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Supplementary Figure from PD-1 Blockade in Solid Tumors with Defects in Polymerase Epsilon

Supplementary Data from PD-1 Blockade in Solid Tumors with Defects in Polymerase Epsilon

Missense mutations in the polymerase epsilon (POLE) gene have been reported to generate proofreading defects resulting in an ultramutated genome and to sensitize tumors to checkpoint blockade immunotherapy. However, many POLE-mutated tumors do not respond to such treatment. To better understand the link between POLE mutation variants and response to immunotherapy, we prospectively assessed the efficacy of nivolumab in a multicenter clinical trial in patients bearing advanced mismatch repair–proficient POLE-mutated solid tumors. We found that only tumors harboring selective POLE pathogenic mutations in the DNA binding or catalytic site of the exonuclease domain presented high mutational burden with a specific single-base substitution signature, high T-cell infiltrates, and a high response rate to anti–PD-1 monotherapy. This study illustrates how specific DNA repair defects sensitize to immunotherapy. POLE proofreading deficiency represents a novel agnostic biomarker for response to PD-1 checkpoint blockade therapy.Significance:POLE proofreading deficiency leads to high tumor mutational burden with high tumor-infiltrating lymphocytes and predicts anti–PD-1 efficacy in mismatch repair–proficient tumors. Conversely, tumors harboring POLE mutations not affecting proofreading derived no benefit from PD-1 blockade. POLE proofreading deficiency is a new tissue-agnostic biomarker for cancer immunotherapy.See related video: https://vimeo.com/720727355This article is highlighted in the In This Issue feature, p. 1397

Detection of cells expressing CD44 and ALDH activity in gastric tumorspheres in vitro.

Effect of verapamil treatment in combination with dose-dependent chemotherapies on gastric cancer cell lines viability.

Detection of ALDH+ population with SP protocol and with ALDEFLUOR protocol.

Supplementary experimental procedures,figure legends and references

Inactivation of beta-2 microglobulin (B2M) is considered a determinant of resistance to immune checkpoint inhibitors (ICPi) in melanoma and lung cancers. In contrast, B2M loss does not appear to affect response to ICPis in mismatch repair–deficient (MMRd) colorectal tumors where biallelic inactivation of B2M is frequently observed. We inactivated B2m in multiple murine MMRd cancer models. Although MMRd cells would not readily grow in immunocompetent mice, MMRd B2m null cells were tumorigenic and regressed when treated with anti–PD-1 and anti-CTLA4. The efficacy of ICPis against MMRd B2m null tumors did not require CD8+ T cells but relied on the presence of CD4+ T cells. Human tumors expressing low levels of B2M display increased intratumoral CD4+ T cells. We conclude that B2M inactivation does not blunt the efficacy of ICPi in MMRd tumors, and we identify a unique role for CD4+ T cells in tumor rejection. Significance: B2M alterations, which impair antigen presentation, occur frequently in microsatellite-unstable colorectal cancers. Although in melanoma and lung cancers B2M loss is a mechanism of resistance to immune checkpoint blockade, we show that MMRd tumors respond to ICPis through CD4+ T-cell activation. This article is highlighted in the In This Issue feature, p. 1601

Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5742.

