SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt lymphoma cells. SMARCA4 therefore facilitates chromatin accessibility for TFs that shape centrocyte trajectories, and loss of fine-control of these programs biases toward centroblast cell-fate, GC hyperplasia and lymphoma.

Inhibition of MALT1 and BCL2 Induces Synergistic Antitumor Activity in Models of B-Cell Lymphoma.

The activated B cell (ABC) subset of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic B-cell receptor signaling and associated with poor outcomes when treated with standard therapy. In ABC-DLBCL, MALT1 is a core enzyme that is constitutively activated by stimulation of the B-cell receptor or gain-of-function mutations in upstream components of the signaling pathway, making it an attractive therapeutic target. We discovered a novel small-molecule inhibitor, ABBV-MALT1, that potently shuts down B-cell signaling selectively in ABC-DLBCL preclinical models leading to potent cell growth and xenograft inhibition. We also identified a rational combination partner for ABBV-MALT1 in the BCL2 inhibitor, venetoclax, which when combined significantly synergizes to elicit deep and durable responses in preclinical models. This work highlights the potential of ABBV-MALT1 monotherapy and combination with venetoclax as effective treatment options for patients with ABC-DLBCL.

FAAH inhibition ameliorates breast cancer in a murine model.

Breast cancer is the leading cancer among females worldwide. Disease outcome depends on the hormonal status of the cancer and whether or not it is metastatic, but there is a need for more efficacious therapeutic strategies where first line treatment fails. In this study, Fatty Acid Amide Hydrolase (FAAH) inhibition and endocannabinoids were examined as therapeutic alternatives. FAAH is an integral membrane enzyme that hydrolyzes endocannabinoids, rendering them inactive, and FAAH inhibition is predicted to increase cancer cell death. To test this, breast cancer cells were probed for FAAH expression using Western blot analysis, treated with FAAH inhibitors, exogenous endocannabinoids, and combinations of the two treatments, and assessed for viability. High levels of FAAH were observed in different breast cancer cell lines. FAAH inhibition was more effective than exogenous endocannabinoid treatment, and the combination of FAAH inhibitors and endocannabinoids was the most effective in inducing apoptosis of breast cancer cells in vitro. In addition, in vivo FAAH inhibition reduced breast cancer growth in immunodeficient mice. FAAH inhibition is a promising approach, and tremendous progress has been made in the field to validate this mechanism as an alternative to chemotherapy. Further research exploring the therapeutic potential and impact of FAAH expression on cancer cells is warranted.

Effect of delayed cell infusion in patients with large B-cell lymphoma treated with chimeric antigen receptor T-cell therapy.

Complications occurring after lymphodepleting chemotherapy (LDC) may delay chimeric antigen receptor (CAR) T-cell infusion. The effect of these delays on clinical outcomes is unclear. We performed a retrospective analysis of 240 patients with relapsed/refractory large B-cell lymphoma treated with standard-of-care axicabtagene ciloleucel (axi-cel) and identified 40 patients (16.7%) who had delay in axi-cel infusion. Of these, 85% had delay due to infection. At time of LDC initiation, patients with delayed infusion had lower absolute neutrophil count (p=0.006), lower platelets (p=0.004), lower hemoglobin (p5 days (4.6 vs. 8.2 months; p=0.036), but not 1 day (5.7 vs. 8.2 months; p=0.238). Following propensity score matching, patients with delayed infusion continued to have shorter median PFS (3.5 vs. 6.0 months; p=0.015). Levels of proinflammatory cytokines on day of infusion were significantly higher in patients with delayed infusion. Together, these findings suggest that delays in CAR T-cell administration after initiation of LDC are associated with inferior outcomes. Further studies are needed to guide strategies to improve efficacy in such patients.

A single-cell atlas of CD19 chimeric antigen receptor T cells.

