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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.

The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (β1–β2–β5), the immunoproteasome (β1i–β2i–β5i) and the two intermediate proteasomes (β1–β2–β5i and β1i–β2–β5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c-­myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three β5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, β5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three β5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.

Background Mutational signatures are specific patterns of somatic mutations introduced into the genome by oncogenic processes. Several mutational signatures have been identified and quantified from multiple cancer studies, and some of them have been linked to known oncogenic processes. Identification of the processes contributing to mutations observed in a sample is potentially informative to understand the cancer etiology. Results We present here SigsPack , a Bioconductor package to estimate a sample’s exposure to mutational processes described by a set of mutational signatures. The package also provides functions to estimate stability of these exposures, using bootstrapping. The performance of exposure and exposure stability estimations have been validated using synthetic and real data. Finally, the package provides tools to normalize the mutation frequencies with respect to the tri-nucleotide contents of the regions probed in the experiment. The importance of this effect is illustrated in an example. Conclusion SigsPack provides a complete set of tools for individual sample exposure estimation, and for mutation catalogue & mutational signatures normalization.

Personalized assessment of immunocompetence and DNA double-strand break (DSB) repair requires methods that are sensitive to genetic and molecular complexity beyond the well-known monogenic disorders. Inspired by decades of research using B cells to study DNA repair processes, here we present SWIBRID (SWItch junction Breakpoint Repertoire IDentification), a tool to systematically profile genomic junctions generated in vivo during antibody class switch recombination (CSR) in B cells. As CSR junctions reflect immune diversity and DNA repair proficiency, SWIBRID detects phenotypic manifestations of deficiencies via a highly scalable, blood-based PCR followed by long-read sequencing and bioinformatic analysis. We show that specific DNA repair defects, including cancer-associated mutations, exhibit distinct CSR junction patterns. Notably, SWIBRID distinguishes different types of DSB repair knockouts and identifies the respective genetic defect in cell lines. In 68 patients, we detect immunodeficiencies and DNA repair defects with high accuracy (area under the curve 0.99 and 0.84, respectively), and identify previously uncharacterized patient groups as well as patient-specific CSR junction signatures. With SWIBRID, we seek to advance the identification of pathogenic defects, support early diagnosis, and address molecular heterogeneity that drives variable clinical outcomes. Class switch recombination (CSR) is a process contributing to antibody diversity and generating characteristic genomic junctions that reflect DNA double-strand break repair efficiency. Here authors present a platform to profile the repertoire of CSR junction patterns; long-read-sequenced PCR products from blood-derived lymphocytes are analysed by a dedicated bioinformatic pipeline to accurately detect immunodeficiencies and DNA repair defects.

A balanced SUMOylation equilibrium safeguards the functional anti‐tumor immune response. Oncogene activation drives SUMOylation, rendering aberrant SUMOylation a hallmark of cancer. To delineate the impact of activated SUMOylation on the tumor‐immune synapse, we applied HLA class‐I‐targeted ligandomics and identified a function of activated SUMOylation in restricting the immunopeptidome landscape. Importantly, aberrant SUMOylation suppressed a unique HLA‐I peptide and oncoprotein‐derived neoepitope repertoire, enabling cancer cells to evade T cell immune surveillance. Mechanistically, SUMOylation impaired the immunoproteasome constitution and proteolytic activity, thus limiting the diversity of the peptide landscape. Further, SUMOylation altered TAP1 transporter binding preferences, thereby mimicking viral immune evasion strategies. As an actionable application, pharmacological inhibition of SUMOylation unmasked the targetable immunopeptidome, enhanced the tumor cell susceptibility to T cell killing and substantially reshaped the immune cell landscape. These findings highlight SUMOylation as a critical regulator of the adaptive anti‐tumor immune response. We propose SUMOylation inhibition as a strategy to enhance immunogenic peptide presentation, thereby improving the efficacy of cancer immunotherapies. Pharmacological SUMOylation inhibition (SUMOi) counteracts tumor immune evasion by unmasking an immunogenic HLA‐I peptide and neoepitope repertoire. By restoring HLA‐I ligand availability through increased antigen processing and presentation, enhanced proteasomal cleavage, and modulated TAP1 peptide affinity, SUMOi boosts tumor immunogenicity, resulting in improved T cell infiltration and cytotoxicity across multiple preclinical cancer models.

Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells, presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study, 20ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35–121.5); P=0.02), number of metastatic sites (hazard ratio 3.87 (1.66–9.03); P=0.02), tumor burden (hazard ratio 5.72 (1.57–20.9); P=0.01), and percentage of NES-expressing CMCs≥35% (hazard ratio 5.73 (1.66–19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis.

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.

BackgroundMelanoma-associated antigen 1 (MAGE-A1) is a cancer-testis antigen with highly selective expression in testis (which is an immune privileged site) and in multiple high unmet medical need cancers. Therefore, it represents an attractive target for T cell receptor (TCR)-based therapies. TK-8001 is a MAGE-A1 directed TCR with optimized affinity and specificity, derived from the huTCR mouse platform,1 introduced by retroviral transduction into autologous patient-derived CD8+ T cells. The anticipated mode of action of TK-8001 is to bind to MAGE-A1-epitope presenting tumor cells and eliminate them via CD8+ cytotoxic activity and interferon-γ release. Preclinical exploration of the TK-8001 TCR has demonstrated potent antitumor activity, even in low-expressing MAGE-1 positive tumor cells, and favorable benchmarking vs. existing MAGE-A1 directed TCRs derived from human donors. This abstract describes the currently launched phase 1/2 trial for TK-8001.MethodsThe IMAG1NE trial (Immunotherapeutic MAGE-A1 directed Neoplasm Elimination) is a phase 1/2, first-in-human, open-label, accelerated titration, two-part clinical trial of TK-8001 (MAGE-A1-directed TCR-transduced autologous CD8+ T cells) in subjects with HLA-A*02:01 genotype and advanced-stage/metastatic, MAGE-A1+ solid tumors that either have no approved therapeutic alternative(s) or are in non-curable state and have received a minimum of two lines of systemic therapy. Major endpoints for the IMAG1NE trial will be safety, pharmacokinetics, pharmacodynamics (e.g. cytokine profiles) as well as preliminary clinical efficacy (degree of tumor mass reduction and duration of response).In Part 1 of the trial, three different doses of TK-8001 will be explored for safety and preliminary clinical efficacy in an accelerated titration design. The starting dose is set at 1x10E8 MAGE-A1 TCR transduced CD8+ T cells followed by two escalation steps. Part 2 of the trial will enroll up to 30 subjects with advanced-stage, MAGE-A1 positive cancer to confirm safety and efficacy.The study is expected to open for enrolment in Q4/2021. For further information please contact T-knife GmbH at info@t-knife.com.ReferencesLi, Liang-Ping, J Christoph Lampert, Xiaojing Chen, Catarina Leitao, Jelena Popović, Werner Müller, and Thomas Blankenstein. Transgenic mice with a diverse human T cell antigen receptor repertoire. Nature Medicine 2010;16: 1029–34.Ethics ApprovalIn progress, expected 11/2021

Bottke, D., Koychev, D., Busse, A., Heufelder, K., Wiegel, T., Thiel, E., Hinkelbein, W. and Keilholz, U. Fractionated Irradiation Can Induce Functionally Relevant Multidrug Resistance Gene and Protein Expression in Human Tumor Cell Lines. Radiat. Res. 170, 41–48 (2008). The molecular basis of radiotherapy-related multidrug resistance (MDR) is still unclear. Here we report on a study investigating the effect of fractionated irradiation on expression of the MDR-associated proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance-related protein (LRP), the respective mRNAs, and the functional consequences. Cells of six colon and five breast cancer cell lines were irradiated with a total dose of 27 Gy, five fractions of 1.8 Gy per week. The mRNA expression was measured by quantitative RT-PCR, protein levels and drug sensitivity to cisplatin, doxorubicin and bendamustine were assessed by flow cytometry. Breast cancer cell lines showed enhancement of the mRNAs encoding for P-gp, MRP1 and LRP in comparison to nonirradiated cells. No up-regulation of the three mRNA species was observed in the colon cancer cell lines. After irradiation, three breast cancer cell lines showed an up-regulation of LRP, one line an up-regulation of MRP1, and four lines a small up-regulation of P-gp. In the colon cancer cell lines, radiation induced significant enhancement of all three proteins. In comparison to controls, the irradiated cells lines showed a significant resistance to cisplatin, doxorubicin and bendamustine. This study confirms the prior reports of enhancement of P-gp and MRP1 after irradiation, which is accompanied by a multidrug resistance phenomenon, but in addition proposes a novel mechanism in the appearance of MDR after radiation-induced enhancement of LRP.