Human B-cell Lymphoma Research Articles

Abstract 2049: A novel, rapid, sensitive, and reproducible in vivo platform to assess efficacy and toxicity of bispecific antibodies

Abstract A bispecific T-cell engager (BiTE) enhances the antitumor capabilities of T-cells by directing them to recognize a tumor-specific antigen. Several BiTEs are currently in clinical trials, and there is one FDA-approved BiTE for cancer therapy at the moment. However, one of the biggest challenges BiTE is currently facing is cytokine release syndrome (CRS). While most patients have mild flu-like symptoms, some patients experience a severe inflammatory syndrome, which could ultimately cause multi-system organ failure and death. Thus, the key is to accurately predict the risk-benefit ratio of each individual before starting the treatment. Unfortunately, there is no reliable in vitro or in vivo model to predict the toxicity and efficacy of BiTE at the moment. Additionally, one of the most challenging factors for pre-clinical evaluation of BiTEs for toxicity and efficacy is the inherent differences among patients’ immune systems. Here, we developed an in vivo PBMC-humanized mouse model that can evaluate both toxicity and efficacy of BiTE and other immune-oncology therapeutics in each individual mouse. The platform was validated using an anti-CD28 superagonist (TGN1412), which none of the pre-clinical in vitro assays and in vivo studies, including non-human primates, executed before clinical trials were able to predict the observed clinical toxicities. This model uses luciferase-tagged human B-cell lymphoma Raji tumor cells to allow the measurement of tumor burden and response to treatment, along with toxicity simultaneously. In addition, we showed that this platform can determine individual PBMC donor differences, and potential adverse drug combinations and drug dose-response with efficacy and safety on individual PBMC donors. The model we developed can potentially be used as a predictive and reproducible platform to identify patient, cancer, and therapy combinations at risk for developing CRS. This model will also help to optimize the therapeutic drug dose range to improve the safety profile of BiTE and other immune-oncology drugs. We believe this platform will greatly benefit not only the scientific community but potentially cancer patients as well. Citation Format: Jiwon Yang, Jing Jiao, Kyle Draheim, Danying Cai, Michael A. Brehm, Leonard D. Shultz, Dale L. Greiner, Mingshan Cheng, James G. Keck. A novel, rapid, sensitive, and reproducible in vivo platform to assess efficacy and toxicity of bispecific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2049.

Abstract LB559: The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis

Abstract The X-linked gene DDX3X encodes an RNA helicase and is mutated at high frequencies in several types of human B-cell lymphoma. Females have two active DDX3X alleles and males carry a DDX3Y homolog on the Y chromosome. We show here that pan-hematopoietic, homozygous deletion of Ddx3x in female mice perturbs erythropoiesis causing early developmental arrest. However, both hemizygous male and heterozygous female embryos develop normally, suggesting that one Ddx3x allele is sufficient for fetal hematopoietic development in females and that the Ddx3y allele can compensate for the loss of Ddx3x in males. In adult mice, loss of DDX3X affects hematopoietic progenitors, early lymphoid development, marginal zone, and germinal center B-cells as well as lymphomagenesis driven by an Eμ-Myc or λ-Myc transgene in a sex-dependent manner. A B-cell-specific depletion of both Ddx3x alleles induces a delay in tumorigenesis in female mice while Ddx3x deletion in male mice does not impact tumorigenesis. However, male Eμ-Myc mice lacking Ddx3x on a pan-hematopoietic way remained for the majority lymphoma-free. The few tumors that appeared in these male mice showed upregulated expression of DDX3Y indicating a requirement of DDX3 activity for lymphomagenesis. Our data reveal sex-specific roles of DDX3X in erythro- and lymphopoiesis as well as in MYC-driven lymphomagenesis, which are important when considering inhibition of DDX3 as a treatment of Myc dependent B-cell lymphoma. Citation Format: Marion Lacroix, Hugues Beauchemin, Julie Ross, Jennifer Fraszczak, Peiman Shooshtarizadeh, Riyan Chen, Tarik Moroy. The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB559.

