Human Follicular Lymphoma Research Articles

Abstract PR01: EZH2 inhibitors improve CAR-T therapy by enhancing lymphoma B cell immunogenicity and CAR-T cell functions

Abstract Although CAR-T19 therapy is widely used for relapsed/refractory B cell lymphomas, approximately half of patients experience post CAR-T relapse. The mechanisms underlying this resistance remain largely unknown and efforts to overcome this limitation are hindered by the lack of suitable pre-clinical models for study. To address these unmet needs, we generated a novel genetically engineered mouse model with conditional expression of Ezh2Y641F and BCL2 in germinal center (GC) B cells. Ezh2/BCL2 mice developed follicular lymphoma (FL) which histologically and transcriptionally recapitulates human FL. Moreover, we established a cell line derived from an Ezh2/BCL2 mouse that developed transformed FL (tFL). Injection of the cell line named “tFL-P6” in immunocompetent C57BL6 recipients resulted in rapid disease progression that histologically and transcriptionally mimics human tFL. Treatment of tFL-P6 with EZH2 inhibitors (EZH2i) didn’t affect proliferation and viability, however, the number of pretreated cells was significantly reduced when co-cultured with T cells. EZH2i pretreated tFL-P6 displayed extended interaction with T cells, as measured by live imaging. EZH2i reprogrammed tFL-P6 to restore T cell engagement genes such as ICOSL, ICAM1, OX40L, and integrins, as well as cytokines and chemokines involved in T cell recruitment, therefore enhancing immunogenicity. Notably, pretreated tFL-P6 cells were rejected by C57BL6 whereas they developed lethal disease in immunodeficient recipients. Pre-treatment of tFL-P6 as well as human DLBCL and PDX-derived cell lines significantly enhanced CAR-T19 cell killing effects in vitro. Administering CAR-T cells in mice engrafted with EZH2i-pretreated tFL-P6 significantly reduced the tumor burden and prolonged their survival (100% vs 30%, p<0.01). To assess the direct interactions between lymphoma cells and CAR-T cells in vivo, we performed intravital 2-photon imaging of popliteal lymph nodes using dTomato labeled CAR-T cells and GFP+ tFL-P6 cells. Pretreatment of tFL-P6 cells with EZH2i doubled the recruitment of CAR-T cells in the microenvironment and enhanced the duration of contact and surface engagement with CAR-T cells. To explore the impact of EZH2 inhibition on CAR-T cells, we manufactured CAR-T cells from splenic T cells of mice treated with EZH2i or vehicle for 14 days. Prior exposure to EZH2i didn’t affect proliferation and transduction during CAR-T production. Ex vivo assays with those CAR-T cells and tFL-P6 cells showed a superior killing effect and expansion of EZH2i-exposed CAR-T (p<0.001). Mice bearing tFL-P6 lymphomas displayed a longer survival when infused with EZH2i-pretreated CAR-T cells (p=0.06). Pretreatment with EZH2i resulted in an increased memory/effector ratio (p<0.01) and reduction of PD1+CD38+ exhausted CD8CAR-T cells (p<0.001). Overall, EZH2i improved CAR-T therapy by enhancing lymphoma cell immunogenicity and CAR-T cell functions. These results prompted the initiation of a clinical trial to evaluate the safety and efficacy of this combination in R/R B cell lymphomas (NCT05934838). Citation Format: Yusuke Isshiki, Xi Chen, Matt Teater, Ioannis Karagiannidis, Henna Nam, Amy Chadburn, Ari Melnick, Wendy Béguelin. EZH2 inhibitors improve CAR-T therapy by enhancing lymphoma B cell immunogenicity and CAR-T cell functions [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR01.

Emergence of Highly-Plastic B Cell States Cooperates with Early Immune Microenvironment Remodeling to Drive Follicular Lymphomagenesis

Follicular lymphoma (FL) is a prototypical example of B cell lymphoma arising from germinal center (GC) B cells. Recent single cell transcriptomic studies investigating the transcriptional heterogeneity of FL suggest that tumor B cells are functionally diverse with cells acquiring an intermediate phenotype between GC and memory (mem), in line with the premalignant FL ontogeny where BCL2 + memory B cells require multiple re-entries into the GC to facilitate the transformation process. Since FL cells often hijack the epigenetic machinery through mutations in histone modifying proteins - notably KMT2D - we questioned here how epigenetic alterations contribute to the emergence of this intratumoral heterogeneity. We developed a genetically engineered mouse FL-like model (GEMM) carrying Kmt2d loss-of-function in B cells and BCL2 overexpression recapitulating different progression stages from preneoplastic hyperplasia to FL-like tumors. We analyzed 45,541 high-quality single cell transcriptomes and immune repertoires (B cell receptor, BCR and T cell receptor, TCR) from 18 mice including immunized-control mice in the context of physiological GC reaction. We developed a computational workflow on mutant B cells combining supervised and unsupervised metaclustering approaches in order to highlight recurrent gene programs active in preneoplastic hyperplasia and the various stages leading to FL-like tumors. We used inferCNV to assess the association between genetics and transcriptomic heterogeneity and inferred cell-cell interactions to characterize the microenvironmental determinants associated with B cell heterogeneity throughout FL progression. By modeling the GC cycle dynamics and building B cell signatures during a normal immune response, we found that our FL GEMM mirrored the desynchronization of the GC reaction program previously observed in human FL. Single cell expression profiles of Kmt2d-BCL2 mutant cells also showed a reproducible increase in phenotypic heterogeneity during FL progression, manifesting as a continuum of cell states from proliferating GC-like to quiescent mem-like cell states. We defined this GC-to-Mem axis as the major source of intra-tumor transcriptional heterogeneity. Single-cell BCR sequencing revealed the emergence of highly-plastic B cell states during FL evolution, co-existing within individual mice and being largely independent of subclonal genetic variation. To further elucidate the emergence and maintenance of these cell state transitions, we explored the phenotypic and transcriptional changes within the immune microenvironment along disease progression. Significant compositional changes were found, including the expansion of T follicular helper cells and T regulatory T cells together with the loss of naive CD4, CD8 T, and NK cell populations, preceding malignant transformation and creating a (pre)tumoral supportive niche persisting in advanced tumors. Prior remodeling of a premalignant supportive immune niche may be crucial in establishing malignant cell heterogeneity. Ongoing efforts aim to impair tumor-immune interactions driving tumor B cell dynamics and survival. In conclusion, our results provide a high-resolution view of events spanning FL progression and indicate a major role of early TME remodeling in establishing a suitable niche for progression. Early intervention aimed to target the B cell-tumor microenvironment interactions driving intra-tumoral heterogeneity may represent a promising therapeutic avenue against early disease and prevention of FL recurrence.

