Activated SUMOylation is a hallmark of cancer. Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionarily conserved function of activated ...SUMOylation, which attenuated the immunogenicity of tumor cells. Activated SUMOylation allowed cancer cells to evade CD8+ T cell-mediated immunosurveillance by suppressing the MHC class I (MHC-I) antigen-processing and presentation machinery (APM). Loss of the MHC-I APM is a frequent cause of resistance to cancer immunotherapies, and the pharmacological inhibition of SUMOylation (SUMOi) resulted in reduced activity of the transcriptional repressor scaffold attachment factor B (SAFB) and induction of the MHC-I APM. Consequently, SUMOi enhanced the presentation of antigens and the susceptibility of tumor cells to CD8+ T cell-mediated killing. Importantly, SUMOi also triggered the activation of CD8+ T cells and thereby drove a feed-forward loop amplifying the specific antitumor immune response. In summary, we showed that activated SUMOylation allowed tumor cells to evade antitumor immunosurveillance, and we have expanded the understanding of SUMOi as a rational therapeutic strategy for enhancing the efficacy of cancer immunotherapies.
Patient‐derived xenograft (PDX) models have shown to reflect original patient tumors better than any other preclinical model. We embarked in a study establishing a large panel of head and neck ...squamous cell carcinomas PDX for biomarker analysis and evaluation of established and novel compounds. Out of 115 transplanted specimens 52 models were established of which 29 were characterized for response to docetaxel, cetuximab, methotrexate, carboplatin, 5‐fluorouracil and everolimus. Further, tumors were subjected to sequencing analysis and gene expression profiling of selected mTOR pathway members. Most frequent response was observed for docetaxel and cetuximab. Responses to carboplatin, 5‐fluorouracil and methotrexate were moderate. Everolimus revealed activity in the majority of PDX. Mutational profiling and gene expression analysis did not reveal a predictive biomarker for everolimus even though by trend RPS6KB1 mRNA expression was associated with response. In conclusion we demonstrate a comprehensively characterized panel of head and neck cancer PDX models, which represent a valuable and renewable tissue resource for evaluation of novel compounds and associated biomarkers.
What's new?
Preclinical drug evaluation in head and neck squamous cell carcinoma (HNSCC) is challenged by the inability of established cell lines to predict clinical impact. It may be possible to overcome that problem with patient‐derived xenografts (PDX), which more closely reflect tumor characteristics. Here, a large collection of PDXs were established for HNSCC and tested for therapeutic response. The mTOR inhibitor everolimus was found to be active in a majority of the models. Biomarkers capable of predicting tumor response to everolimus were not identified, though increased expression of RPS6KB1, a member of the mTOR pathway, was common among responders.
According to tumor heterogeneity, enrichment or loss of individual tumor cell types during model generation, some occurring cancer-related mutations were not detected in every sample of the ...respective model. In turn, analyzing the transcriptomes of patient metastases and derived models according to cancer hallmark gene signatures (including DNA repair in general), showed similar patterns of gene set enrichments at transcriptome and proteome level (Fig. Furthermore, gene set enrichment analysis (GSEA) of combined PDX models showing treatment response to 5-FU versus resistant models resulted in significantly enriched gene sets indicating DNA repair (ES = 0.44, p = 0.009), specifically NER (ES = 0.42, p = 0.002), and response to veliparib (ES = 0.62, p < 0.001; Fig. 2F, Fig. For response analysis of combination treatment of the PARP inhibitor olaparib with either 5-FU or trametinib in vitro, we selected pmCRC models according to the list of identified predictive biomarkers (Table S12) and employed different approaches: first we used single cell suspensions of PDX tumor tissues, applied a drug concentration matrix (Fig. 2G) and measured cell cytotoxicity over time.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Patient-derived xenograft (PDX) tumor models are essential for identifying new biomarkers, signaling pathways and novel targets, to better define key factors of therapy response and resistance ...mechanisms. Therefore, this study aimed at establishing pancreas carcinoma (PC) PDX models with thorough molecular characterization, and the identification of signatures defining responsiveness toward drug treatment. In total, 45 PC-PDXs were generated from 120 patient tumor specimens and the identity of PDX and corresponding patient tumors was validated. The majority of engrafted PDX models represent ductal adenocarcinomas (PDAC). The PDX growth characteristics were assessed, with great variations in doubling times (4 to 32 days). The mutational analyses revealed an individual mutational profile of the PDXs, predominantly showing alterations in the genes encoding KRAS, TP53, FAT1, KMT2D, MUC4, RNF213, ATR, MUC16, GNAS, RANBP2 and CDKN2A. Sensitivity of PDX toward standard of care (SoC) drugs gemcitabine, 5-fluorouracil, oxaliplatin and abraxane, and combinations thereof, revealed PDX models with sensitivity and resistance toward these treatments. We performed correlation analyses of drug sensitivity of these PDX models and their molecular profile to identify signatures for response and resistance. This study strongly supports the importance and value of PDX models for improvement in therapies of PC.
