Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) are used to treat BRCA-mutated (BRCAm) cancer patients; however, resistance has been observed. Therefore, biomarkers to indicate PARPi resistance and combination therapy to overcome that are urgently needed. We identified a high prevalence of activated FGF receptor 3 (FGFR3) in BRCAm triple-negative breast cancer (TNBC) cells with intrinsic and acquired PARPi resistance. FGFR3 phosphorylated PARP1 at tyrosine 158 (Y158) to recruit BRG1 and prolong chromatin-loaded MRE11, thus promoting homologous recombination (HR) to enhance PARPi resistance. FGFR inhibition prolonged PARP trapping and synergized with PARPi in vitro and in vivo. High-level PARP1 Y158 phosphorylation (p-Y158) positively correlated with PARPi resistance in TNBC patient-derived xenograft models, and in PARPi-resistant TNBC patient tumors. These findings reveal that PARP1 p-Y158 facilitates BRG1-mediated HR to resolve the PARP-DNA complex, and PARP1 p-Y158 may indicate PARPi resistance that can be relieved by combining FGFR inhibitors (FGFRis) with PARPis. In summary, we show that FGFRi restores PARP trapping and PARPi antitumor efficacy in PARPi-resistant breast cancer by decreasing HR through the PARP1 p-Y158/BRG1/MER11 axis, suggesting that PARP1 p-Y158 is a biomarker for PARPi resistance that can be overcome by combining FGFRis with PARPis.

Sequential cancer therapy presents a critical challenge, as the impact of prior treatments on immunotherapy remains unclear. Here, we demonstrate that therapeutic stress from prolonged cetuximab exposure induces tumor-intrinsic resistance to immune checkpoint blockade (ICB) in head and neck squamous cell carcinoma (HNSCC). In a multicenter analysis, extended cetuximab treatment correlates with poor ICB response and survival. Mechanistically, chronic therapeutic stress provokes an initial inflammatory response that transitions into immune resistance. A previously unknown post-translational modification, STAT1 lysine 637 acetylation, serves as the molecular switch driving this process. Triggered by treatment-induced tumor necrosis factor alpha (TNF-α), this acetylation impairs STAT1 dimerization and transcriptional activity, while treatment-induced interferon (IFN)-β promotes STAT1 phosphorylation at tyrosine 701 and subsequent degradation. These modifications disrupt tumor IFN-γ responsiveness. Importantly, STAT1 acetylation in pre-treatment tumor samples predicts ICB efficacy, underscoring its potential as a clinically relevant biomarker for guiding immunotherapy decisions.

Gli1-binding proteins identified through mass spectrum.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Enzymes from list of Gli1-binding proteins.

Supplemental Materials and Methods

Supplementary Table 1. Relationships between expression of p-PKM2, EGFR and Ki-67 in surgical specimens of triple-negative breast cancer patients. Supplementary Table 2. Relationships between expression of p-PKM2, EGFR and Ki-67 in surgical specimens of non-triple-negative breast cancer patients. Supplementary Figure S1. EGF signaling enhances aerobic glycolysis in TNBC cells. Supplementary Figure S2. PKM2 is a novel binding partner of EGFR and its activity is inhibited by EGFR via Tyr phosphorylation. Supplementary Figure S3. EGFR reprograms cancer cell metabolism by PKM2 phosphorylation in TNBC cells. Supplementary Figure S4. Upregulation of HK2 expression by EGF signaling also contributes to aerobic glycolysis in TNBC cells. Supplementary Figure S5. Glycolysis metabolite, F1,6BP, binds directly to and enhances the activity of EGFR. Supplementary Figure S6. The combination of EGFR and glycolysis inhibitors synergistically suppresses TNBC cell proliferation. Supplementary Figure S7. EGF signaling-induced lactate inhibits cytotoxic T cell activity in TNBC cells.