Distinct Minor Neoplastic Follicular T-Cell Subsets Dictate Lymphoma Fates through Functional Specialization

Background: The presence and role of follicular T-cell populations other than T follicular helper (Tfh) cells, such as T follicular regulatory (Tfr) and cytotoxic (Tfc) cells, are gaining increasing attention in certain pathological states. However, the ecosystem of follicular T cells in the tumor microenvironment (TME) has not been fully elucidated. In particular, the significance of minor follicular T-cell subsets in the neoplastic follicular environment remains elusive. Here, we aimed to reveal the landscape of follicular T-cell alterations in various cancers, with a particular emphasis on the follicular lymphoma (FL) TME. Methods: We analyzed single-cell RNA/TCR sequencing data of >500,000 human T cells from FL (obtained from four cohorts) and 25 other cancer types, as well as homeostatic and reactive lymph nodes (LNs), to construct a comprehensive single-T-cell atlas. We investigated differentially expressed genes, RNA velocity, and TCR clonality using this atlas. To determine the functions of neoplastic follicular regulatory (Tnfr) and cytotoxic (Tnfc) T cells, we performed in vitro cytokineproduction and co-culture assays, in combination with cell activation/suppression, cell division, and apoptosis assays, using human FL samples. With the PhenoCycler-Fusion system, we conducted multiplex digital spatial profiling (DSP) of 169 FL samples from two independent cohorts (now being extended to 242 FL samples from three cohorts) for >25 antibodies. We also performed single-cell spatial and protein expression profiling and prognostic analysis. Results: In FL, distinct minor neoplastic follicular T-cell subsets-Tnfr and CD4 (Tnfc4) and CD8 (Tnfc8) Tnfc cells-increased relative to homeostatic LNs. The TCR repertoire analysis revealed that Tnfr cells shared clonotypes with conventional effector regulatory T (Trg) and Tfh cells, whereas Tnfc4 and Tnfc8 cells shared clonotypes with Tfh cells and effector and exhausted (Tcex) cytotoxic CD8 T cells, respectively. In line with these findings, the RNA velocity survey suggested that Tnfr, Tnfc4, and Tnfc8 cells originated from Trg, Tfh, and naïve-like CD8 T cells, respectively. Tnfr and Tnfc cells expressed higher levels of effector genes, including those involved in cytokine release, chemokine response, migration, and PD-1 signaling, than their reactive LN counterparts. The pan-cancer survey revealed that Tfr and CD4 Tfc cells were exclusive to FL, whereas the prevalence and gene expression profiles of CD8 Tfc cells varied across cancers. Tnfr cells were marked by abundant expression of IL10 and IL21, whereas Tnfc cells displayed a unique phenotype, as they concomitantly expressed markers of effector Tfh (e.g., CXCL13, CXCR5, and PDCD1), naïve/stem (e.g., CCR7 and TCF7), central memory (e.g., CD27, CD28, and SELL), and tissue-resident memory (e.g., ITGAE) cells. Hierarchical clustering demonstrated that Tnfc8 cells had transcriptional profiles similar to those of melanoma TCF1 +PD-1 +CD8 + stem-like T cells. DSP of FL detected Tnfr and Tnfc cells frequently localized within and around neoplastic follicles, forming a cellular neighborhood that allowed them to interact closely. Tnfr cells were distributed predominantly near Tfh cells. The functional co-culture assays demonstrated that Tnfr cells suppressed Tfh-cell activation and division, thereby inhibiting Tfh-mediated malignant B-cell activation and survival. Tnfc8 cells showed a higher cell division capability than that of Tcex cells, suggesting that Tnfc8 cells function as a pool of CD8 T cells in neoplastic follicles. The prognostic analysis revealed that Tnfr and Tnfc cell proportions correlated with early disease relapse (i.e., POD24) and predicted a significantly longer time-to-relapse ( P <0.05 for Tnfr and <0.001 for Tnfc cells) in FL. In the multivariate analysis, the prognostic impact of these two cell subsets was independent of the FLIPI. The prognostic analysis findings were confirmed using a validation cohort. Conclusions: Our multi-omics approach identified the expansion of minor neoplastic follicular T-cell subsets that carry unique transcriptional and functional profiles and robust prognostic impacts. These findings deepen our understanding of the biological and immunological roles of non-Tfh follicular T cells in the lymphoma TME and highlights their clinical potential for patient risk stratification and future therapeutic interventions.

Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency

The discovery of recurrent mutations in subunits and regulators of the vacuolar-type H+-translocating ATPase (V-ATPase) in follicular lymphoma (FL) highlights a role for macroautophagy/autophagy, amino-acid, and nutrient-sensing pathways in the pathogenesis of this disease. Here, we report on novel mutations in the ER-resident chaperone VMA21, which is involved in V-ATPase assembly in 12% of FL. Mutations in a novel VMA21 hotspot (p.93X) result in the removal of a C-terminal non-canonical ER retrieval signal thus causing VMA21 mislocalization to lysosomes. The resulting impairment in V-ATPase function prevents full lysosomal acidification and function, including impaired pH-dependent protein degradation as shown via lysosomal metabolomics and ultimately causes a degree of amino acid depletion in the cytoplasm. These deficiencies result in compensatory autophagy activation, as measured using multiple complementary assays in human and yeast cells. Of translational significance, the compensatory activation of autophagy creates a dependency for survival for VMA21-mutated primary human FL as shown using inhibitors to ULK1, the proximal autophagy-regulating kinase. Using high-throughput microscopy-based screening assays for autophagy-inhibiting compounds, we identify multiple clinical grade cyclin-dependent kinase inhibitors as promising drugs and thus provide new rationale for innovative clinical trials in FL harboring aberrant V-ATPase.

The molecular ontogeny of follicular lymphoma: gene mutations succeeding the BCL2 translocation define common precursor cells

Relapsed follicular lymphoma (FL) can arise from common progenitor cells (CPCs). Conceptually, CPC-defining mutations are somatic alterations shared by the initial and relapsed tumours, mostly B-cell leukaemia/lymphoma 2 (BCL2)/immunoglobulin heavy locus (IGH) translocations and other recurrent gene mutations. Through complementary approaches for highly sensitive mutation detection, we do not find CPC-defining mutations in highly purified BCL2/IGH-negative haematopoietic progenitor cells in clinical remission samples from three patients with relapsed FL. Instead, we find cells harbouring the same BCL2/IGH translocation but lacking CREB binding protein (CREBBP), lysine methyltransferase 2D (KMT2D) and other recurrent gene mutations. Thus, (i) the BCL2/IGH translocation can precede CPC-defining mutations in human FL, and (ii) BCL2/IGH-translocated cells can persist in clinical remission.

Follicular Lymphoma-associated BTK Mutations are Inactivating Resulting in Augmented AKT Activation.

On the basis of the recent discovery of mutations in Bruton tyrosine kinase (BTK) in follicular lymphoma, we studied their functional properties. We identified novel somatic BTK mutations in 7% of a combined total of 139 follicular lymphoma and 11 transformed follicular lymphoma cases, none of which had received prior treatment with B-cell receptor (BCR) targeted drugs. We reconstituted wild-type (WT) and mutant BTK into various engineered lymphoma cell lines. We measured BCR-induced signal transduction events in engineered cell lines and primary human follicular lymphoma B cells. We uncovered that all BTK mutants destabilized the BTK protein and some created BTK kinase-dead mutants. The phospholipase C gamma 2 (PLCγ2) is a substrate of BTK but the BTK mutants did not alter PLCγ2 phosphorylation. Instead, we discovered that BTK mutants induced an exaggerated AKT phosphorylation phenotype in anti-Ig-treated recombinant lymphoma cell lines. The short hairpin RNA-mediated knockdown of BTK expression in primary human nonmalignant lymph node-derived B cells resulted in strong anti-Ig-induced AKT activation, as did the degradation of BTK protein in cell lines using ibrutinib-based proteolysis targeting chimera. Finally, through analyses of primary human follicular lymphoma B cells carrying WT or mutant BTK, we detected elevated AKT phosphorylation following surface Ig crosslinking in all follicular lymphoma B cells, including all BTK-mutant follicular lymphoma. The augmented AKT phosphorylation following BCR crosslinking could be abrogated by pretreatment with a PI3Kδ inhibitor. Altogether, our data uncover novel unexpected properties of follicular lymphoma-associated BTK mutations with direct implications for targeted therapy development in follicular lymphoma.See related commentary by Afaghani and Taylor, p. 2123.

Abstract IA13: Oncogenic Rag GTPase signaling links cellular nutrients with the FL microenvironment

Abstract During the humoral response, B cells undergo a sudden anabolic shift that requires high cellular nutrient levels to sustain the subsequent proliferative burst. Follicular lymphoma (FL) originates from B cells that have participated in the humoral response, and 15% of FL samples have selected for point, activating mutations in RRAGC, a member of the Rag GTPase family that controls the activation of the mechanistic target of rapamycin complex 1 downstream of the sensing of cellular nutrients. S74C and T89N, two of the most frequent activating single-amino acid changes in RRAGC, when targeted to the endogenous Rragc locus in mice, confer only a partial insensitivity to nutrient deprivation but strongly exacerbate B-cell responses and accelerate lymphomagenesis. Surprisingly, this moderate increase in nutrient signaling affected the interaction of B cells with the cellular microenvironment, synergizing their effects on mTORC1 activation with paracrine cues from the supportive T-cell microenvironment that activate B cells via the PI3K–Akt–mTORC1 axis. Hence, Rragc mutations sustain induced germinal centers and murine and human FL in the presence of decreased T-cell help. From a therapeutic standpoint, Rragc mutations impose a selective vulnerability to pharmacologic inhibition of mTORC1. Our results support a model in which activating mutations in the nutrient signaling pathway foster lymphomagenesis by corrupting a nutrient-dependent control over paracrine signals from the T-cell microenvironment. While pharmacologic inhibition of mTORC1 with rapamycin yielded exciting preclinical responses in murine lymphomas with activating mutations in Rragc, targeting the nutrient signaling cascade itself, instead of using such allosteric, partial inhibition of mTOR, may constitute a more efficacious intervention. Because nutrient signaling inhibitors are still in development phase, their efficacy and safety remain unproven. Previous genetic approaches to investigate the consequences of inhibition of Rag GTPase signaling relied on deletion of the Rags in mice and led to severe phenotypes and death. Incomplete inhibition of nutrient signaling, an approach that would mirror more closely the effect of small molecules, has not been pursued to support both their efficacy and safety. We have generated knock-in mice endogenously expressing a point-mutant form of RagC (Q119L) that partially suppresses nutrient signaling. RagCQ119L/Q119L mice are not viable, but RagCQ119L/+ mice show minimal phenotypic alterations with partially decreased nutrient signaling. While B-cell development was unaffected, B-cell activation and the humoral response were impaired in RagCQ119L/+ in a B cell-intrinsic manner. When bred to the FL- and autoimmunity-prone strain VavP-Bcl2, RagCQ119L/+ mice were protected against development of both FL and autoimmunity. No obvious systemic tradeoff for the suppression of nutrient signaling seems to occur, because RagCQ119L/+ mice show normal physiology and longevity with a similar age-dependent health decline. Altogether, our work supports the oncogenicity of activating mutations in components of the nutrient signaling pathway, such as RagC, and both the efficacy and safety of a moderate inhibition of nutrient signaling against pathologic B cells without detrimental systemic effects. Citation Format: Ana Ortega-Molina, Cristina Lebrero-Fernández, Nerea Deleyto-Seldas, Alba Sanz, Alejo Efeyan. Oncogenic Rag GTPase signaling links cellular nutrients with the FL microenvironment [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA13.