BackgroundAdoptive transfer of engineered T cells has shown remarkable success in B-cell malignancies. However, the most common strategy of targeting lineage-specific antigens can lead to undesirable ...side effects. Also, a substantial fraction of patients have refractory disease. Novel treatment approaches with more precise targeting may be an appealing alternative. Oncogenic somatic mutations represent ideal targets because of tumor specificity. Mutation-derived neoantigens can be recognized by T-cell receptors (TCRs) in the context of MHC–peptide presentation.MethodsHere we have generated T-cell lines from healthy donors by autologous in vitro priming, targeting a missense mutation on the adaptor protein MyD88, changing leucine at position 265 to proline (MyD88 L265P), which is one of the most common driver mutations found in B-cell lymphomas.ResultsGenerated T-cell lines were selectively reactive against the mutant HLA-B*07:02-restricted epitope but not against the corresponding wild-type peptide. Cloned TCRs from these cell lines led to mutation-specific and HLA-restricted reactivity with varying functional avidity. T cells engineered with a mutation-specific TCR (TCR-T cells) recognized and killed B-cell lymphoma cell lines characterized by intrinsic MyD88 L265P mutation. Furthermore, TCR-T cells showed promising therapeutic efficacy in xenograft mouse models. In addition, initial safety screening did not indicate any sign of off-target reactivity.ConclusionTaken together, our data suggest that mutation-specific TCRs can be used to target the MyD88 L265P mutation, and hold promise for precision therapy in a significant subgroup of B-cell malignancies, possibly achieving the goal of absolute tumor specificity, a long sought-after dream of immunotherapy.
Abstract
Background: The preclinical evaluation of novel immune checkpoint modulators is dependent on models with functional human immune cells. In previous experiments, we have demonstrated, that we ...can use either peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells (HSC) to establish a humanized immune system on highly immunodeficient mice with functional T, B or NK cells. Furthermore, we determined PD-L1 expression as a predictive marker and target for immunotherapy on different patient-derived xenografts (PDX). By co-transplantation of PDX, we successfully generated a fully human tumor-immune-cell model in mice. Finally, we evaluated the functionality of the model by the treatment with checkpoint inhibitors and for combination therapies i.e. with chemotherapy or radiation.
Methods: HSC-humanized mice (HIS) were generated by i.v. transplantation of HSC. Engraftment of immune cells was monitored by FACS analysis. For PBMC-humanized mice, immune cells were implanted i.v. PD-L1 expression on PDX was determined by FACS and immunohistochemistry (IHC). PDX from 13 different entities (i.e. melanoma, lymphoma) were transplanted on HIS mice and treated with Ipilimumab (Ipi) and Nivolumab (Nivo) alone or in combination with radiation.
Results: 14 weeks after HSC transplantation up to 20% of the human immune cells in the blood were T-cells. We have transplanted more than 40 different PDX from 13 different tumor entities on HIS mice. Most of the investigated PDX (>70%) successfully engrafted and showed no significant difference in tumor growth compared non-humanized mice. However, for some PDX we observed a delayed tumor growth or a complete rejection. Engraftment delay seems to correlate with the PD-L1 expression of PDX (the higher PD-L1, then the higher growth delay). Treatment with Ipi or Nivo alone or in combination led to a minor tumor growth delay and an increased percentage of T-cells in the blood and the tumor. Response showed a correlation to innate immune response and PD-L1 expression of PDX and could further be increased by combination with radiotherapy.
Conclusions: Our humanized immune-PDX models enable appropriate preclinical translational research on tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy.
Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Konrad Klinghammer, Korinna Jöhrens, Wolfgang Walther, Jens Hoffmann. Preclinical models for translational immuno-oncology research: patient-derived xenografts on humanized mice abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2713.