Correlation between Gli1 and PRMT1 Levels (S1); Identification of a PRMT1-Methylated Site in Gli1 (S2); Effects of R597 on Gli1 Transcriptional Activity (S3); Tumorigenicity of Wild-Type Gli1 and R597K-Mutated Gli1 (S4); Regulation of Gli1 by PRMT1 Is Independent of SMO/TGF-B/Kras (S5).

<p>Supplementary Figure S1. EGF signaling enhances aerobic glycolysis in TNBC cells. Supplementary Figure S2. PKM2 is a novel binding partner of EGFR and its activity is inhibited by EGFR via Tyr phosphorylation. Supplementary Figure S3. EGFR reprograms cancer cell metabolism by PKM2 phosphorylation in TNBC cells. Supplementary Figure S4. Upregulation of HK2 expression by EGF signaling also contributes to aerobic glycolysis in TNBC cells. Supplementary Figure S5. Glycolysis metabolite, F1,6BP, binds directly to and enhances the activity of EGFR. Supplementary Figure S6. The combination of EGFR and glycolysis inhibitors synergistically suppresses TNBC cell proliferation. Supplementary Figure S7. EGF signaling-induced lactate inhibits cytotoxic T cell activity in TNBC cells.</p>

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), which induce DNA damage by inhibiting PARP1 enzymatic activity and trapping PARP on the damaged DNA, are used to eliminate BRCA1/2-mutated (BRCAm) cancer. However, clinical observations suggest that BRCAm tumors develop PARPi resistance. Current strategies to overcome PARPi resistance include impeding multiple DNA repair pathways to induce excessive DNA damage. Here, we propose a novel strategy targeting oncogenic receptor tyrosine kinases to enhance PARP trapping. By developing triple-negative breast cancer (TNBC) cells with acquired talazoparib resistance, we observed a high prevalence of activated fibroblast growth factor receptor 3 (FGFR3) kinase in these cells through kinase antibody array analysis. Mass spectrometry analysis and in vitro kinase assay suggested that FGFR3 phosphorylated PARP1 at tyrosine residues 158 and 176. Biochemistry studies suggested that only PARP1 tyrosine 158 phosphorylation contributes to PARPi resistance in the cells we developed. We then developed a monoclonal antibody against tyrosine 158 phosphorylated PARP1, and found that high-level PARP1 tyrosine 158 phosphorylation positively correlated with PARPi resistance in breast cancer patient-derived xenograft models. We further demonstrated that the combination of FGFR inhibitor and PARPi delayed DNA repair with prolonged PARP trapping. Moreover, synergy between PARPi and FGFR inhibition was observed in multiple TNBC cell lines with PARPi resistance in vitro. The combination of PARPi and FGFR inhibitor also showed synergism in vivo, and treatment with the combination of PARPi and FGFR inhibitor was tolerated in mouse models. These findings reveal that PARP1 tyrosine 158 phosphorylation facilitates resolving of the PARPi-induced PARP-trapping, and that the tyrosine 158 phosphorylated PARP1 may be an effective biomarker to indicate FGFR3 mediated PARPi resistance. Citation Format: MeiKuang Chen, Yuan Gao, Weiya Xia, Yu-Han Wang, Jennifer K. Litton, Yu-Yi Chu, Funda Meric-Bernstam, Helen Piwnica-Worms, Banu Arun, Jordi Rodon Ahnert, Yongkun Wei, Wei-Chao Chang, Hung-Ling Wang, Coya Tapia, Constance T. Albarracin, Shao-Chun Wang, Ying-Nai Wang, Gabriel N. Hortobagyi, Chunru Lin, Liuqing Yang, Dihua Yu, Mien-Chie Hung. FGFR3 mediated PARP1 tyrosine 158 phosphorylation promotes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1792.

Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated promising clinical activity in multiple cancers. However, resistance to PARP inhibitors remains a substantial clinical challenge. In the present study, we report that anaplastic lymphoma kinase (ALK) directly phosphorylates CDK9 at tyrosine-19 to promote homologous recombination (HR) repair and PARP inhibitor resistance. Phospho-CDK9-Tyr19 increases its kinase activity and nuclear localization to stabilize positive transcriptional elongation factor b and activate polymerase II-dependent transcription of HR-repair genes. Conversely, ALK inhibition increases ubiquitination and degradation of CDK9 by Skp2, an E3 ligase. Notably, combination of US Food and Drug Administration-approved ALK and PARP inhibitors markedly reduce tumor growth and improve survival of mice in PARP inhibitor-/platinum-resistant tumor xenograft models. Using human tumor biospecimens, we further demonstrate that phosphorylated ALK (p-ALK) expression is associated with resistance to PARP inhibitors and positively correlated with p-Tyr19-CDK9 expression. Together, our findings support a biomarker-driven, combinatorial treatment strategy involving ALK and PARP inhibitors to induce synthetic lethality in PARP inhibitor-/platinum-resistant tumors with high p-ALK–p-Tyr19-CDK9 expression.

Abstract Investigations into various immunotherapies combined with conventional anticancer drugs are ongoing to increase therapeutic efficacy. However, combination therapy generally increases the risk of side effects. To achieve high efficacy with minimal side effects, nontoxic adjuvants should be identified and appropriate combinations should be designed based on the functional mechanism. In this regard, metformin can be an attractive candidate for immunotherapeutic adjuvants. Metformin is a widely used oral medication for type 2 diabetes (T2D) and has been recognized as a safe and well-tolerated drug through several decades of clinical experience. Interestingly, metformin also exhibits antitumor effects as several case-control studies for T2D patients indicated that metformin reduces the incidence of various cancer types. However, the functions and the detailed mechanism of metformin related to cancer immunity are not fully understood. In this study, we investigated the antitumor effects of metformin in relation to cancer immunity in the tumor microenvironment. Our data showed that AMPK activated by metformin decreases the expression of PD-L1 in the cancer cells, blocking PD-L1’s ability to aid cancer cells in escaping immune surveillance. This is caused by the mechanism in which phosphorylation of PD-L1 at S195 induces an abnormal glycan structure that leads to endoplasmic reticulum-associated degradation. In addition, we have obtained human breast tumor tissues from a previous clinical trial investigating metformin as treatment for breast cancer patients. The data from human tumor tissues also provided strong support to our current conclusion, namely AMPK activated by metformin reduces the level of PD-L1. On the basis of these results, we validated the possibility of metformin as an adjuvant to boost the efficacy of previous immunotherapy without toxicity. Our findings suggest that metformin has strong potential to be used as an adjuvant for immunotherapy. Metformin is expected to have synergistic effect with various non-PDL1/PD-1 targeting immune therapies without additional toxicity. Citation Format: Jong-Ho Cha, Wen-Hao Yang, Weiya Xia, Yongkun Wei, Li-Chuan Chan, Seung-Oe Lim, Chia-Wei Li, Jennifer Hsu, Hung-Ling Wang, Chu-Wei Kuo, Wei-Chao Chang, Sirwan Hadad, Colin Purdie, Aaron McCoy, Jennifer Litton, Elizabeth Mittendorf, Stacy Moulder, William Symmans, Alastair M Thompson, Helen Piwnica-Worms, Chung-Hsuan Chen, Kay-Hooi Khoo, Mien-Chie Hung. Metformin is a potential nontoxic adjuvant to enhance the efficacy of non-PDL1/PD-1 targeting immune therapies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A16.