Abstract IA40: Defining precursors in low-grade lymphomas that seed second lymphomas

Abstract Most patients with low-grade lymphoma will achieve a clinical remission (CR) with standard chemoimmunotherapy. Yet, relapse from minimal residual disease (MRD), either as low-grade or high-grade disease, remains the leading cause of death. Several studies have provided consistent evidence showing the strong prognostic value of measurable MRD using flow cytometry, PCR, next-generation sequencing, or other approaches. While MRD includes the malignant cells that remain in a patient who achieves CR, not all MRD cells may have the functional capability to proliferate into relapse. We recently introduced a nomenclature to more precisely distinguish cells included within MRD. In this nomenclature, M-REC (Minimal-RElapsable Cancer) denotes fully transformed cells capable of driving a relapse; MN-REC (Minimal Non-RElapsable Cancer) denotes fully-transformed cells incapable of driving a relapse; and MRP (Minimal Residual Precursors) denotes residual cells that harbor somatic and/or phenotypic alterations but are not fully malignant, such as those causing a dysplastic “field-effect” in cancers like those of the bladder, head and neck, or esophagus. Studies that defined the genetic phylogeny of lymphoma using diagnostic, relapsed, and transformed B-cell lymphomas from the same patients have identified examples where MRPs repeatedly evolve into clinical lymphomas. This conflicts with the traditional model of cancer evolution, in which “relapses” result from M-RECs that persist after therapy. There are two immediate implications to this paradigm-shifting concept: 1) Many “relapses” are actually second lymphomas, and 2) defining and targeting the vulnerabilities of MRPs may be essential for improving lymphoma-free survival among patients who achieve a CR. It is important to note that the vulnerabilities of MRD may differ within the bone marrow, blood, lymph node, and other sites, so sampling of each is essential. To make this possible, we have advanced two strategies. First, we have established xenografts of human follicular lymphoma explants within immunocompromised mice that capture both malignant and nonmalignant cellular components, as well as predictable transformation to large-cell lymphoma in vivo. Second, we have initiated rapid autopsies of patients with disseminated lymphoma to obtain specimens from multiple organs and thereby define the heterogeneity of environmental niches that support the persistence of MRD. Citation Format: Abner Louissaint, David Weinstock. Defining precursors in low-grade lymphomas that seed second lymphomas [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA40.

Mutant EZH2 Induces a Pre-malignant Lymphoma Niche by Reprogramming the Immune Response

Follicular lymphomas (FLs) are slow-growing, indolent tumors containing extensive follicular dendritic cell (FDC) networks and recurrent EZH2 gain-of-function mutations. Paradoxically, FLs originate from highly proliferative germinal center (GC) B cells with proliferation strictly dependent on interactions with T follicular helper cells. Herein, we show that EZH2 mutations initiate FL by attenuating GC B cell requirement for Tcell help and driving slow expansion of GC centrocytes that become enmeshed with and dependent on FDCs. By impairing Tcell help, mutant EZH2 prevents induction of proliferative MYC programs. Thus, EZH2 mutation fosters malignant transformation by epigenetically reprograming B cells to form an aberrant immunological niche that reflects characteristic features of human FLs, explaining how indolent tumors arise from GC B cells.

Cathepsin S Alterations Induce a Tumor-Promoting Immune Microenvironment in Follicular Lymphoma

Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ Tcells invitro. Tumors with hyperactive CTSS showed increased CD4+ Tcell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.