Abstract
Background
The preclinical evaluation of novel immune therapies for cancer remains a challenge, as models require both, engraftment of human tumor cells and a matching human immune cell ...population. In previous experiments, we have demonstrated, that we can use either peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells (HSC) to establish a humanized immune system on highly immunodeficient mice. In this study, we transplanted patient-derived xenografts (PDX) from leukaemia and lymphoma on these humanized mice. Further, we validated the models by evaluating treatments with checkpoint inhibitors Nivolumab (Nivo) and Ipilimumab (Ipi) or standard of care therapies.
Methods
HSC were transplanted i.v. on immunodeficient mice. Engraftment of human immune cells was monitored by FACS analysis of blood samples. Three lymphoma PDX and one acute myeloid leukaemia (AML) model were s.c. transplanted on humanized mice. To model systemic leukaemia, three AML models were transplanted i.v. on humanized mice. Engraftment of the PDX was compared to growth on non-humanized mice. After successful engraftment, mice were treated with Nivo, Ipi or in combination with standard therapies and irradiation.
Results
We confirmed successful engraftment of HSC in the humanized mice. Within 14 weeks after HSC inoculation, FACS analysis revealed an established functional human immune system with up to 20% T-cells, characterized by a high PD-1 expression. The s.c. transplanted leukaemia and lymphoma PDX engrafted on the humanized mice and tumor growth was measured with calipers. Engraftment of i.v. transplanted AML models was confirmed by FACS measurement of human CD33+ AML cells in the blood of the humanized mice. Treatment with Ipi or Nivo alone or in combination led to a minor growth delay. FACS analysis confirmed an increased percentage of activated T-cells in the blood and in the s.c. tumors. Response to checkpoint inhibitors was in correlation to PD-L1 expression on the lymphoma and AML cells. Combination of check point inhibitors with radiotherapy provided additive effects in our models.
Conclusions
We developed a humanized immune-PDX model for different blood cancers enabling appropriate preclinical translational research on tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy. These novel model are currently optimized for the preclinical evaluation of new bispecific immune cell engagers (BITE) and cell therapies (CART).
Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Antje Siegert, Bernadette Brzezicha, Anja Sterner-Kock, Wolfgang Walther, Jens Hoffmann. Leukaemia and lymphoma patient-derived xenografts (PDX) engraft on humanized mice and respond to immune therapy with check point inhibitors 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 5631.
Abstract
Malignant mesothelioma is a rare but aggressive disease with few therapeutic options and a poor prognosis. The main cause of the disease is the exposition to asbestos, even after 50 years of ...latency. Intended therapeutic goals, such as increase of patient survival and improvement of quality of life have only been achieved to a limited extent. Curative treatment is currently still not possible. Therefore, there is a strong clinical need for translational models. For this, we have characterized patient-derived xenograft models (PDX) of mesothelioma to evaluate their suitability for preclinical research. For PDX establishment, tumor tissue from surgically resected pleura or peritoneum mesotheliomas, was implanted subcutaneously into immunodeficient mice and serially passaged for at least 3 in vivo-passages. Established PDX models were characterized towards sensitivity to standard of care drugs. PDX tissue was collected for RNA sequencing and immunohistochemistry. The generated molecular profiles (comparative gene expression and sequence variations) of the models were analyzed comprehensively. A panel of 6 mesothelioma PDX has been established. In these PDX models, characteristic histological structure of the original patient tumor was preserved. The PDX models responded individually to standard of care drugs like pemetrexed, cisplatin, and gemcitabine as well as combinations thereof. The combination of pemetrexed or gemcitabine with cisplatin showed a stronger tumor growth inhibition than the respective monotherapies. The analyzed mutational and gene expression landscape resembles the known pattern of mesotheliomas. In summary, our established mesothelioma PDX will serve as a preclinical tool to investigate cellular and molecular mechanisms of drug sensitivity and resistance. They will provide a valuable preclinical model system to identify and validate biomarkers and to develop new therapeutic approaches.