Abstract 2816: A novel in vivo model to simultaneously assess efficacy and toxicity of chimeric antigen receptor T-cell immunotherapy

Abstract Over the past few years, chimeric antigen receptor T (CAR T)-cell therapy has emerged as a novel treatment option for certain hematologic malignancies. Several CAR T-cell therapies produced highly efficacious responses from patients with hematologic malignancies. However, wider adoption of CAR T-cell therapy is challenged due to the potential development of life-threatening toxicities including cytokine release syndrome (CRS). CRS symptoms can range from mild fever to life-threatening events, including death. Preclinical animal models to assess the efficacy and toxicity of CAR T-cell therapies have been lacking. Here, we developed a novel mouse model to assess the efficacy and toxicity of CAR T-cell therapy simultaneously. Two different PBMC humanized NSG™ variants were used. NSG-MHC Class I/II double knock-out strain is known to have a delayed onset of GvHD, and NSG-SGM3xIL15 strain shows a higher engraftment level of human NK and myeloid cells, in addition to human T cells. Mice were humanized using PBMCs and treated with autologous or allogeneic CAR T cells, and efficacy and toxicity were assessed. Compared to the control treatment, CD19 CAR T-cells caused a decrease in the human CD19+ cell population in the blood and spleen, and induced cytokine release. Both T-cell and myeloid cytokine releases, including IFNgamma, IL-10, RANTES, and MIP-1alpha, were induced in our animal models. The in vivo platform can also be used to determine individual PBMC/CAR T donor differences and show the extent of efficacy and toxicity of each PBMC donor treated with autologous and allogeneic CAR T-cell therapy. We further confirmed anticancer efficacy using luciferase-tagged human B-cell lymphoma Raji tumor cells, which express a high level of CD19. In summary, we have developed a novel in vivo model to test the efficacy and toxicity of both autologous and allogeneic CAR T-cell therapy simultaneously. Citation Format: Jiwon Yang, Won Lee, Jing Jiao, Danying Cai, Heather Gustafson, Rebecca Gardner, Mingshan Cheng, James G. Keck. A novel in vivo model to simultaneously assess efficacy and toxicity of chimeric antigen receptor T-cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2816.

Anticancer activity of IRAK-4 inhibitors against canine lymphoid malignancies.

The interleukin-1 receptor-related kinase 4 (IRAK4), downstream of myd88, plays an essential role in hyperactive TLR signalling seen in some B-cell lymphomas. In particular, efficient IRAK4 inhibitors of activated B-cell subtype of human diffuse large B-Cell lymphoma (DLBCL) are being developed. However, the anticancer effect of IRAK-4 inhibitors in veterinary medicine has not been elucidated. It is therefore explored in this study involving the GL-1 and CL-1 canine lymphoma cell lines in vitro. MyD88 expression was analysed using polymerase chain reaction. GL-1 and CL-1 cells were subjected to concentration- and time-dependent treatment with an IRAK-4 inhibitor and assessed for viability, TLR signalling association and apoptosis using a cell counting Kit-8 assay, Western blotting and flow cytometry. The GL-1 and CL-1 cells exhibited enhanced MyD88 expression, however, canine peripheral blood mononuclear cells (cPBMCs) did not. The IRAK-4 inhibitor reduced cell viability in a dose- and time-dependent manner, significantly reduced the phosphorylation of molecules associated with TLR signalling at IC50 such as IRAK1, IRAK4, NF-κB and STAT3, and induced apoptosis in GL-1 and CL-1 cells. The anticancer effect of the IRAK-4 inhibitor on canine lymphoma cells is mediated by apoptosis via downregulation of TLR signalling.

Transcriptomics-Based Investigation of Molecular Mechanisms Underlying Apoptosis Induced by ZnO Nanoparticles in Human Diffuse Large B-Cell Lymphoma.

