Search

Supplementary Data from In Vivo Syngeneic Tumor Models with Acquired Resistance to Anti–PD-1/PD-L1 Therapies

Antibodies targeting PD-1 and PD-L1 have produced durable responses in a subset of patients with cancer. However, a majority of these patients will ultimately relapse due to acquired resistance. To explore the underlying mechanisms of this secondary resistance, we developed five syngeneic murine tumor variants with acquired resistance to anti–PD-1 and/or PD-L1 antibodies in vivo. Resistant in vivo models were obtained by serial treatment/reimplantation cycles of the MC38 colorectal, MB49 and MBT2 bladder, and RENCA kidney and TyrNras melanoma models. Tumor immune infiltrates were characterized for wild type and resistant tumors using spectral cytometry and their molecular alterations analyzed using RNA sequencing analyses. Alterations in the tumor immune microenvironment were strongly heterogeneous among resistant models, involving select lymphoid and/or myeloid subpopulations. Molecular alterations in resistant models included previously identified pathways as well as novel candidate genes found to be deregulated in several resistant models. Among these, Serpinf1, coding for pigment epithelial-derived factor (PEDF) was further explored in the MC38 and the MBT2 models. Overexpression of Serpinf1 induced resistance to anti–PD-1 antibodies in the MC38 model, whereas knockdown of Serpinf1 sensitized this model as well as the primarily resistant MBT2 model. Serpinf1 overexpression was associated with increased production of free fatty acids and reduced activation of CD8+ cells, while orlistat, a compound that reduces the production of free fatty acids, reversed resistance to anti–PD-1 therapy. Our results suggest that a panel of syngeneic resistant models constitutes a useful tool to model the heterogeneity of resistance mechanisms encountered in the clinic.

The underlying mechanisms of acquired resistance to the immune checkpoint inhibitory (ICI) PD-1 and PD-L1 antibodies remain largely unknown and need to be further explored. In this study, we established and analyzed robust ICI resistance in in vivo models in order to identify new mechanisms and genes involved in acquired resistance. Resistant in vivo models were obtained by serial treatment/reimplantation cycles in immunocompetent mice bearing MC38, MB49, MBT2, TyrNRas or RENCA tumors. Spectral cytometry was applied to characterize modifications in the tumor immune microenvironment. RNAseq analyses were performed to identify differences in gene expression profiles between sensitive parental models compared to their resistant counterparts. Alterations in the tumor immune microenvironment were highly heterogeneous amongst resistant models, and are thus considered representative for the diversity expected in patients. Each resistant model displayed multiple modifications in the tumor immune infiltrate in comparison to the respective wild type model, involving selected lymphoid and/or myeloid subpopulations. For instance, in anti-PD-1 and anti-PD-L1 resistant MC38 models, we observed a remarkable increase of FoxP3+CD4+ T cells while CD8+ Tem cells were decreased in anti PD-1 resistant models. We also found that the type 2 macrophage content was increased in the anti PD-1 resistant MB49 model. Gene profiling analyses demonstrated the variability associated with acquisition of a resistance phenotype. Alterations of pathways previously reported to be associated with resistance to ICIs were observed in some of our models while other genes appeared to be highly de-regulated in several models. For instance, in the anti PD-L1 resistant MBT2 model many signaling pathways were found to be up-regulated, such as HIF1α, TGFβ, and ERK/MAPK signaling. Several therapeutic combinations, in particular targeting alternative immune checkpoints, were found to reverse the resistance phenotype in selected models. Moreover, in accordance with flow cytometry results, in the anti PD-1 resistant MC38 model we observed a significant delay in tumor growth when a TNFR1 antibody was combined with a PD-1 antibody (p Citation Format: Morgane Denis, Chloé Grasselly, Pierre-Antoine Choffour, Anne Wierinckx, Doriane Mathé, Kamel Chettab, Anne Tourette, Nolan Talhi, Fabian Birzele, Elsa Kress, Lars Petter Jordheim, Christian Klein, Eva-Laure Matera, Charles Dumontet. Characterization of acquired resistant models to therapies targeting the PD-1/PD-L1 axis demonstrates model-dependent mechanisms [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 4200.

International audience; Phosphatidylinositide 3-kinases (PI3Ks) are widely expressed enzymes involved in membrane signalization pathways. Attempts to administer inhibitors with broad activity against different isoforms have failed due to toxicity. Conversely the PI3K delta isoform is much more selectively expressed, enabling therapeutic targeting of this isoform. Of particular interest PI3K delta is expressed in human basophils and its inhibition has been shown to reduce anti-IgE induced basophil degranulation, suggesting that PI3K delta inhibitors could be useful as anti-allergy drugs. Herein, we report for the first time the activity of compounds derived from chalcone scaffolds as inhibitors of normal human basophil degranulation and identified the most active compound with anti-PI3K delta properties that was investigated in preclinical models. Compound 18, namely 1-[2-hydroxy-4,6-dimethoxy-3-(N-methylpiperidin-4-yl)pheny1]-3(2,4,6-trimethoxypheny1)-prop-2-en-1-one, was found to inhibit normal human basophil degranulation in a dose-dependent manner. In a murine model of ovalbumin-induced asthma, compound 18 was shown to reduce expiratory pressure while its impact on the inflammatory infiltrate in alveolar lavage and total lung was dependent on the route of administration. In a DNFB-induced model of atopic dermatitis compound 18 administered systemically proved to be as potent as topical betamethasone. These results support the anti-atopic and allergic properties of the title compound and warrant further clinical development. (C) 2018 Elsevier Masson SAS