Leveraging compromised DNA damage repair (DDR) pathways commonly found in tumor cells, a classic strategy in cancer therapy is inducing excessive DNA damage to cause cancer cell death. Small molecule poly(ADP-ribose) polymerase (PARP) inhibitors (PARP-is) have been approved for clinical use in treating breast cancer and ovarian cancer patients bearing DDR-deficient tumors with mutations in breast cancer susceptibility proteins (BRCAm). However, accumulating evidences show that both intrinsic and acquired resistances to PARP-is exist in clinic and pre-clinical animal models. Therefore, we developed panels of cells with acquired PARP-is resistance from PARP-is-sensitive triple negative breast cancer (TNBC) and estrogen receptor positive breast cancer cell lines, and used these cells to screen for common traits that can be targeted with feasible therapeutic agent combinations to overcome PARP-is resistance. Since TNBC lacks of effective targeted therapy so far, we focused on using the panel of PARP-is-resistant TNBC cells in this study. Among the molecular mechanisms known contribute to PARP-is resistance, oncogenic kinase activations, including several hyper-activated receptor tyrosine kinases (RTKs), are involved in enhancing DNA damage repair and decreasing affinity of PARP-is to PARP1. In this study, we systematically screened for activated RTKs in the PARP-is-resistant cells we developed by antibody arrays. We then identified that activations of MET, Axl and EphA2 were common traits in TNBC cells with acquired PARP-is resistance, but not in estrogen receptor positive cells. Among the three RTKs, MET has more small molecules inhibitors that can target it, and thus, we made it a priority in investigating synergism between MET inhibitor and PARP inhibitor in multiple cancer types including TNBC with intrinsic and acquired PARP-is-resistance, high-grade serous ovarian cancer (HGSOC) and liver cancer cells. Here, we demonstrated that combinations of PARP-is and MET inhibitors (MET-is) possess moderate to strong synergism in the different cancer types we studied. As we previously reported that MET translocates into cell nucleus and phosphorylates PARP1 at tyrosine (Y) 907 residue in TNBC with intrinsic PARP-is resistance, we found that this MET-mediated PARP1-Y907 phosphorylation also exist in and can serve as marker to indicate PARP-is resistance among the HGSOC, liver cancer cells and the TNBC cells with acquired PARP-is resistance. We further found that MET phosphorylation is high (immunohistological staining H-score greater than 200) in breast cancer (23 out of 31, 70%) and ovarian cancer (8 out of 23, 35%) patient-derived xenograft mouse model tissue microarrays, suggesting that the combination of MET-is and PARP-is is likely to benefit a huge population of cancer patient in multiple cancer types. Citation Format: Mei-Kuang Chen, Weiya Xia, Qiongzhu Dong, Yi Du, Hung-Ling Wang, Coya Tapia, Yongkun Wei, Ye Han, Yu-Yi Chu, Clinton Yam, Yuan Gao, Yu-Han Wang, Funda Meric-Bernstam, Jinsong Liu, Shao-Chun Wang, Dihua Yu, Mien-Chie Hung. Synergism of PARP inhibitor and MET inhibitor in multiple cancer types with intrinsic and acquired PARP inhibitor resistances [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5682.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are promising targeted therapeutics for breast and ovarian cancers bearing a germline BRCA1/2 mutation (BRCAm), and several have already received regulatory approval in the United States. In patients with a BRCAm cancer, PARPi can increase the burden of unrepaired DNA double-strand breaks by blocking PARP activity and trapping PARP1 onto damaged DNA. Resistance to PARP inhibitors can block the formation of DNA double-strand breaks through BRCA-related DNA repair pathway. MET is a hyper-activated receptor tyrosine kinase expressed in multiple cancer types and the activation contributes to resistance to DNA damage-inducing therapeutic drugs. Our previous study showed that MET inhibition by pan-kinase inhibitors has synergism with PARPi in suppressing growth of breast cancer in vitro and in xenograft tumor models. In this study, we validated the inhibitory effect of novel inhibitors, HS10241 (selective MET inhibitor) and HS10160 (PARPi), to their target respectively in triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSOC) cells. We further demonstrated that these two inhibitors function synergistically in eliminating TNBC and HGSOC cells; combining with HS10241 increased DNA double-strand breaks induced by HS10160 in cancer cells; and PARP1 tyrosine (Y)-907 phosphorylation (PARP1 p-Y907) can be an effective biomarker as an indicator of MET-mediated PARPi in HGSOC. Our results suggest that the combination of HS10241 and HS10160 may benefit patients bearing tumors overexpressing MET as well as those resistant to single-agent PARPi treatment.