Follicular Lymphoma Organoids for Investigating the Tumor Microenvironment

Background: Current in vitro lymphoma models, including three-dimensional organoids, generally contain exclusively neoplastic lymphocytes and require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of syngeneic tumor-infiltrating lymphocytes (TILs) alongside endogenous primary malignant lymphocytes could be useful for modeling complex interactions in the TME, and for immunological maneuvers and therapies relying on TILs. However, such conditions for maintaining lymphomas in their syngeneic TME as a cohesive unit have remained elusive. Methods: We adapted an air-liquid interface (ALI) method that we previously described (Neal JT et al 2019 Cell) for propagating patient-derived organoids (PDOs) from primary human follicular lymphomas. Surgically excised lymphoma samples were tested for the ability to maintain lymphoma cell viability in vitro using a lymph node organoid technique. Lymph nodes containing lymphoma cells (and in one case, a PBMC sample including circulating lymphoma cells) were processed into a single cell suspension and frozen until use. Samples were thawed and prepared into immune organoids (see figure). We assessed cell composition by flow cytometry on day 7, and in a subset of samples, up to 21 days post-thaw. Results: A total of 6 patients were profiled for PDO formation, PDO composition and stability, and PDO longevity. 4 of 6 samples showed good cell viability at day 7 post-culture and in a subset of samples, up to 21 days post-culture. Cell composition was well-maintained over time, with presence of lymphoma cells (CD19+ CD10+ CD5-) easily detectable and maintenance of supporting cells of the lymph node such as T follicular helper cells (CD3+ CD4+ CXCR5+ PD-1+) and non-B, non-T cells. Supporting lymph node cells were not detected in the PBMC sample, suggesting the cell composition is related to the initial composition and not due to differentiation in vitro. Genotyping, gene expression phenotyping, and T-cell/B-cell receptor profiling data will be presented at the meeting, including accuracy of PDOs for preserving the original spectrum of these indices. Conclusions: Propagation of PDOs of primary lymphomas with endogenous immune stroma is feasible and maintains cohesive elements of the TME. This system should allow immunoncology investigations within the TME and to facilitate personalized immunotherapy testing. Fig 1: Human lymphoma organoid cultures as a model to study tumor microenvironments and immune responses in vitro. (A) Experimental schema for preparing lymphoma organoids from tumor explants. In an adapted workflow optimized for ex vivo culture of human tonsillar germinal centers (Wagar L et al, submitted), we subject cryopreserved FL samples to organoid culture. (B) An example of follicular lymphoma organoid reorganization in vitro after four days in culture. (C) Total cell viability in FL organoids for up to 21 days in culture. Six samples were tested. (D) Frequency of major cell types in FL samples after organoid culture. Although there is variation among donors' samples, an individual's cell composition is well maintained for at least 14 days in most organoids. Figure 1 Disclosures Khodadoust: Corvus Pharmaceuticals: Research Funding. Davis:Vir Biotechnology: Consultancy, Equity Ownership, Honoraria; PACT Bio: Consultancy, Equity Ownership, Honoraria; Adicet Inc: Consultancy, Equity Ownership, Honoraria; Chuga Pharmabody: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Atreca: Consultancy, Equity Ownership, Honoraria; Juno: Consultancy, Equity Ownership, Honoraria. Alizadeh:Pfizer: Research Funding; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Roche: Consultancy.

Oncogenic Rag GTPase signaling enhances B cell activation and drives follicular lymphoma sensitive to pharmacological inhibition of mTOR.

The humoral immune response demands that B cells undergo a sudden anabolic shift and high cellular nutrient levels which are required to sustain the subsequent proliferative burst. Follicular lymphoma (FL) originates from B cells that have participated in the humoral response, and 15% of FL samples harbor point, activating mutations in RRAGC, an essential activator of mTORC1 downstream of the sensing of cellular nutrients. The impact of recurrent RRAGC mutations in B cell function and lymphoma is unexplored. RRAGC mutations, targeted to the endogenous locus in mice, confer a partial insensitivity to nutrient deprivation, but strongly exacerbate B cell responses and accelerate lymphomagenesis, while creating a selective vulnerability to pharmacological inhibition of mTORC1. This moderate increase in nutrient signaling synergizes with paracrine cues from the supportive T cell microenvironment that activates B cells via the PI3K-Akt-mTORC1 axis. Hence, Rragc mutations sustain induced germinal centers and murine and human FL in the presence of decreased T cell help. Our results support a model in which activating mutations in the nutrient signaling pathway foster lymphomagenesis by corrupting a nutrient-dependent control over paracrine signals from the T cell microenvironment.

CXCR5+CD8+ T cells are a distinct functional subset with an antitumor activity.

CXCR5 mediates homing of both B and follicular helper T (TFH) cells into follicles of secondary lymphoid organs. We found that CXCR5+CD8+ T cells are present in human tonsils and follicular lymphoma, inhibit TFH-mediated B-cell differentiation, and exhibit strong cytotoxic activity. Consistent with these findings, adoptive transfer of CXCR5+CD8+ T cells into an animal model of lymphoma resulted in significantly greater antitumor activity than CXCR5−CD8+ T cells. Furthermore, RNA-Seq-based transcriptional profiling revealed a 77-gene signature unique to CXCR5+CD8+ T cells. The upregulated 33 genes among the 77-gene signature correlated with improved survival in follicular lymphoma patients. We also showed that CXCR5+CD8+ T cells could be induced and expanded ex vivo using IL-23 plus TGF-β, suggesting a possible strategy to generate these cells for clinical application. In summary, our study identified CXCR5+CD8+ T cells as a distinct T-cell subset with ability to suppress TFH-mediated B-cell differentiation, exert strong antitumor activity, and confer favorable prognosis in follicular lymphoma patients.