IntroductionZinc oxide nanoparticles (ZnO NPs) show anti-cancer activity. Diffuse Large B-cell Lymphoma (DLBCL) is a type of B-cell malignancies with unsatisfying treatment outcomes. This study was set to assess the potential of ZnO NPs to selectively induce apoptosis in human DLBCL cells (OCI-LY3), and to describe possible molecular mechanisms of action.MethodsThe impact of ZnO NPs on DLBCL cells and normal peripheral blood mononuclear cells (PBMCs) was studied using cytotoxicity assay and flow-cytometry. Transcriptomics analysis was conducted to identify ZnO NPs-dependent changes in the transcriptomic profiles of DLBCL cells.ResultsZnO NPs selectively induced apoptosis in DLBCL cells, and caused changes in their transcriptomes. Deferential gene expression (DGE) with fold change (FC) ≥3 and p ≤ 0.008 with corrected p ≤ 0.05 was identified for 528 genes; 125 genes were over-expressed and 403 genes were under-expressed in ZnO NPs-treated DLBCL cells. The over-expressed genes involved in biological processes and pathways like stress response to metal ion, cellular response to zinc ion, metallothioneins bind metals, oxidative stress, and negative regulation of growth. In contrast, the under-expressed genes were implicated in DNA packaging complex, signaling by NOTCH, negative regulation of gene expression by epigenetic, signaling by WNT, M phase of cell cycle, and telomere maintenance. Setting the FC to ≥1.5 with p ≤ 0.05 and corrected p ≤ 0.1 showed ZnO NPs to induce over-expression of anti-oxidant genes and under-expression of oncogenes; target B-cell receptor (BCR) signaling pathway and NF-κB pathway; and promote apoptosis by intrinsic and extrinsic pathways.DiscussionOverall, ZnO NPs selectively induced apoptosis in DLBCL cells, and possible molecular mechanisms of action were described.

The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis

Abstract The X-linked gene DDX3X encodes an RNA helicase and is mutated at high frequencies in several types of human B-cell lymphoma. Females have two active DDX3X alleles and males carry a DDX3Y homolog on the Y chromosome. We show here that pan-hematopoietic, homozygous deletion of Ddx3x in female mice perturbs erythropoiesis causing early developmental arrest. However, both hemizygous male and heterozygous female embryos develop normally, suggesting that one Ddx3x allele is sufficient for fetal hematopoietic development in females and that the Ddx3y allele can compensate for the loss of Ddx3x in males. In adult mice, loss of DDX3X affects hematopoietic progenitors, early lymphoid development, marginal zone and germinal center B-cells and lymphomagenesis driven by an Em-Myc transgene in a sex-dependent manner. Loss of one Ddx3x allele slightly accelerated MYC-driven lymphomagenesis in female mice, while male Em-Myc mice lacking Ddx3x remained almost lymphoma-free. However, the few tumors that appeared in male mice showed upregulated expression of DDX3Y indicating a minimal requirement for DDX3 activity for lymphomagenesis. Our data reveal sex specific roles of DDX3X in erythro- and lymphopoiesis as well as in MYC-driven lymphomagenesis, which are important when considering inhibition of DDX3 as a treatment of B-cell lymphoma. Tarik Möröy holds a Canada Research Chair (Tier 1), a Foundation grant from the CIHR (FDN-148372) and support from the Cancer Research Society (CRS). Marion Lacroix is supported by fellowships from the Cole Foundation and the IRCM.

Comprehensive analysis of the expression and significance of CXCLs in human diffuse large B-cell lymphoma

CXCL chemokines (CXCLs) are small cytokines or signal proteins secreted by cells that have been proven to be linked to the occurrence and development of many kinds of cancer. However, the expression and diagnostic and prognostic value of CXCLs in diffuse large B-cell lymphoma (DLBCL) remain to be further studied. We obtained CXCL transcription and survival data of patients with DLBCL from Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), The Cancer Genome Atlas (TCGA), TIMER and cBioPortal databases. R software, STRING and EXCEL were used to process the data. This study discovered that the expression levels of CXCL9-14 in DLBCL were higher than those in normal tissues, while CXCL4, CXCL7 and CXCL8 were lower in tumor than in normal tissues. The expression levels of CXCL2, CXCL10 and CXCL11 were related to tumor stage. CXCL9-14 could be used as an auxiliary molecular marker for the diagnosis of DLBCL. CXCL17 might be a potential prognostic marker of DLBCL.

Circulating Endocannabinoids in Canine Multicentric Lymphoma Patients.