Background: Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). Due to western way of life, NASH incidence is rising and predicted to become the leading cause of liver transplant in 2020 and of HCC in the next decades. There is an urgent need for robust animal models fully recapitulating the NASH-related HCC carcinogenesis due to this changing in HCC etiology. In this study, we develop and characterize specific diet-induced variants from our transgenic HCC mouse model, focusing on immune landscape. Methods: We used in the whole study ASV-B mice: a transgenic mouse model (C57BL/6J) spontaneously developing a reproducible stage-defined HCC (hyperplasia at week(W)8, nodular stage at W12, and diffuse carcinoma at W16-20). Livers were characterized for angiogenesis and immune populations using immunostaining (IHC and IF), and qRT-PCR (LightCycler, Roche). To mimic NASH, ASV-B mice were exposed to 5 different diets. Ten ASV-B and 5 control mice were fed as follows: classic diet as control (yellow), or a high-fat diet (blue), a diet enriched with saturated fatty acids + 1.25% cholesterol (green), a diet containing 22% of vegetal oil + 0.2% cholesterol (orange), and a 1.25% cholesterol diet containing 21% of milkfat (red). All mice fed with special diets also received 30% fructose in the drink water. A second experiment was performed on ASV-B and C57BL/6J wild type mice, using control and Nash-inducing regimen, to confirm our first results. Results: ASV-B model showed an increase in liver volume and angiogenesis, HCC livers harboring marked arterialization and capillarization as compared to control. Assessing immune markers on 7 evaluable tumor specimens, we observed an increase in CD8, Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A and IL17F mRNA expressions, as frequently observed in human inflammatory HCC. In addition, IHC staining showed intratumoral infiltration of lymphocytes (CD8+) and macrophages (F4/80+, a well-characterized and extensively referenced mouse macrophage marker). ASV-B mice receiving yellow, blue, and green regimens showed similar liver volumes and weights. By macroscopic analysis, we observed increased liver steatosis, and fibrosis in the red and orange regimen compared to others. Moreover, we observed a 40% mortality rate in the orange regimen, and a 20% mortality rate in the blue and green regimens. Interestingly, by microscopic analysis, we observed liver steatosis and inflammation, in 100%, and 75% of the mice, respectively. These results indicate that the green regimen is the most suitable to induce NASH-underlyning disease in our transgenic HCC model. At the conference, we will show in the diet-variants, the immune landscape of the livers and the results of our ongoing second experiment. Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including inflammatory reaction and immune cell infiltration. In the ASV-B model, we have been able to develop specific-diets variants mimicking NASH characteristics that could be used for drug testing. Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Philippe Bonnin, Benoit Rousseau, Aurélie Rodrigues, Fouad Ladfil, Marc Pocard, Armand de Gramont, Eric Raymond, Sandrine Faivre, Valérie Paradis, Clarisse Eveno. Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B) [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 1636.

Introduction: MBC cancer patients may have prolonged survival, and MBC cancers can be considered as a chronic disease. The main goal of the present study was to describe the clinico-biological features of patients surviving more than two years with MBC. Method: During 4 months, we conducted a national multicentric survey about patients aged ≥ 18 in metastatic setting (all solid tumors) for more than 24 months. Clinico-biological data from 200 patients were collected in 39 French centers. Preliminary results of MBC patients (N=88, 87 women/one man) are presented. Results: Most of them were ductal carcinoma (88%), expressing hormonal receptor HR (77%). 43% of tumors overexpressed HER2 (HER2+ tumors: 43%; Triple negative tumors: 6%). Median age at MBC diagnosis was 53 years [29-85]. 18% had metastatic disease at diagnosis and 82 % were localized with a disease-free survival of 65 months [4-312]. Median time of MBC disease was 4,5 years [2-20]. At the time of MBC diagnosis, 64% of patients were not single; 55% were working while 30% were retired. At data collection, 89% of non-single patients were not separated, and 43 % of working patients at diagnosis were still working. Mean number of treatment lines in advanced disease was 4.7 [1-13]. 89% of MBC patients received at least one chemotherapy, 68% hormonotherapy, 70% targeted therapy and 38% had been included in at least one clinical trial. 97% of patients had a local treatment of their primary tumor. Concerning metastasis, 23% had a surgical treatment and 40% radiotherapy treatment. 80% of patients remain with PS of 0 or 1. Only 9% of patients were followed by a palliative care team, 24% by a psychologist and 23% by a nutritionist. Conclusion: Our preliminary results of suggest that an important proportion of MBC cancer patients who live more than 2 years are young, have been treated with chemotherapy, hormonal and targeted therapy, have participated to clinical trials and still have good performance status. No change in marital status was observed. Half of working patients at MBC diagnosis continue to work. Few of them received palliative care. This study may help to better describe long-term survivors with MBC, and socio-medical burden as cancer became a chronic disease. Citation Format: Delphine Loirat, Camille Tlemsani, Jennifer Arrondeau, Audrey Bellesoeur, Christophe Le Tourneau, Benoit Rousseau. Clinico-biological characteristics of patients surviving more than two years with metastatic breast cancer (MBC): Results of a transversal national multicentric survey [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-15-12.

