitor (AZD1775) as single agent showed anti-tumor 1Cancer Research Institute, Seoul National effects in PC cells, however, targeting WEE1 upregulated p-ATM level. Here, we observed University College of Medicine, Seoul, that co-targeting of WEE1 and ATM acted synergistically to reduce cell proliferation and 2Department of Internal Medicine, Seoul migration, and to induce DNA damage in vitro. Notably, inhibition of WEE1 or WEE1/ATM National University Hospital, Seoul, Korea downregulated programmed cell death ligand 1 expression by blocking glycogen synthase kinase-3! serine 9 phosphorylation and decrease of CMTM6 expression. In Capan-1 mouse xenograft model, AZD1775 plus AZD0156 (ATM inhibitor) treatment reduced tumor growth and downregulated tumor expression of programmed cell death ligand 1, CMTM6, CD163, and CXCR2, all of which contribute to tumor immune evasion. + + + + + + + + + + + + + + + + + + + + Conclusion Correspondence: Do-Youn Oh, MD, PhD Dual blockade of WEE1 and ATM might be a potential therapeutic strategy for PC. Taken + D +e p+a r +tm +e n +t o +f I n+t e +rn +al M+ e+d i +ci n +e , + + + + + + + + + + + + + + + + + + + + + + + + + + together, our results support further clinical development of DDR-targeting strategies for + S +e o +u l +N a+ti o+n a+l U +n i+v e +rs i+ty +C o +ll e +g e + o f + M +ed +ic i n+e ,+ + 1 +0 1 + D a+e h+a k +-r o+, J+o n +g n +o - g+u ,+ S e+o u +l 0 +3 0 +80 ,+ K +or e+a + PC. + T +e l :+ 8 2+-2 -+2 0 +7 2 +-0 7+0 1 + + + + + + + + + + + + + F +a x +: 8 2+- 2 +-7 6+2 - +96 6+2 + + + + + + + + + + + + + E +-m +a i +l: o +h d +o y +o u +n @ +s n+u .+ac .+k r + + + + + + + + + + + + + + + + + + + + + + + + + + + + Key words + R +e c +ei v +e d + A +p r+il 5+, 2 +0 1 +9 + + + + + + + + + + + A +c c +ep +te d + J +un +e 2 +1, 2+0 1+9 + + + + + + + + + + Pancreatic neoplasms, DNA damage response, WEE1, ATM, PD-L1 + P +u b +li s +h e d+ O +n l+in e+ J+u n +e 2 +5 , +20 1+9 + + + + + + + + + + + + + + + + + + + + + + + + + + + Introduction telangiectasia mutated (ATM) and partner and localizer of BRCA2 (PALB2) are present in PC patients with frequencies of 5% (germline mutations) and 12% (somatic mutations) [1]. Pancreatic cancer (PC) is one of the most lethal diseases These genes play critical roles in the DNA damage response worldwide, and there is an urgent need to develop effective (DDR), which signals the presence of strand breaks and other therapies for this disease [1,2]. Recently, genomic analyses forms of DNA damage and coordinates their repair. Para- have revealed that many cancer susceptibility genes are fre- doxically, mutations in genes that compromise the DDR can quently mutated in PC, including KRAS (92% of cases) and both cause and protect against cancer. On the one hand, TP53 (78%). In addition, germline and somatic mutations in defects in the DDR can lead to genomic instability and the genes encoding breast cancer 1 and 2 (BRCA1, BRCA2), ataxia accumulation of mutations that increase the probability of │ https://www.e-crt.org │ Copyright ⓒ 2020 by the Korean Cancer Association 149 This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Cancer Res Treat. 2020;52(1):149-166 cancer. On the other hand, DDR pathway dysfunction can Increasing evidence suggests the existence of crosstalk render tumor cells susceptible to chemotherapeutic agents between the DDR signaling network and immune pathways that damage DNA and/or impair alternative DDR pathways [13,14]. For example, recent studies have demonstrated that [3]. Thus, targeting of specific molecules in the DDR is a the DDR regulates PD-L1 expression in cancer cells via a potential strategy for the development of new drugs for can- pathway involving activation of signal transducer and acti- cers with urgent unmet needs, including PC. vator of transcription (STAT) signaling and inactivation of The cellular response to DNA damage, including single- glycogen synthase kinase-3! (GSK-3!) [15,16]. However, stranded or double-stranded DNA breaks, is controlled by a such interactions between the DDR and immune signaling network of proteins that include