The endocannabinoid system is increasingly being implicated in the pathogenesis and progression of various human cancers. Specifically, increased levels of 2-arachidonoylglycerol (2-AG) and oleoythanolamide (OEA) have been demonstrated in human diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) patients, respectively. The objectives of this paper were to compare 2-AG, OEA, N-arachidonoylethanolamine (AEA), and palmitoylethanolamide (PEA) levels between dogs with multicentric lymphoma and healthy control dogs. In addition, evaluate 2-AG, OEA, AEA, and PEA levels as biomarkers for progression free interval (PFI) and overall survival time (OST) in the dogs with lymphoma. The study consisted of 26 dogs with multicentric B cell lymphoma, 14 dogs with multicentric T cell lymphoma, and 12 healthy control dogs. Serum 2-AG, OEA, AEA, and PEA levels were measured using liquid chromatography combined with tandem mass spectrometry (LC—MS/MS) in dogs with lymphoma and in healthy dogs. OEA, AEA, and PEA levels were significantly elevated in dogs with lymphoma compared to healthy controls (p < 0.05). Total AG was significantly higher in healthy control dogs (p = 0.049). There was no significant difference between dogs with B cell and T cell lymphoma for any of the measured endocannabinoids. Elevated PEA was significantly associated with decreased PFI (p = 0.04) in dogs with lymphoma with a hazards ratio of 1.816 [95% Confidence Interval (CI): 1.020–3.232]. Overall, dogs with lymphoma have elevated levels of OEA, AEA, and PEA. PEA levels have the potential to be a prognostic biomarker.

TET deficiency perturbs mature B cell homeostasis and promotes oncogenesis associated with accumulation of G-quadruplex and R-loop structures.

Enzymes of the TET family are methylcytosine dioxygenases that undergo frequent mutational or functional inactivation in human cancers. Recurrent loss-of-function mutations in TET proteins are frequent in human Diffuse Large B-Cell Lymphoma (DLBCL). Here we investigate the role of TET proteins in B-cell homeostasis and development of B cell lymphomas with features of DLBCL. We show that deletion of Tet2 and Tet3 genes in mature B cells in mice perturbs B-cell homeostasis and results in spontaneous development of germinal center-derived B cell lymphomas with increased G-quadruplexes and R-loops. At a genome-wide level, G-quadruplexes and R-loops were associated with increased DNA double strand breaks at immunoglobulin switch regions. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented expansion of germinal center B cells, diminished the accumulation of G-quadruplexes and R-loops, and delayed B lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. CRISPR-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss-of-function might predispose to development of B cell malignancies.

Shared and Distinct Genetic Features in Human and Canine B-Cell Lymphomas

IntroductionAnimal models of human cancers are an important tool for the development and preclinical evaluation of new treatments. Canine B-cell lymphoma (cBCL) is an appealing alternative to murine preclinical models due to its frequent, spontaneous incidence and its clinical and histological similarity to human B-cell non-Hodgkin lymphoma (NHL). The potential utility of cBCL as a veterinary model of human B-cell lymphomas would be bolstered by a more complete understanding of the genetic features found in cBCL.MethodsTo study the genetics of cBCL, we obtained fresh frozen and matched plasma/serum from 86 patients from the Canine Comparative Oncology Genomic Consortium(CCOGC) with 65 confirmed as B-cell lymphomas by immunophenotyping. Tumor DNA was prepared into libraries using the QIAseq FX DNA Library Kit (Qiagen). Plasma and serum DNA was prepared into libraries using the NebNext Ultra II DNA Library Prep Kit. Targeted hybridization enrichment was performed on the libraries using our custom baits and sequencing reads were aligned to canFam3.1 using Geneious and each mutation was visually confirmed. Variants were annotated with Variant Effect Predictor and human-dog pairwise alignments were extracted from Ensembl to identify the orthologous human amino acid for all canine variants.ResultsOur analysis confirmed the previously reported high frequency of mutations in TRAF3 and FBXW7. We also observed mutations in POT1, TP53, and SETD2 at similar frequencies to those reported in previous studies. DDX3X was mutated in 20% of cases, which is substantially higher than previously reported. MYC mutations were also more frequent (13%) than has been previously described in cBCL.In human lymphomas, MYC is commonly deregulated by translocation to a potent enhancer and these events are often associated with point mutations in MYC that are induced by aberrant somatic hypermutation (aSHM). Interestingly, we identified a more focal pattern of MYC mutations in cBCL that implies they do not result from aSHM and are likely functional. This finding implicates the conserved MYC phosphodegron sequence, a motif commonly mutated among additional aSHM-associated mutations, as the target of bona fide driver mutations in both human and cBCLs. Mutations in FBXW7 primarily affected the substrate recognition domain responsible for MYC degradation. The observation that MYC and FBXW7 mutations did not co-occur in any canine patient is consistent with the notion that FBXW7 mutations operate as an alternative path to MYC stabilization which is not frequently observed in human NHL.DDX3X was one of the most frequently mutated genes in our cohort (20%). DDX3X mutations are common in human Burkitt lymphoma and, though less abundant in hDLBCL, tend to be observed in samples with MYC translocations. In Burkitt lymphoma, these mutations display a sex-specific pattern, wherein females show mainly missense mutations, while males are affected by loss-of-function mutations. Interestingly, all DDX3X mutations in cBCL are missense variants and are presumed to be dominant acting. This lack of sex difference in DDX3X mutations is an important distinction between human and canine B-cell lymphomas that warrants further exploration.ConclusionsOur study has revealed key differences in the mutational profiles of canine and human B-cell lymphomas and provides an impetus for enhanced genomic characterization of canine lymphomas as a model for human NHL, particularly in clinical trial settings. DisclosuresGrande: Sage Bionetworks: Current Employment. Alcaide: GA Diagnostics AB: Current Employment. Morin: Celgene: Consultancy; Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees; Epizyme: Patents & Royalties. Coyle: Allakos, Inc.: Consultancy.