SAT-008 Overcoming BCL-2 Resistance in Glucocorticoid-Treated Tumor Lymphoid Cells

Glucocorticoids are prescribed in many chemotherapy protocols for neoplasms of lymphoid origin based on their ability to inhibit lymphocyte proliferation and promote cell death (apoptosis). Lymphocytes have been shown to be profoundly sensitive to glucocorticoid-induced apoptosis, however glucocorticoid resistance restricts the efficacy of these stress hormones and reduces their clinical value. Bcl-2 is an anti-apoptotic protein that promotes cell survival and resistance to cell death during glucocorticoid therapy. Predictably, Bcl-2, as well as other anti-apoptotic Bcl-2 family members, have been found to be overexpressed in a variety of human cancers, including in over 90% of human follicular lymphomas and 95% of chronic lymphocytic leukemias. Unfortunately, approaches to overcome Bcl-2 resistance in cancer cells including anti-sense oligonucleotides, drugs that inhibit Bcl-2 function, and BH3 mimics have not been effective. We took a unique approach to overcome Bcl-2 resistance in tumor cells expressing this anti-apoptotic protein by first identifying points along glucocorticoid-induced cell death signaling program that are explicitly inhibited by Bcl-2. We then intervened at this stage that bypasses the point of inhibition. Provocatively, drugs such as CCCP and several microbial toxins which disrupt the mitochondrial membrane potential, when used in combination with glucocorticoids overcome the resistance afforded by Bcl-2 and kills the cells. Our data suggests drugs which alter the mitochondrial membrane potential in combination with glucocorticoids provide an effective approach in killing lymphocytic tumor cells expressing Bcl-2. This combination should be considered in accompanying current chemotherapeutic drug combinations to provide additional therapeutic benefit in overcoming Bcl-2 resistant tumors.

Desynchronization of the Germinal Center Dynamics and Remodeling of the Tumor Microenvironment Characterize KMT2D-Driven Lymphomagenesis

Follicular lymphoma (FL) is a prototypical example of germinal center (GC) derived B-cell lymphoma. Using a mouse model recapitulating the sporadic occurrence of the FL hallmark BCL2/IGH translocation in healthy individuals, our previous work demonstrated that FL genesis is a dynamic process that requires multiple re-entries of BCL2+ memory B-cells into the GC to ultimately accumulate in lymphoid organs. In line with this, using single-cell gene expression analysis of human FL vs normal GC B-cells, we recently discovered that FL cells are not ‘frozen’ at a particular GC maturation stage but instead exhibit a major desynchronization of the GC-specific expression program (Milpied et al. Nat Immunol in press). Since KMT2D loss-of-function mutations and BCL2 translocations are the 2 main alterations in FL, we hypothesized these 2 genetic events might explain the GC program desynchronization we observe in humans.To explore the in vivo consequences of Kmt2d inactivation with Bcl2 overexpression in regulating GC/memory dynamics, we transduced bone marrow progenitors carrying B-cell-specific conditional Cd19-Cre Kmt2dflox/flox alleles with a retrovirus encoding human BCL2 or reporter alone, followed by iv transplantation into lethally irradiated WT recipients. Only double-mutant Kmt2d-/-Bcl2+ mice manifested with GC-derived lymphomas in chronically challenged animals, recapitulating histological and phenotypic features associated with human FL progression from early preneoplastic lesions to overt FL-like tumors. We used integrative single-cell analysis of surface phenotype (10-color panel), gene expression (88-gene panel by microfluidic RT-qPCR) and IGH clonality to deconvolute cellular heterogeneity of flow-sorted GC and memory B-cells after acute (day 10) or chronic T-cell dependent immunization in double-mutant vs. single Bcl2+, Kmt2d-/- or WT mice, retaining >4000 cells for downstream analysis. Populations of WT GC B-cells were molecularly heterogeneous and spanned a cyclic continuum of transitional B-cell states polarized between the light and dark zone where synchronized expression of gene modules characterized mouse GC functional identity. In acute responses, single and double-mutant mice formed GC similar to control chimera mice, and single GC B-cells from all genotypes clustered together with WT GCs, suggesting unperturbed GC transcriptional dynamics upon first antigen encounter. However, preneoplastic/tumoral Kmt2d-/-Bcl2+ mice after chronic challenge showed massive GC hyperplasia and single B-cells expressed a distinct transcriptional signature that clustered separately from WT GC or memory cells. Specifically, murine lymphoma B-cells sorted with a GC-like phenotype accumulated in transitional cell states where the synchrony of most GC-specific co-expression patterns was progressively lost whereas expression of cytokines (Lta) or surface markers linked to GC to memory transition (Gpr183, Cxcr3) became markedly expressed, indicating that Kmt2d-deficient lymphomas were not blocked at a particular GC stage.Given the importance of T-cell help for the fate ‘decisions’ of GC-to-memory B-cells, we further explored whether tumor cell-intrinsic factors may affect immune cell phenotypes thereby supporting the GC gene expression desynchronization. Using flow cytometry, we found that Kmt2d inactivation instructed a progressive remodeling of the tumor microenvironment with an increased recruitment of CD4+ T-follicular helper (TFH) (n=21, p<0.01). Using droplet-based single-cell RNA-seq to profile total spleens from 2 WT mice and 2 lymphomas (>9000 cells), we revealed a TFH cluster with an activation signature distinct from normal TFH and a concomitant expansion of exhausted CD8+ T-cells strongly co-expressing inhibitory receptors (Lag3, Tim3, Pdcd1), thereby indicating that Kmt2d inactivation in B-cells may favor lymphoma formation by shaping the FL tumor supportive niche and contributing to immune evasion.In summary, our integrative single-cell analyses in murine lymphomas revealed that Kmt2d mutations in FL not only instruct B-cell intrinsic effects involving the desynchronization of the normal GC expression program, but also trigger a concomitant re-education of a tumor-supportive immune microenvironment, establishing for the first time a key link between the most frequent epigenetic alteration and the FL microenvironment. DisclosuresSalles:F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; Janssen: Honoraria, Other: Advisory Board; Novartis: Consultancy, Honoraria; Takeda: Honoraria; Servier: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Merck: Honoraria; BMS: Honoraria, Other: Advisory Board; Morphosys: Honoraria; Acerta: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Other: Advisory Board; Pfizer: Honoraria; Servier: Honoraria; Abbvie: Honoraria.

