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Initiation and sustainment of oncogenic signaling is a hallmark of cancer evolution and progression. In renal clear cell carcinoma, loss of von Hippel-Lindau protein causes stabilization of hypoxia-inducible transcription factors (HIF) evoking a pseudo-hypoxic response, perturbing epithelial homeostasis and leading to cancer development. Although genetic polymorphisms link the EPAS1 oncogene (coding for HIF-2α) to renal cancer and anti-HIF-2 compounds emerge as renal tumor therapies, little is known about transcriptional dysregulation of this factor in renal malignancies. We use genetic, epigenetic and transcriptomic data from large patient cohorts and cell models to dissect mechanisms of augmented EPAS1 transcription in clear cell renal cell carcinoma. We define an oncogenic enhancer of EPAS1 which operates depending on the presence of HIF and renal lineage-specific factors, thereby providing evidence for an auto-regulatory feed-forward circuit of HIF-2α regulation which promotes renal cancer growth. The mechanisms of increased EPAS1 transcription in clear cell renal cell carcinoma remain to be explored. Here, the authors propose a regulatory circuitry of EPAS1 expression which is dependent on HIF and lineage-specific transcription factors.

Chimeric antigen receptor (CAR) T-cell therapy has become a standard-of-care in oncology, yet standardized monitoring of circulating CAR T cells remains a major challenge due to diverse CAR constructs and limited availability of detection reagents. The antibody domain of the CAR commonly consists of the heavy and light chain connected through a linker, typically either 4x glycine and 1x serine (G4S) or a defined amino acid sequence (Whitlow/218). Here, we evaluated the novel monoclonal antibodies (mAbs) targeting the linker sequence as a universal tool for CAR detection. Using flow cytometry, we compared anti-linker mAbs with conventional reagents, including anti-idiotype CD19.FMC63 mAb, CD19 and BCMA antigen-based detection reagents (Ag), and anti-F(ab') mAb. Analyses were performed on commercial CD19- and BCMA-directed CAR T-cell products and an investigational Claudin-6 (CLDN6) CAR T-cell product. Performance was further assessed across diverse experimental platforms for clinical monitoring and in a murine model to evaluate sensitivity and translational relevance. Linker-mAbs detected all tested CAR constructs with high specificity and sensitivity, matching target-specific binding with conventional Ag reagents. Anti-linker mAbs demonstrated minimal background and low limits of quantification both comparable to Ag reagents. Longitudinal monitoring in lymphoma and myeloma patients revealed consistent CAR T-cell kinetics between anti-linker mAbs and Ag reagents. High performance of linker-based CAR-detection was demonstrated in high-dimensional, multi-parameter flow cytometry, in immunofluorescence imaging and in a murine model of anti-CD19 CAR T cells. These findings establish anti-Whitlow/218 and anti-G4S mAbs as sensitive, specific, and universal reagents for CAR detection across multiple targets, constructs, and species, providing a standardized platform for harmonization of CAR T-cell monitoring in preclinical, clinical trial, and diagnostic settings.

Cellular stress responses including the unfolded protein response (UPR) decide over the fate of an individual cell to ensure survival of the entire organism. During physiologic UPR counter-regulation, protective proteins are upregulated to prevent cell death. A similar strategy induces resistance to UPR in cancer. Therefore, we hypothesized that blocking protein synthesis following induction of UPR substantially enhances drug-induced apoptosis of malignant cells. In line, upregulation of the chaperone BiP was prevented by simultaneous arrest of protein synthesis in B cell malignancies. Cytotoxicity by immunotoxins—approved inhibitors of protein synthesis—was synergistically enhanced in combination with UPR-inducers in seven distinct hematologic and three solid tumor entities in vitro. Synergistic cell death depended on mitochondrial outer membrane permeabilization via BAK/BAX, which correlated with synergistic, IRE1α-dependent reduction of BID, accompanied by an additive fall of MCL-1. The strong synergy was reproduced in vivo against xenograft mouse models of mantle cell lymphoma, Burkitt’s lymphoma, and patient-derived acute lymphoblastic leukemia. In contrast, synergy was absent in blood cells of healthy donors suggesting a tumor-specific vulnerability. Together, these data support clinical evaluation of blocking stress response counter-regulation using inhibitors of protein synthesis as a novel therapeutic strategy.