Citation Format: Michael Becker, Bernadette Brzezicha, Jana Rolff, Theresia Conrad, Beate Rau, Wolfgang Walther, Jens Hoffmann. Preclinical models from rare aggressive tumors: Establishment, drug sensitivity and genomic analyses of mesothelioma PDX as models for oncology drug development abstract. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A006. doi:10.1158/1535-7163.TARG-19-A006
Abstract
Background: The preclinical evaluation of novel immune modulators for cancer treatment remains a challenge, as models require both, engraftment of human tumor cells and a compatible human ...immune cell population. In previous experiments, we have demonstrated, that we can use either peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells (HSC) to establish a humanized immune system with functional T-, B-, and NK cells, monocytes, and dendritic cells on highly immunodeficient mice. However these models are limited by rarely matching HLA isotypes between tumor and immune cells. In this case study, we established a patient-derived xenograft (PDX) model from a patient with head and neck squamous cell cancer (HNSCC). Furthermore, we collected blood samples and isolated PBMCs. After transplantation and engraftment of HNSCC PDX, patients PBMC were used to humanize mice. By this procedure we successfully generated a patient-specific human tumor-immune cell model in mice with 100% HLA-match. Model development included the comparison of PDX engraftment on mice with either HLA-matching or non HLA-matching PBMC’s from different donors. Finally, we further validated the model by comparing treatment effects with the checkpoint inhibitor Nivolumab in the autologous immune cell PDX model with the heterologous models. Methods: The HNSCC PDX HN15239 was transplanted on NOG mice. After tumor engraftment mice were randomized in 6 groups, receiving PBMCs by i.v. transplantation either from the patient or from 5 well characterized donors (PDX patient PBMCs – 100% HLA matching, 5 donors with different grade of HLA matching). Blood and tumor samples were analysed by FACS and IHC for immune cell infiltration and activation. Results:In the autologous huPBMC model, no interference with the proliferation of HN15239 PDX was seen. However, on mice humanized with donor PBMC’s with a high HLA match, a strong stimulation of tumor proliferation compared to non-humanized mice was observed. Surprisingly, treatment with Nivolumab did not induce a significant tumor growth inhibition in the autologous model system. On the mice humanized with PBMC from different donors, we observed a correlation of treatment effects with HLA match, with strong tumor growth inhibition in the mice with the best match. In the PDX tumors, infiltrating immune cells were detected by FACS and IHC analyses. Conclusions: We developed a humanized immune-PDX model enabling appropriate preclinical translational research on tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy. Furthermore, results showed a correlation between immune therapy effect and HLA matching in preclinical models.
Citation Format: Maria Stecklum, Konrad Klinghammer, Annika Wulf-Goldenberg, Bernadette Brzezicha, Korinna Jöhrens, Jens Hoffmann. Preclinical case study: Patient-derived head and neck cancer xenograft on mice humanized with autologous immune cells, a model for personalized immuno-oncology research abstract. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A013. doi:10.1158/1535-7163.TARG-19-A013
Abstract
Breast cancer is a highly heterogeneous disease, with a heterogeneous prognosis. Patient-derived xenograft models (PDX) can reflect this heterogeneity and can be used to support the ...development of novel therapeutic strategies against breast cancer. Preclinical experiments with larger cohorts of breast cancer PDX can be used to model a clinical phase II study with new drugs or drug combinations. The predictive value of these preclinical trials is currently evaluated in co-clinical trials, where treatment effects are compared between patients and their corresponding PDX. We present results from our translational pre- and co-clinical studies in breast cancer immune therapy.
As part of the EFRE-POP Project, we established PDX by collecting breast cancer tissue samples from surgery and engrafting them on immunodeficient mice. The tissues were obtained from patients with disease progression after chemotherapy with three to four drugs. PDXs have been successfully engrafted, phenotypically characterized and drug-screened. The PDXs were tested for response to the standard of care therapies. The staining for estrogen/progesterone/androgen/Her2 receptors, Ki-67, and CK5/6 of the original tumor and the PDX were comparable. The patient tumors and the breast cancer PDX models were further analyzed for the expression of PD-L1. We identified four patients that had received PD-1 or PD-L1 antibodies after a sample (used to establish a corresponding PDX) was obtained.
To evaluate the response of our preclinical PDX models to immune checkpoint inhibitors, we engrafted mice with a human immune system either by transplanting allogeneic hematopoietic stem cells (HSC) or allogenic PBMC's from healthy donors. Besides, we were able to obtain PBMC's from the patients to set up an autologous model. The patient-derived tumors were transplanted to these humanized mice and after successful engraftment treated with the PD-1 or PD-L1 antibodies corresponding with the clinical treatment of the patient.
In summary, humanized PDX of refractory tumors have been established and the first pre-clinical evaluation of response to immune therapy provides evidence that xenografts respond in a qualitatively similar manner as the patient. This suggests a translational relevance of the described pre-clinical models.
Citation Format: Annika Wulf-Goldenberg, Verena Kiver, Maria Stecklum, Bernadette Brzezicha, Philipp Jurmeister, Caroline Schweiger, Jens-Uwe Blohmer, Ulrich Keilholz, Jens Hoffmann. Breast cancer patient-derived xenograft models for pre- and co-clinical investigation of immune based therapies 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 5059.
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