Glycosylation of immune receptors and ligands, such as T cell receptor and coinhibitory molecules, regulates immune signaling activation and immune surveillance. However, how oncogenic signaling initiates glycosylation of coinhibitory molecules to induce immunosuppression remains unclear. Here we show that IL-6-activated JAK1 phosphorylates programmed death-ligand 1 (PD-L1) Tyr112, which recruits the endoplasmic reticulum-associated N-glycosyltransferase STT3A to catalyze PD-L1 glycosylation and maintain PD-L1 stability. Targeting of IL-6 by IL-6 antibody induced synergistic T cell killing effects when combined with anti-T cell immunoglobulin mucin-3 (anti-Tim-3) therapy in animal models. A positive correlation between IL-6 and PD-L1 expression was also observed in hepatocellular carcinoma patient tumor tissues. These results identify a mechanism regulating PD-L1 glycosylation initiation and suggest the combination of anti-IL-6 and anti-Tim-3 as an effective marker-guided therapeutic strategy.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Abstract The oncogenic transcription factor Gli1 is a critical effector in the Hedgehog (Hh) pathway, which is necessary for the development and progression of pancreatic ductal adenocarcinoma (PDAC). Although TGFβ and K-Ras are known regulators of Gli1 gene transcription in this setting, it is not understood how Gli1 functional activity is regulated. Here, we report the identification of Gli1 as a substrate for the protein arginine N-methyltransferase PRMT1 in PDAC. We found that PRMT1 methylates Gli1 at R597, promoting its transcriptional activity by enhancing the binding of Gli1 to its target gene promoters. Interruption of Gli1 methylation attenuates oncogenic functions of Gli1 and sensitizes PDAC cells to gemcitabine treatment. In human PDAC specimens, the levels of both total Gli1 and methylated Gli1 were correlated positively with PRMT1 protein levels. Notably, PRMT1 regulated Gli1 independently of the canonical Hh pathway as well as the TGFβ/Kras-mediated noncanonical Hh pathway, thereby signifying a novel regulatory mechanism for Gli1 transcriptional activity. Taken together, our results identified a new posttranslational modification of Gli1 that underlies its pivotal oncogenic functions in PDAC. Cancer Res; 76(23); 7049–58. ©2016 AACR.

Abstract Oncogenic signaling reprograms cancer cell metabolism to augment the production of glycolytic metabolites in favor of tumor growth. The ability of cancer cells to evade immunosurveillance and the role of metabolic regulators in T-cell functions suggest that oncogene-induced metabolic reprogramming may be linked to immune escape. EGF signaling, frequently dysregulated in triple-negative breast cancer (TNBC), is also associated with increased glycolysis. Here, we demonstrated in TNBC cells that EGF signaling activates the first step in glycolysis, but impedes the last step, leading to an accumulation of metabolic intermediates in this pathway. Furthermore, we showed that one of these intermediates, fructose 1,6 bisphosphate (F1,6BP), directly binds to and enhances the activity of the EGFR, thereby increasing lactate excretion, which leads to inhibition of local cytotoxic T-cell activity. Notably, combining the glycolysis inhibitor 2-deoxy-d-glucose with the EGFR inhibitor gefitinib effectively suppressed TNBC cell proliferation and tumor growth. Our results illustrate how jointly targeting the EGFR/F1,6BP signaling axis may offer an immediately applicable therapeutic strategy to treat TNBC. Cancer Res; 76(5); 1284–96. ©2016 AACR.

Nat. Med. 22, 194–201 (2016); published online 18 January 2016; corrected after print 25 August 2016 In the version of this article initially published, the concentrations of H2O2 were incorrectly labeled as micromolar (μM) instead of millimolar (mM) in the legends of Figures 1h, 3c–f and Supplementary Figures 3, 8f–h, 10g,j and 12c,d.