Netrin-1 was first described as an axonal chemoattractant in peripheral nervous system leading to axon outgrowth. Later on, after well characterizing its receptors, deleted in colorectal cancer (DCC) and uncoordinated-5 (UNC5), and their role in apoptosis, they were classified as dependence receptors. Dependence receptors belong to a family of receptors distinguished from other classical receptors by their capability of inducing cell death in the absence of their respective ligands. Their importance in tumorigenesis suggest that this family of receptors along with their ligands could constitute a novel target in cancer therapy. Recent studies showed upregulation of netrin-1 and its receptors in lung cancer tumors after treatment with conventional chemotherapies. The aim of our study was to analyze the expression of netrin 1 and its receptors UNC5B and DCC in multiple myeloma and to determine the impact of a monoclonal antibody targeting netrin-1 on myeloma cell lines in vitro and in vivo. This antibody, NP137, produced by NetrisPharma, is now in early phase clinical trials. In the first place, we performed a screening of various human myeloma cell lines, including AMO-1, LP-1, MM1S, RPMI8226, U266 and OPM-2, for netrin-1 and its different receptors using RT-qPCR and western blotting. We also analyzed the expression of netrin 1 and its receptors in fresh bone marrow aspirates by flow cytometry and in bone marrow biopsies using immunohistochemistry obtained from patients with multiple myeloma. Our results showed that myeloma cell lines expressed netrin-1 and its receptors DCC and UNC5B as determined both by RTqPCR and by western blotting. This expression was also analyzed in patient samples with 16 bone marrow biopsies using immunohistochemistry and in 10 fresh bone marrow aspirates by flow cytometry. Netrin-1 was identified by IHC in 6/16 samples while DCC and UNC5B were identified in all and in 13 samples, respectively. In fresh samples, both netrin-1 and its receptors were found to be more highly expressed than in normal lymphocytes. The exposure of myeloma cell lines to anti-netrin-1 humanized antibody in vitro did not induce a significant decrease in cell viability in vitro, whereas treatment of SCID mice with established RPMI8226 myeloma tumors lead to a significant reduction of tumor size in comparison to controls, suggesting a possible targeting of netrin 1 in this disease. Combination of NP137 with conventional anti-myeloma agents will be presented. In conclusion, these results suggest that netrin 1 signaling could constitute a novel original target in multiple myeloma. Citation Format: David Fahed, Kamel Chettab, Patrick Mehlen, Doriane Mathe, Anne Tourette, Morgane Denis, Alexandra Traverse-Glehen, Lionel Karlin, Charles Dumontet. Netrin-1 expression and targeting in multiple myeloma [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 5291.

Immune checkpoint inhibitors (ICIs) show impressive response rate in multiple cancer types. Nivolumab and atezolizumab has already been approved by the FDA. However, a large proportion of patients do not show any benefits or will develop acquired resistance phenomenon. In order to increase long term survival, alternative strategies are focused on rationale combination of immunotherapies with well-known therapeutic agents such as chemotherapies. In this present project, we described the impact of various chemotherapies regimen in combination with anti-PD1 and anti-PDL-1 on preclinical bladder, colorectal and metastatic breast cancers models. We performed a study of PD-1 or PDL-1 blockade in combination with reference chemotherapies in four fully immunocompetent mouse models of cancer: we used the MVAC (methotrexate, vinblastine, doxorubicin, cisplatin) regimen as standard chemotherapy in bladder cancer MBT-2 and MB49, cyclophosphamide and doxorubicin in breast cancer 4T1, and capecitabine with oxaliplatin in colorectal cancer MC38. We analyzed the in vivo antitumor response and collected tumor samples four days after first treatment administration to analyze the immune infiltrate in tumors. In vivo phenotype study reveals variable responsiveness to ICP blockade. We found strong effects of the combinations in two of the models studied: bladder cancer MB49 and colorectal cancer MC38, a lack of effect in breast cancer 4T1, and an inhibition of immune checkpoint antibody activity in MBT-2 preclinical model. Analysis using flow cytometry showed strong differences both at baseline between mice models as well as after exposure to ICIs, chemotherapy or the combination of both types of treatment. All the preclinical models studied at baseline were highly infiltrated with cells considered as « immunosuppressive », particularly MDSCs. We showed that the balance between antitumor effector cells and immunosuppressive cells could be inverted with some of the treatment combinations. As an example, in the MB49 model the most impressive modifications induced by the combination was an increase of the M1/M2 ratio. However, profound alterations of tumors microenvironment were not systematically correlated with a favorable in vivo phenotype. Indeed, we observed no benefit of the combination in the 4T1 model, although the combination profoundly modified the immune microenvironment with a strong decrease in MDSCs and a large increase in the CD4 and CD8 infiltrates. Our results suggest that modulation of immune cells infiltrate by cytotoxic agents could impact on the potency of ICIs when these are co-administered, with either an enhance or a reduced antitumor activity of the combination. However this impact appears to be model- and combination-dependent. The present investigations should help to select appropriate chemotherapies to combine with target monoclonal antibodies for clinical trials. Citation Format: Chloé Grasselly, Morgane Denis, Aurore Bourguignon, Nolan Talhi, Anne Tourette, Doriane Mathe, Laurent Serre, Lars Petter Jordheim, Charles Dumontet. In vivo activity of combinations of cytotoxic regimens with anti-PD1 and anti-PDL1 in various syngeneic cancer models [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 2723.