Li et al. present a resource of single-cell RNA sequencing (scRNA-seq) data from the infusion products of relapsed or refractory large B cell lymphoma (rrLBCL) patients treated with standard-of-care axicabtagene ciloleucel and identify features that are significantly different between products from responders and non-responders at 3-month followup by PET/CT, an important landmark for long-term outcomes.

Prolonged cytopenia following CD19 CAR T cell therapy is linked with bone marrow infiltration of clonally expanded IFNγ-expressing CD8 T cells.

Autologous anti-CD19 chimeric antigen receptor T cell (CAR T) therapy is highly effective in relapsed/refractory large B cell lymphoma (rrLBCL) but is associated with toxicities that delay recovery. While the biological mechanisms of cytokine release syndrome and neurotoxicity have been investigated, the pathophysiology is poorly understood for prolonged cytopenia, defined as grade ≥3 cytopenia lasting beyond 30 days after CAR T infusion. We performed single-cell RNA sequencing of bone marrow samples from healthy donors and rrLBCL patients with or without prolonged cytopenia and identified significantly increased frequencies of clonally expanded CX3CR1hi cytotoxic T cells, expressing high interferon (IFN)-γ and cytokine signaling gene sets, associated with prolonged cytopenia. In line with this, we found that hematopoietic stem cells from these patients expressed IFN-γ response signatures. IFN-γ deregulates hematopoietic stem cell self-renewal and differentiation and can be targeted with thrombopoietin agonists or IFN-γ-neutralizing antibodies, highlighting a potential mechanism-based approach for the treatment of CAR T-associated prolonged cytopenia.

Follicular Lymphoma Microenvironment Characteristics Associated with Tumor Cell Mutations and MHC Class II Expression.

Follicular lymphoma (FL) is a B-cell malignancy with a complex tumor microenvironment that is rich in nonmalignant immune cells. We applied single-cell RNA sequencing to characterize the diverse tumor and immune cell populations of FL and identified major phenotypic subsets of FL T cells, including a cytotoxic CD4 T-cell population. We characterized four major FL subtypes with differential representation or relative depletion of distinct T-cell subsets. By integrating exome sequencing, we observed that somatic mutations are associated with, but not definitive for, reduced MHC expression on FL cells. In turn, expression of MHCII genes by FL cells was associated with significant differences in the proportions and targetable immunophenotypic characteristics of T cells. This provides a classification framework of the FL microenvironment in association with FL genotypes and MHC expression, and informs different potential immunotherapeutic strategies based upon tumor cell MHCII expression. SIGNIFICANCE: We have characterized the FL-infiltrating T cells, identified cytotoxic CD4 T cells as an important component that is associated with tumor cell-intrinsic characteristics, and identified sets of targetable immune checkpoints on T cells that differed from FLs with normal versus low MHC expression. See related commentary by Melnick, p. 374. This article is highlighted in the In This Issue feature, p. 369.

Detecting Förster resonance energy transfer in living cells by conventional and spectral flow cytometry.

Assays based on Förster resonance energy transfer (FRET) can be used to study many processes in cell biology. Although this is most often done with microscopy for fluorescence detection, we report two ways to measure FRET in living cells by flow cytometry. Using a conventional flow cytometer and the "3-cube method" for intensity-based calculation of FRET efficiency, we measured the enzymatic activity of specific kinases in cells expressing a genetically-encoded reporter. For both AKT and protein kinase A, the method measured kinase activity in time-course, dose-response, and kinetic assays. Using the Cytek Aurora spectral flow cytometer, which applies linear unmixing to emission measured in multiple wavelength ranges, FRET from the same reporters was measured with greater single-cell precision, in real time and in the presence of other fluorophores. Results from gene-knockout studies suggested that spectral flow cytometry might enable the sorting of cells on the basis of FRET. The methods we present provide convenient and flexible options for using FRET with flow cytometry in studies of cell biology.

Overexpression of CD200 is a Stem Cell-Specific Mechanism of Immune Evasion in AML.