Background: Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). Due to western way of life, NASH incidence is rising and is predicted to become the leading cause of HCC in the next decades. Therefore, there is an urgent need for robust animal models fully recapitulating the NASH-related HCC carcinogenesis. In this study, we develop and characterize specific diet-induced variants from our transgenic HCC mouse model, focusing on immune landscape. Methods: To mimic NASH, ASV-B, a transgenic mouse model (C57BL/6J) that spontaneously develops a reproducible stage-defined HCC (hyperplasia at week(W)8, nodular stage at W12, and diffuse carcinoma at W16-20) was exposed to 5 different diets. Ten ASV-B and 5 control mice were fed as follows: classic diet as control (yellow), or a high-fat diet (blue), a diet enriched with saturated fatty acids + 1.25% cholesterol (green), a diet containing 22% of vegetal oil + 0.2% cholesterol (orange), and a 1.25% cholesterol diet containing 21% of milkfat (red). All mice fed with special diets also received 30% fructose in the drink water. RNA was extracted from frozen livers at W20 for 40 immune markers analysis using qRT-PCR (LightCycler, Roche). Immune populations were assessed using automated immunohistochemistry (IHC) (Bond Max, Leica). Results: ASV-B model shows an increase in liver volume and angiogenesis, ASV-B livers harboring marked arterialization and capillarization as compared to control. Assessing immune markers on 7 evaluable tumor specimens, we observed an increase in CD8, Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A and IL17F mRNA expression, as frequently observed in human inflammatory HCC. In addition, IHC staining showed intratumoral infiltration of lymphocytes (CD8+) and macrophages (F4/80+, a well-characterized and extensively referenced mouse macrophage marker). ASV-B mice receiving yellow, blue, and green regimens showed similar liver volumes and weight. By macroscopic analysis, we observed increased liver steatosis, and fibrosis in the red and orange regimen compared to others. Moreover, we observed a 40% mortality rate in the orange regimen, and a 20% mortality rate in the blue and the green regimens. At the conference, we will show the morphologic changes of the livers using HPS staining and the immune landscape in the livers of the diet-variants. Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including inflammatory reaction and immune cell infiltration. In the ASV-B model, we have been able to develop specific-diets variants aiming at mimicking NASH that could be used for drug testing. Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Benoit Rousseau, Patricia Hainaud, Philippe Bonnin, Aurélie Rodrigues, Fouad Ladfil, Marc Pocard, Valérie Paradis, Armand de Gramont, Eric Raymond, Evelyne Dupuy, Clarisse Eveno, Sandrine Faivre. Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 91.

