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Direct cell death induction, in addition to immune-effector cell-mediated mechanisms, is one of the key mechanisms of action of anti-CD20 antibodies, and yet the signaling pathways implicated remain poorly investigated. Here we show that the transcription factor EGR-1 is rapidly induced by anti-CD20 antibodies and is a key mediator for CD20-induced cell death. EGR-1 induction results from an increased calcium influx induced by anti-CD20 antibodies. We show that both rituximab and obinutuzumab induce calcium influx, albeit through different mechanisms, and this influx is crucial for cell death induction. Inhibition of the calcium flux with calcium channel blockers (CCB) abolished EGR-1 induction and impaired the efficacy of anti-CD20 antibodies in preclinical in vitro and in vivo models. Finally, we investigated the impact of CCBs in patients treated with anti-CD20 antibodies included in the clinical trials GOYA and REMARC, and found that patients simultaneously receiving CCBs and anti-CD20 therapy have a shorter progression-free survival and overall survival. These results reveal EGR-1 as a key mediator of the direct cytotoxic activity of anti-CD20 antibodies and provide a rationale to evaluate EGR-1 expression as a new biomarker to predict response to anti-CD20 treatment. In addition, our findings show that calcium influx is required for anti-CD20–mediated tumor cell death and suggest that simultaneous administration of calcium channel blocking agents could be deleterious in patients receiving anti-CD20–based immunotherapy.

Supplementary Figures

The role of fusion genes and cancer driver genes in malignant transformation has traditionally been explored using transgenic or chimeric mouse models. It has been challenging to develop models that fully resemble the characteristics and morphology of human cancers. This applies to anaplastic large-cell lymphoma (ALCL), a malignancy classified as a peripheral T-cell lymphoma. It is still unclear at which stage of T-cell development ALCL can occur, as well as the early molecular events required for malignant transformation. In this issue of Cancer Research, Pawlicki and colleagues introduced the NPM–ALK fusion gene and mutant variants into primary T cells from healthy donors. By monitoring transduced T-cell clones over time, they demonstrated that transformed T cells undergo a progressive loss of T-cell identity accompanied with upregulation of epithelial-to-mesenchymal transition program and reemergence of an immature, thymic profile. Introduction of NPM–ALK was, however, not sufficient to convert healthy T cells to malignant clones, as this process required activation of T-cell receptor signaling. The study sets the stage for modeling early genetic changes in human tumors. See related article by Pawlicki et al., p. 3241

Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 + T cells, represent a key challenge in the tumor microenvironment by limiting potent antitumor immune responses. While high densities of tumor-infiltrating Tregs are associated with poor prognosis in patients with various types of solid cancers, their prognostic impact in B-cell non-Hodgkin lymphoma (NHL) remains unclear. Emerging studies suggest substantial heterogeneity in the phenotype and suppressive capacities of Tregs, emphasizing the importance of understanding Treg diversity and the need for additional markers to identify highly suppressive Tregs. Our in-depth characterization of Tregs in NHL tumors could open new paths for rational drug design, facilitating selective therapeutic manipulation of Tregs to reduce immunosuppression and improve anti-tumor immunity. Methods: Single-cell suspensions from NHL patients (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and healthy donors (tonsils and peripheral blood)) were analyzed by fluorescent flow- and mass cytometry to characterize Tregs, focusing on their expression of co-stimulatory and co-inhibitory checkpoint receptors. The immunosuppressive capacity of Tregs was measured by in vitro co-culture of FACS-sorted subsets of Tregs together with autologous CellTrace Violet-labelled T effector cells as responder cells, using samples from FL and tonsils. Live CD4 + T cells were obtained by FACS sorting from DLBCL (n = 3), FL (n = 3) and healthy donor tonsils (n = 3) and subjected to single-cell RNA sequencing (scRNA-seq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) and scTCR-seq by the 10X Genomics platform. The computational framework of CIBERSORTx was used to generate unique signature matrices for the three Treg subsets identified by scRNA-seq, to facilitate validation in separate scRNA-seq cohorts (King, Sci Immunol 2021; Roider, Nat Cell Biol 2020), and to impute frequencies of the Treg subsets in cohorts with bulk RNA-seq data (Chapuy, Nat Med 2018; Schmitz, NEJM 2018; Pastore, Lancet Oncol 2015). Results: Immunophenotyping by mass cytometry revealed a subset of activated Tregs identified by co-expression of TIGIT, CTLA-4, PD-1, ICOS and OX40, and higher expression of FOXP3, CD25 and CD45RO, that was present in DLBCL and tonsils, but lacking in peripheral blood. This was validated by fluorescent flow cytometry, demonstrating significantly higher frequencies of activated Tregs in NHL tumors compared to PBMCs and tonsils from healthy donors. The phenotypic heterogeneity of intratumoral Tregs reflected different suppressive capacities as activated Tregs more potently suppressed the proliferation of autologous effector CD4 + and CD8 + T cells than naïve Tregs. For global transcriptomic profiling of CD4 + T cells from FL, DLBCL and tonsillar samples, we integrating scRNA-seq and CITE-seq data from 17,774 cells, revealing 13 distinct cellular states including three states of Tregs: naïve, activated and non-conventional LAG3 +FOXP3 - Tregs. Activated Tregs had higher expression of checkpoint receptors (TNFRSF4, TNFRSF18, ICOS), phosphatases (DUSP2, DUSP4), NF-κB pathway (NFKBIA, TNFAIP3, NFKBIZ, REL), chemokine receptors (CXCR4) and transcription factors (JUNB, IRF1, STAT3) as compared to naïve Tregs. We next used a computational approach to develop unique signature matrices for each Treg subset. This approach demonstrated strong concordance between CIBERSORTx estimated cell abundances of the three Treg subsets and the ground truth, and was validated in two external scRNA-seq cohorts. The development of unique signature matrices for Treg subsets facilitated imputation of their frequencies in bulk RNA-seq datasets. These analyses revealed that higher frequency of activated Tregs was enriched in the germinal B cell subtype of DLBCL and was associated with adverse outcome in FL. Conclusion: This study demonstrates that Tregs infiltrating NHL tumors are transcriptionally and functionally diverse and include highly immunosuppressive activated Tregs co-expressing several checkpoint receptors, which distinguish them from peripheral blood Tregs. Activated intratumoral Tregs could hamper clinical responses to checkpoint blockade, and identifying and targeting their vulnerabilities has the potential to improve anti-tumor immune responses. Disclosures Holte: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Nordic: Membership on an entity's Board of Directors or advisory committees; Nanovector: Membership on an entity's Board of Directors or advisory committees, Other: lectures honorarias; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Alizadeh: Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Bristol Myers Squibb: Research Funding.

Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 T cells, are enriched in B-cell non-Hodgkin lymphoma (NHL) and constitute a barrier to potent antitumor immune responses. Despite extensive studies, the significance of tumor-infiltrating Tregs on disease outcome is unclear and while Tregs may express co-inhibitory and co-stimulatory receptors, the role of intratumoral Tregs in the context of immune checkpoint therapy remains elusive. Emerging evidence suggests heterogeneity among Tregs and their suppressive capacities in cancer, emphasizing the need for additional markers to identify highly suppressive Tregs. Therefore, an in-depth characterization of Treg heterogeneity in NHL could provide important insight into the disease pathogenesis and have implications for rational drug design. Methods: Expression of checkpoint receptors in Tregs was characterized by fluorescence flow cytometry and mass cytometry analysis of single-cell suspensions from diffuse large B-cell lymphoma (DLBCL; n = 16), follicular lymphoma (FL; n = 8), mantle cell lymphoma (MCL; n = 10), marginal zone lymphoma (MZL; n = 2), chronic lymphocytic lymphoma (CLL; n = 7), as well as tonsils (n = 8) and peripheral blood (n = 4) from healthy donors. Functional characterization of intratumoral Tregs was performed by a proliferation assay using FACS-sorted Tregs as suppressor cells and autologous CellTrace Violet-labelled T effector cells as responder cells. Single-cell RNA sequencing (scRNA-seq) was performed on FACS-sorted CD4 T cells from 3 DLBCL, 3 FL and 3 healthy donor tonsils using the 10X Genomics single cell 5' based library construction and VDJ libraries for TCR-sequencing. Additionally, for simultaneous profiling of phenotypic features with the mRNA expression in single cells, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq) was applied. The Treg compartment was characterized by clustering into distinct transcriptional Treg states and differential expression of marker genes. Results: TIGIT and CTLA-4 were identified as common markers of intratumoral Tregs, in addition to FOXP3 and CD25. Unsupervised computational analysis revealed two distinct Treg subsets, based on contrasting expression of PD-1, OX40, CD226 and ICOS (Figure 1A). One subset displayed a checkpoint receptorlow phenotype that corresponded to peripheral blood Tregs. The second subset had a checkpoint receptorhigh phenotype with elevated levels of PD-1, OX40, ICOS, TIGIT, CTLA-4 and increased levels of activation markers CD28, CD69 and CD95/Fas. The frequency of checkpoint receptorhigh Tregs was significantly increased in NHL tumors, compared to PBMCs and tonsils from healthy donors. FL tumors had the highest frequency of Tregs with receptorhigh phenotype among the NHL entities (median frequency of 86%, range 71-92%) and DLBCL had the highest donor-to-donor variation (median frequency of 77%, range 35-98%) (Figure 1B). This phenotypic heterogeneity of the Treg compartment reflected different suppressive capacities of the two subsets. Checkpoint receptorhigh Tregs were more potent mediators of immunosuppression in terms of suppressing the proliferation of autologous effector CD4 and CD8 T cells (Figure 1C). Furthermore, transcriptomic analysis of CD4 T cells by scRNA-seq and CITEseq revealed distinct transcriptomic signatures of the checkpoint receptorhigh and -receptorlow subsets. In addition, a third subset of Tregs, characterized by increased expression of LAG3 and immunosuppression-associated genes (CTLA-4, IL10, CD38, KLRB1) but lack of FOXP3, was identified (Figure 1D-E). Analysis of scTCR-sequences to compare TCR repertoires and to identify developmental trajectories will further add to our knowledge of intratumoral Tregs. Conclusions: These results reveal heterogeneity within the Treg compartment in NHL based on expression of checkpoint receptors, transcriptional profiles and suppressive capacities. As intratumoral Treg phenotypes differ from peripheral blood Tregs, this presents new therapeutic opportunities. Specific targeting of intratumoral Tregs would lead to stronger antitumor effects while limiting immune-related adverse events. A deeper understanding of Treg heterogeneity within the tumor microenvironment could therefore open new paths for rational design of immune checkpoint therapy. Disclosures Kolstad: Merck: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