Targeting Histone Acetyltransferase Gene Inactivation in Diffuse Large B Cell Lymphoma

Diffuse Large B-cell Lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma, accounting for ~30% of de-novo diagnoses and also arising as a frequent clinical evolution of indolent lymphomas. Although curable in a substantial fraction of cases, one third of patients do not achieve durable remissions, highlighting the need for novel, targeted therapies. Over the past decade, we and others have identified the CREBBP acetyltransferase and, less frequently, its paralogue EP300 as highly recurrent targets of inactivating somatic mutations/deletions in DLBCL and follicular lymphoma (FL) (30% and 60% of patients, respectively), indicating a prominent role in the pathogenesis of these tumors (Pasqualucci et al., Nature 2011). In most cases, mutations are heterozygous and the residual wildtype (WT) allele is expressed, suggesting a haploinsufficient tumor suppressor role. Indeed, germinal center (GC)-specific loss of Crebbp perturbs the expression of genes that are relevant to the normal biology of this structure, i.e. the lymphoma cell of origin, and cooperates with BCL2 deregulation to increase the incidence of tumors recapitulating the features of the human disease (Zhang et al., Cancer Discovery 2017). Intringuingly, while CREBBP binds to virtually all GC-specific superenhancers, no detrimental effects were observed upon its deletion in mice, suggesting the existence of compensatory mechanisms. Consistent with this hypothesis, inactivation of CREBBP and EP300 rarely coexist in human DLBCL and FL, suggesting that cells require a certain amount of acetyltransferase activity.To investigate whether EP300 compensates for CREBBP loss in the GC, we analyzed the GC responses in compound mouse models engineered to specifically delete these two genes (alone and in combination) upon SRBC immunization and induction of a Cγ1-driven Cre-recombinase. While CrebbpKO mice showed a mild increase in GC formation, as reported, loss of Ep300 led to ~40% reduction in the percentage of GC cells (mean: 1.8% vs 3.1% in WT littermates; p<0.05), documenting that these two enzymes play non-entirely overlapping roles in this population. Importantly, GC formation was completely abrogated in CrebbpKOEp300KO mice and dramatically impaired in CrebbpHETEp300KO mice, as compared to both WT and single EP300KO mice. These data suggest that GC B cells require a minimum amount of acetyltransferase activity, and reveal a potential therapeutically exploitable dependency of Crebbp-mutated GC B cells on Ep300.In order to probe if a similar dependency exists in neoplastic GC B cells, we used an inducible CRISPR/Cas9 system to delete EP300 (or a control non-genic region) in 4 DLBCL cell lines representative of the various CREBBP genotypes found in DLBCL, and monitored cell proliferation and survival in competition assays over 12 days. Compared to CREBBPWT, CREBBP heterozygous and homozygous mutant cells were significantly counter-selected from the total population following doxycycline induced EP300 deletion (~30% at day 7). Moreover, no EP300-edited clones were recovered from the CREBBP mutant lines in single cell plating assays, compared to CREBBP WT (p<0.01). Thus, DLBCL cells remain addicted to the residual EP300 aceyltransferase activity, supporting the existence of a therapeutic window for EP300 inhibitors.To explore this concept further, we generated isogenic DLBCL clones carrying WT or defective CREBBP alleles (n=4 each), and performed drug-sensitivity assays with 2 novel small molecule inhibitors that specifically target the CREBBP/EP300 HAT or BRD domain. While, at higher doses, both inhibitors interfered with cell growth in all clones, CREBBPKO cells were significantly more sensitive than their isogenic WT pairsat low nanomolar ranges (IC50: 60nM vs 300nM). Importantly, we were able to design an in vitro protocol that was toxic to CREBBPKO cells but tolerated by CREBBPWT cells, providing a proof of concept for therapeutically targeting these molecules.In conclusion, we show that CREBBP and EP300 have differential roles in normal GC B cell development and that CREBBP mutated cells are addicted to the residual EP300 activity. This dependency is maintained in DLBCL cells, providing the basis for the potential application of acetyl transferase inhibition into the clinical settings. DisclosuresNo relevant conflicts of interest to declare.

PO-222 Molecular characterisation of low grade lymphoma in northern sea otters (Enhydra lutris kenyoni)

IntroductionLymphoid cancers are a biologically diverse group of neoplasms. Disease aetiology is multifactorial, implicating viral and infectious agents, immune dysfunction, and genetic factors. Lymphoid cancers affect domestic and laboratory animals; among members of Mustelidae, lymphoma has been reported in domestic ferrets, yet rarely in sea otters, with only 3 reported cases since 2002. However, since 2006, 3 sea otters at the Vancouver Aquarium (British Columbia, Canada) developed lymphoma. All 3 sea otters were rescued as young animals and have lived at the aquarium for the majority of their lives. A 21 year old female was diagnosed with chronic lymphocytic leukaemia, while 2 males, aged 12 and 16, were diagnosed with lymphoma. Both males received chemotherapy before being humanely euthanized due to declining quality of life. A high rate of blood cancers in this population motivated the molecular characterisation of the most recent lymphoma.Material and methodsWhole genome sequencing was performed on peripheral blood and a lymphoma needle biopsy. The tumour biopsy was sequenced on the Illumina HiSeq X and Oxford Nanopore MinION, generating 264.5 and 4.63 passed filtered Gbp, respectively. De novo assembly was performed on the Illumina DNA and RNA data using ABySS 1.3.4. Structural variants were called with trans-abyss 1.4.10. Somatic variants were called by Strelka (v1.0.15) using matched tumour-normal samples aligned against the Enhydra lutris kenyoni reference genome. Variants were annotated by snpEFF (v4.3) using an in-house custom gene annotation database. The mutations in the sea otter tumour were subsequently compared to human and canine lymphomas.Results and discussionsThe sea otter genome contains 24 129 genes, of which 17 421 were mapped to the human genome. We identified several mutated genes with a known involvement in human lymphomas. We observed a stop-gain mutation in EP400, a paralog of human EP300. EP300 is a tumour suppressor which has been previously implicated in human Diffuse Large B-Cell and Follicular lymphomas. We also observed an indel in SPEN, a NOTCH pathway regulator, the deregulation of which is associated with human hematopoietic neoplasms.ConclusionThese observations suggest gene and pathway deregulation are a shared genetic commonality between sea otter and human lymphomas. Comparing the mutational spectrum in human, canine, and sea otter lymphoid cancers may elucidate aetiology and treatment options, and facilitate a One Health approach to neoplasia.