BACKGROUND: Acute myeloid leukemia (AML) stem cells (LSCs) are capable of surviving current standard chemotherapy and are the likely source of deadly, relapsed disease. While stem cell transplant serves as proof-of-principle that AML LSCs can be eliminated by the immune system, the translation of existing immunotherapies to AML has been met with limited success. Consequently, understanding and exploiting the unique immune-evasive mechanisms of AML LSCs is critical. METHODS: Analysis of stem cell datasets and primary patient samples revealed CD200 as a putative stem cell-specific immune checkpoint overexpressed in AML LSCs. Isogenic cell line models of CD200 expression were employed to characterize the interaction of CD200+ AML with various immune cell subsets both in vitro and in peripheral blood mononuclear cell (PBMC)-humanized mouse models. CyTOF and RNA-sequencing were performed on humanized mice to identify novel mechanisms of CD200-mediated immunosuppression. To clinically translate these findings, we developed a fully humanized CD200 antibody (IgG1) that removed the immunosuppressive signal by blocking interaction with the CD200 receptor while also inducing a potent Fc-mediated response. Therapeutic efficacy of the CD200 antibody was evaluated using both humanized mice and patient-derived xenograft models. RESULTS: Our results demonstrate that CD200 is selectively overexpressed in AML LSCs and is broadly immunosuppressive by impairing cytokine secretion in both innate and adaptive immune cell subsets. In a PBMC-humanized mouse model, CD200+ leukemia progressed rapidly, escaping elimination by T cells, compared with CD200- AML. T cells from mice with CD200+ AML were characterized by an abundance of metabolically quiescent CD8+ central and effector memory cells. Mechanistically, CD200 expression on AML cells significantly impaired OXPHOS metabolic activity in T cells from healthy donors. Importantly, CD200 antibody therapy could eliminate disease in the presence of graft-versus-leukemia in immune competent mice and could significantly improve the efficacy of low-intensity azacitidine/venetoclax chemotherapy in immunodeficient hosts. CONCLUSIONS: Overexpression of CD200 is a stem cell-specific marker that contributes to immunosuppression in AML by impairing effector cell metabolism and function. CD200 antibody therapy is capable of simultaneously reducing CD200-mediated suppression while also engaging macrophage activity. This study lays the groundwork for CD200-targeted therapeutic strategies to eliminate LSCs and prevent AML relapse.

Upregulation of Proinflammatory Bradykinin Peptides in Systemic Lupus Erythematosus and Rheumatoid Arthritis.

Our recent study has implicated bradykinin (BK) signaling as being of pathogenic importance in lupus. This study aims to investigate the biomarker potential of BK peptides, BK and BK-des-arg-9, in lupus and other rheumatic autoimmune diseases. Sera from systemic lupus erythematosus (SLE) patients and healthy subjects were screened for BK and BK-des-arg-9 by liquid chromatography-mass spectrometry metabolomics. Serum from 6-mo-old C57BL/6 mice and three murine lupus strains were also screened for the two peptides by metabolomics. Given the promising initial screening results, validation of these two peptides was next conducted using multiple reaction monitoring in larger patient cohorts. In initial metabolomics screening, BK-des-arg-9 was 22-fold higher in SLE serum and 106-fold higher in mouse lupus serum compared with healthy controls. In validation assays using multiple reaction monitoring and quadrupole time-of-flight mass spectrometry, BK and BK-des-arg-9 showed significant elevations in SLE serum compared with controls (p < 0.0001; area under the curve = 0.79-0.88), with a similar but less pronounced increase being noted in rheumatoid arthritis serum. Interestingly, increased renal SLE disease activity index in lupus patients was associated with reduced circulating BK-des-arg-9, and the reasons for this remain to be explored. To sum, increased conversion of BK to the proinflammatory metabolite BK-des-arg-9 appears to be a common theme in systemic rheumatic diseases. Besides serving as an early marker for systemic autoimmunity, independent studies also show that this metabolic axis may also be a pathogenic driver and therapeutic target in lupus.

Tonic B-cell receptor signaling in diffuse large B-cell lymphoma.

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.