Direct cell death induction, in addition to immune-effector cell-mediated mechanisms, is one of the key mechanisms of action of anti-CD20 antibodies, and yet the signaling pathways implicated remain poorly investigated. Here we show that the transcription factor EGR-1 is rapidly induced by anti-CD20 antibodies and is a key mediator for CD20-induced cell death. EGR-1 induction results from an increased calcium influx induced by anti-CD20 antibodies. We show that both rituximab and obinutuzumab induce calcium influx, albeit through different mechanisms, and this influx is crucial for cell death induction. Inhibition of the calcium flux with calcium channel blockers (CCB) abolished EGR-1 induction and impaired the efficacy of anti-CD20 antibodies in preclinical in vitro and in vivo models. Finally, we investigated the impact of CCBs in patients treated with anti-CD20 antibodies included in the clinical trials GOYA and REMARC, and found that patients simultaneously receiving CCBs and anti-CD20 therapy have a shorter progression-free survival and overall survival. These results reveal EGR-1 as a key mediator of the direct cytotoxic activity of anti-CD20 antibodies and provide a rationale to evaluate EGR-1 expression as a new biomarker to predict response to anti-CD20 treatment. In addition, our findings show that calcium influx is required for anti-CD20–mediated tumor cell death and suggest that simultaneous administration of calcium channel blocking agents could be deleterious in patients receiving anti-CD20–based immunotherapy.

Tubulin is the target for many small-molecule natural compounds, which alter microtubules dynamics, and lead to cell cycle arrest and apoptosis. One of these compounds is colchicine, a plant alkaloid produced by Colchicum autumnale. While C. autumnale produces a potent cytotoxin, colchicine, and expresses its target protein, it is immune to colchicine’s cytotoxic action and the mechanism of this resistance is hitherto unknown. In the present paper, the molecular mechanisms responsible for colchicine resistance in C. autumnale are investigated and compared to human tubulin. To this end, homology models for C. autumnale α-β tubulin heterodimer are created and molecular dynamics (MD) simulations together with molecular mechanics Poisson–Boltzmann calculations (MM/PBSA) are performed to determine colchicine’s binding affinity for tubulin. Using our molecular approach, it is shown that the colchicine-binding site in C. autumnale tubulin contains a small number of amino acid substitutions compared to human tubulin. However, these substitutions induce significant reduction in the binding affinity for tubulin, and subsequently fewer conformational changes in its structure result. It is suggested that such small conformational changes are insufficient to profoundly disrupt microtubule dynamics, which explains the high resistance to colchicine by C. autumnale.

Bispecific antibodies are emerging as novel approach for immunotherapies by combining two antigen-recognizing elements into a single construct that is able to simultaneously bind to two distinct targets. Bispecific antibodies can be applied to recruit immunological effector cells for killing of tumor cells or to simultaneously block two signaling pathways or cytokines. Subsequently, they have prompted significant interest for a number of therapeutic applications, both in cancer and in other indications. The growing interest in therapeutic antibodies along with the rapid progress in antibody engineering have yield a multitude of bispecific antibodies formats and derived molecules that differ in size, shape and function. Presently, two major classes of bispecific antibodies are most widely studied: immunoglobulin-like and small single chain Fv (scFv)-based bispecific antibodies. This review summarizes selected key methods used to generate bispecific antibodies, reports new concepts developed for immunotherapy and discusses their potential development and therapeutic benefits.

A variety of topics are reviewed in the area of mathematical and computational modeling in biology, covering the range of scales from populations of organisms to electrons in atoms. The use of maximum entropy as an inference tool in the fields of biology and drug discovery is discussed. Mathematical and computational methods and models in the areas of epidemiology, cell physiology and cancer are surveyed. The technique of molecular dynamics is covered, with special attention to force fields for protein simulations and methods for the calculation of solvation free energies. The utility of quantum mechanical methods in biophysical and biochemical modeling is explored. The field of computational enzymology is examined.