The combination of TRAIL and MG-132 induces apoptosis in both TRAIL-sensitive and TRAIL-resistant human follicular lymphoma cells

We have previously shown that the human follicular lymphoma cell line, HF28GFP, is sensitive to TRAIL-mediated apoptosis. Nevertheless, when the same cells overexpress anti-apoptotic Bcl-2 family protein, Bcl-xL (HF28Bcl-xL), they become resistant to TRAIL. Thus, these cell lines help us to investigate the action of novel apoptosis inducing candidate drugs. In the present study, we examined the effects of MG-132 (a proteasome inhibitor), LiCl (a glycogen synthase kinase-3 inhibitor) and/or TRAIL on pro-apoptotic Bcl-2 family proteins such as Bim and Bid. Here we demonstrate that the combination of MG-132 and TRAIL induced significant apoptotic cell death in both cell lines, HF28GFP and HF28BclxL. Apoptosis correlated with a decrease of phospho-ERK1/2, the accumulation of Bim and translocation of truncated Bid (tBid) and jBid. In addition, the combination of MG-132 and TRAIL seemed to target other apoptotic factors, which led to the accumulation of active capsase-3. Furthermore, co-stimulation of LiCl and TRAIL induced apoptosis in HF28GFP cells. However, HF28Bcl-xL cells were far less sensitive to the combinatorial effects of LiCl and TRAIL. Interestingly, we observed that LiCl did not target Bim and Bid proteins. In conclusion, these data show that targeting of pro-apoptotic Bcl-2 family proteins simultaneously through a selective proteasome inhibition might help to overcome TRAIL resistance caused by overexpression of anti-apoptotic Bcl-2 family proteins. Moreover, the data may provide new strategies to develop targeted therapies against lymphomas.

RIP1 has a role in CD40-mediated apoptosis in human follicular lymphoma cells

CD40 is a cell surface receptor which belongs to tumor necrosis factor receptor (TNFR) family members. It transmits signals that regulate diverse cellular responses such as proliferation, differentiation, adhesion molecule expression and apoptosis. Unlike other TNFR family members (TRAIL-R, Fas-R and TNFR1), the CD40 cytoplasmic tail lacks death domain. However, CD40 is capable of inducing apoptosis in different types of cancer cells including lymphoma. The apoptotic effect of CD40 is linked to the involvement of Fas, TRAIL or receptor interacting protein 1 (RIP1) kinase. We have previously shown that CD40 activation has anti-apoptotic or apoptotic effect in follicular lymphoma (FL) cell lines. In this study, we investigated the mechanism by which CD40 mediates apoptosis in a follicular lymphoma cell line, HF4.9. We show here that CD40-induced apoptosis was dependent on caspase-8 activation because caspase-8 specific inhibitor, Z-IETD-FMK completely prevented apoptosis. Therefore, the involvement of TRAIL, Fas and RIP1 in caspase-8 activation was examined. The exogenous TRAIL-induced apoptosis was fully prevented by anti-TRAIL neutralizing antibody. However, the antibody had no effect on CD40-induced apoptosis indicating that CD40 did not induce the expression of endogenous TRAIL in HF4.9 cells. Moreover, the cells were not sensitive to Fas-mediated apoptosis. Interestingly, RIP1 specific inhibitor, necrostatin-1 decreased CD40-induced apoptosis, which showed that RIP1 has a role in caspase-8 activation. In conclusion, the survival or apoptotic effects of CD40-mediated signaling might be related to the differentiation stages of FL cells.

Crebbp loss cooperates with Bcl2 overexpression to promote lymphoma in mice

AbstractCREBBP is targeted by inactivating mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Here, we provide evidence from transgenic mouse models that Crebbp deletion results in deficits in B-cell development and can cooperate with Bcl2 overexpression to promote B-cell lymphoma. Through transcriptional and epigenetic profiling of these B cells, we found that Crebbp inactivation was associated with broad transcriptional alterations, but no changes in the patterns of histone acetylation at the proximal regulatory regions of these genes. However, B cells with Crebbp inactivation showed high expression of Myc and patterns of altered histone acetylation that were localized to intragenic regions, enriched for Myc DNA binding motifs, and showed Myc binding. Through the analysis of CREBBP mutations from a large cohort of primary human FL and DLBCL, we show a significant difference in the spectrum of CREBBP mutations in these 2 diseases, with higher frequencies of nonsense/frameshift mutations in DLBCL compared with FL. Together, our data therefore provide important links between Crebbp inactivation and Bcl2 dependence and show a role for Crebbp inactivation in the induction of Myc expression. We suggest this may parallel the role of CREBBP frameshift/nonsense mutations in DLBCL that result in loss of the protein, but may contrast the role of missense mutations in the lysine acetyltransferase domain that are more frequently observed in FL and yield an inactive protein.