Driver molecular alterations are found in >20% of non-small cell lung cancers (NSCLCs). They specifically target the RAS-MAPK pathway, including the EGFR, KRAS, and BRAF oncogenes. NF1 is a tumor suppressor gene that encodes neurofibromin, an inhibitor of the RAS-MAPK pathway. NF1 mutation detection is challenging owing to its large size, the presence of numerous pseudogenes, and the absence of mutation hotspot. According to The Cancer Genome Atlas data (TCGA), NF1 somatic mutations are found in ~15% of lung cancer. However, NF1 mutations in NSCLCs are not extensively explored in NSCLC to date. We hypothesized that NF1 alterations could define a specific NSCLC population with distinct clinical and molecular profiles. We performed NF1 analysis using next-generation sequencing in NSCLC surgical specimens with known KRAS, EGFR, TP53, BRAF, HER2, and PIK3CA status. We evaluated the specificities of NF1-mutated NSCLCs. Then, we established of NF1-mutated cellular models with different NF1 wild-type (WT) cell lines. We chose two NSCLC cell lines (A549 and NCI H-1703, ATCC), and one nontumorigenic human bronchial epithelial cell line (HBE4-E6/E7-C1, ATCC). Mono- and biallelic NF1 mutations were established using CRISPR-Cas9 and nickase CRISPR-Cas9 technologies. In vitro functional tests and drug screening were performed using these isogenic cell models. In our series of 138 lung adenocarcinoma specimens, 25 tumors showed NF1 mutations (18%) and 11 showed NF1 deletions (8%). NF1 mutations were rarely associated with other mutations. Most of patients with NF1 alterations were males (72%) and smokers (75%). Overall survival and disease-free survival were statistically better in patients with NF1 alteration patients (N=35) than in KRAS mutated patients (N=30) in univariate analysis. There were more NF1 mutations in patients treated by neoadjuvant chemotherapy (p = 0.01). Then, we established cellular models of NF1-mutated NSCLC, using nickase and CRISPR-Cas9 technology. In HBE4-E6/E7-C1 cells, mono- and biallelic NF1 mutations were generated. Loss of NF1 expression was confirmed by Western blot: partial and total loss-of-expression of neurofibromin was found in monoallelic and biallelic NF1 mutated cell lines, respectively. Using Western blot, we showed that pERK/ERK ratio was higher in NF1-mutated cell lines versus WT cell lines, confirming that NF1 loss-of-function triggered RAS-MAPK pathway activation. Transcriptome analysis confirmed this activation. Pharmacologic screen (including MEK inhibitors) in this isogenic NSCLC model will enable us to assess specific vulnerabilities due to NF1 mono- or biallelic mutations. Our results confirm that NF1 is frequently mutated and represents a distinct subtype of NSCLCs. A better comprehension of functional consequences of NF1 mutations, including mono- and biallelic alterations, may open new avenues for NSCLC therapy. Citation Format: Camille Tlemsani, Nicolas Pecuchet, Aurélia Gruber, Audrey Mansuet-Lupo, Diane Damotte, Marco Alifano, Jennifer Varin, Ingrid Laurendeau, Armelle Luscan, Benoit Rousseau, Beatrice Parfait, Ivan Bieche, Audrey Briand, Benoit Terris, Helene Blons, Karen Leroy, Dominique Vidaud, Michel Vidaud, Pierre Laurent-Puig, Eric Pasmant. Characterization of molecular and functional consequences of somatic NF1 mutations in non-small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5511.

We describe herein a new transgenic mouse tumor model in which fibroblast growth factor (FGF) receptor activity is selectively inhibited. Tyrp1-Tag mice that develop early vascularized tumors of the retinal pigment epithelium were crossed with tyrp1-FGFR1-DN mice that express dominant-negative FGF receptors in the retinal pigment epithelium to generate bigenic mice. Initial angiogenesis-independent tumor growth progressed equally in tyrp1-Tag and bigenic mice with no significant differences in the number of dividing and apoptotic cells within the tumor. By contrast, at a later stage when tyrp1-Tag tumors rapidly expanded to fill the entire eye posterior chamber and migrate along the optic nerve toward the chiasma, bigenic tumors remained small and were poorly vascularized. Secondary tumors of small size developed in only 20% of bigenic mice by 1 month. Immunohistochemical analysis of secondary tumors from bigenic mice showed a reduction of angiogenesis and an increase in apoptosis in tumor cells. Tumor cells from bigenic mice expressed high levels of truncated FGF receptors and did not induce endothelial tube formation in vitro. All in all, this indicates that the tyrp1-Tag mouse may be a useful model to study selective tumor inhibition and the effect of antitumor therapy that targets a specific growth factor pathway. FGF receptors are required at the onset of tumor invasion and angiogenesis in ocular tumors and are good therapeutic targets in this model. The bigenic mouse may also constitute a useful model to answer more fundamental questions of cancer biology such as the mechanism of tumor escape.