Tumors represent heterogeneous networks consisting not only of tumor cells but also of various cellular and non-cellular components, known as the tumor microenvironment (TME). The composition of the TME is highly variable and depends on tumor entity and tumor site. Immune cells are a key component of the TME. At early stages, immune cells can potently eliminate cancer cells, whereas the TME of established tumors is often immunosuppressive and prevents anti-tumor immune responses. Therefore, novel therapies targeting the inhibitory TME, like immune checkpoint inhibitors, have been developed to (re-)activate immune cells against the cancer. Apart from direct modulation of immune cells, tumor cell-targeted therapies, like immunotoxins, can stimulate immune responses by induction of immunogenic cell death. A promising antigen to target B cell lymphoma is CD22. However, currently established CD22-targeted therapies only recognize the human and not the murine protein. Consequently, these drugs have exclusively been studied in immunocompromised xenograft models, despite the importance of the TME for treatment response. The aim of this thesis was the establishment of a human CD22+ lymphoma mouse model to enable the preclinical study of CD22-targeted therapies within immune-competent TMEs. To this end, mice carrying a [Special character(s) omitted]-MYC translocation were crossbred with mice expressing a chimeric human-murine CD22 (h/mCD22). The resulting offspring spontaneously developed murine h/mCD22+ B cell lymphoma, which were re-injected subcutaneously or systemically in syngeneic mice. While stable engraftment of these lymphoma clones was achieved independently of injection route, only systemically growing lymphoma recapitulated organ-specific immune infiltration that resembled human disease. Upregulation of the immune checkpoints PD-1/PD-L1 and the presence of myeloid-derived suppressor cells also suggested an immunosuppressive TME. Treatment with the CD22-targeted immunotoxin Moxetumomab pasudotox (Moxe) resulted in significant tumor regression in bone marrow and spleen, while tumor response in lymph nodes strongly varied among lymphoma clones. Moreover, Moxe induced an expansion of pro-inflammatory myeloid cells. Despite persistence of these cells after treatment, acute inflammation subsided and tumors relapsed. Hence, Moxe was combined with the immune-stimulatory checkpoint inhibitor anti-PD-L1, which led to an additional activation of T cells and prolonged survival. However, consistent with clinical experience, anti-PD-L1 did not achieve long-lasting anti-tumor immune responses. In conclusion, the established human CD22+lymphoma model represents the first mouse model to study CD22-targeted drugs within immune-competent and organ-specific TMEs. Furthermore, it provides a unique platform to explore the combination of targeted therapy with immune-modulatory drugs for future clinical translation.

Targeted immunotherapies have greatly changed treatment of patients with B cell malignancies. To further enhance immunotherapies, research increasingly focuses on the tumor microenvironment (TME), which differs considerably by organ site. However, immunocompetent mouse models of disease to study immunotherapies targeting human molecules within organ-specific TME are surprisingly rare. We developed a myc-driven, primary murine lymphoma model expressing a human-mouse chimeric CD22 (h/mCD22). Stable engraftment of three distinct h/mCD22+ lymphoma was established after subcutaneous and systemic injection. However, only systemic lymphoma showed immune infiltration that reflected human disease. In this model, myeloid cells supported lymphoma growth and showed a phenotype of myeloid-derived suppressor cells. The human CD22-targeted immunotoxin Moxetumomab was highly active against h/mCD22+ lymphoma and similarly reduced infiltration of bone marrow and spleen of all three models up to 90-fold while efficacy against lymphoma in lymph nodes varied substantially, highlighting relevance of organ-specific TME. As in human aggressive lymphoma, anti-PD-L1 as monotherapy was not efficient. However, anti-PD-L1 enhanced efficacy of Moxetumomab suggesting potential for future clinical application. The novel model system of h/mCD22+ lymphoma provides a unique platform to test targeted immunotherapies and may be amenable for other human B cell targets such as CD19 and CD20.