damage-sensing proteins have not yet been studied in PC. such as poly(ADP-ribose) polymerase (PARP); transducers Here, we evaluated the anti-tumor effects of targeting the such as the kinases ATM and ataxia telangiectasia and Rad3 DDR using a WEE1 kinase inhibitor (AZD1775) and an ATM related (ATR); and effectors such as the nuclear kinase WEE1, kinase inhibitor (AZD0156) in PC cells in vitro and in a mouse which is a key protein in cell cycle checkpoint control and xenograft model. We also examined the expression and acti- inhibits entry into mitosis [3]. Ultimately, the DDR results in vation of a number of DDR-related and immune signaling- one of three outcomes for the cell: successful DNA repair, cell related molecules to identify potential crosstalk between the cycle arrest, or apoptosis. DDR and immune system in PC. Therapeutic targeting of the DDR pathway has been exam- ined in diverse tumor types [3]. Notably, inhibition of PARP, ATM, ATR, or WEE1 has been shown to abrogate DNA repair via homologous recombination (HR) in many cancers Materials and Methods with a genetically defective DDR, thus leading to synthetic lethality [4-7]. WEE1 acts as a gatekeeper of the G2/M cell cycle checkpoint, and its activity increases during the S and 1. Human cell lines and reagents G2 phases; thus, WEE1 inhibition can induce growth arrest in S phase [8]. In contrast, cancer cells expressing mutant Ten human PC cell lines were employed in this study: TP53 lack a functional G1 checkpoint, and DNA damage AsPC-1, Capan-1, Capan-2, MIA PaCa-2, PANC-1, SNU213, must be repaired during the G2/M transition. Given that SNU324, and SNU410 were purchased from the Korean Cell TP53 is often mutated in PC [8], PC is the good candidate for Line Bank (Seoul, Korea), and SNU2913 and SNU2918, pati- the development of the DDR-acting agents. ent-derived cell lines, were successfully established from The tumor microenvironment plays a critical role in cancer patient. Cells were cultured in medium (MIA PaCa-2 and progression [9]. PC is unique compared with other tumor PANC-1 cells in Dulbecco's modified Eagle's medium, all types in being surrounded by strong stroma. Abundant other cell lines in RPMI-1640, both from Welgen Inc., Gyeong- immunosuppressive cells reside in the tumor microenviron- san, Korea) supplemented with 10% fetal bovine serum and ment, including regulatory T cells, myeloid-derived suppres- 10 µg/mL gentamicin and were maintained at 37°C in a 5% sor cells (MDSCs), M2-type macrophages, and cancer-asso- CO2 atmosphere. The WEE1 inhibitor AZD1775 and ATM ciated fibroblasts (CAFs) [9,10]. Recruitment of these cells inhibitor AZD0156 were kindly provided by AstraZeneca establishes a barrier to the anti-tumor immune response (Macclesfield, UK). [9,10]. In addition, signaling via the chemokine receptor CXCR2 can drive PC growth by recruiting MDSCs and 2. PD-L1 expression analysis by flow cytometry tumor-associated neutrophils and by enhancing the metasta- tic process [10]. Programmed cell death ligand 1 (PD-L1) is a Cells (2!105) were seeded in 60-mm dishes and incubated negative regulator of the immune response and acts by bind- with AZD1775 and/or AZD0156 for 72 hours. The adherent ing to its receptor programmed cell death 1 (PD-1) on T cells, cells were harvested, resuspended in cell staining buffer which inactivates the cells and thus allows the tumor to (#420201, BioLegend, San Diego, CA), and incubated with escape immune surveillance [11]. Data from genomic analy- anti–PD-L1 antibody (#329708, BioLegend) for 30 minutes at ses indicate that immunogenic subtype of PC, which exhibits room temperature. Cells were then washed once with the high levels of PD-L1, cytotoxic T-lymphocyte-associated pro- same buffer and analyzed on a FACSCalibur. The results are tein 4 and CXCR2 among several subtypes [1]. Notably, the presented as the means of three independent experiments. chemokine-like factor-like MARVEL transmembrane domain containing family member 6 (CMTM6) has been suggested as one of the mechanisms of regulation of PD-L1 through preventing PD-L1 degradation by lysosome [12].