The Ublituximab-Umbralisib (U2) Drug Regimen Potentiates the Activity of the Novel CD47-CD19 Bispecific Antibody, TG-1801, through the Activation of the G Protein-Coupled Receptor EBI2/GPR183

Introduction: TG-1801 is a novel, bispecific anti-CD47 and anti-CD19 fully human IgG1 antibody that targets CD47 selectively on CD19+ B-cells, sparing red blood cells or platelets and blocking the CD47-SIRPα macrophage checkpoint on mature B cells. TG-1801 is currently in clinical trial as a single agent or in combination with ublituximab, a glyco-engineered CD20 antibody, in B-cell non-Hodgkin lymphoma (B-NHL). The doublet therapy of ublituximab with the dual PI3Kδ/CK1e inhibitor umbralisib (“U2” regimen), provides a non-chemotherapy backbone regimen on which several novel multidrug combinations are being explored clinically. Here we explored in vitro and in vivo potential synergies between TG-1801 and ublituximab, umbralisib, and the U2 combination, in preclinical models of B-NHL.Methods: A panel of n=12 human B-cell lymphoma cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells (BMSCs), M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 +/- U2 treatments was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). In vivo, drug efficacy was determined in a Raji xenograft model, by dosing tumor-bearing mice for 17 days with ublituximab (5mg/kg, qw) +/- umbralisib (150mg/kg, bid), the combination of both and/or TG-1801 (5mg/kg, qw).Results: Here we show on a panel of lymphoma cells lines that the number of receptors per cell and the ratio CD47/CD19 do not impact TG-1801-mediated ADCC or phagocytosis (ADCP). In addition, we show that TG-1801 potentiated ublituximab-mediated ADCC and ADCP and exhibited a similar additive effect when added to U2 combination. In vivo, ublituximab alone displayed a tumor growth inhibition (TGI) of 88%, with 3/8 mice harboring a barely palpable tumor, while the umbralisib alone treatment arm showed a TGI of 50%, with 2/8 mice lacking detectable tumors. TG-1801 exhibited a 76% TGI with 1/8 tumor free-mouse. Most importantly, the combination of TG-1801 with umbralisib alone, ublituximab alone, and U2 achieved TGI of 85%, 93% and 93% respectively, with more tumor-free mice 35 days after the last dose in these three groups. Interestingly, this superior anti-tumor effect of the different TG-1801 combinations was associated with a higher infiltration of mouse macrophages within the tumors as assessed by F4/80 IHC labeling. RNA-seq analysis of the Raji xenografts and of n=4 representative in vitro B-NHL co-cultures treated with TG-1801, U2 or the triple combination uncovered the upregulation of the G-protein coupled receptor EBI2/GRP183 as a common event associated with the synergistic antitumor effects of TG-1801 and U2 in vitro and in vivo. A critical role of EBI2 in the regulation of macrophage activity, B cell migration and in the promotion of a pro-inflammatory phenotype was demonstrated upon exposure of the co-cultures with the EBI2 small molecule inhibitor NIBR189, which impaired the U2/TG-1801-evoked ADCP, B-cell cytoskeleton remodeling and inflammatory cytokine production.Conclusion: The data presented here set the preclinical rationale and support a combination strategy of the novel CD47-CD19 bispecific antibody TG-1801 in B-NHL with other B-cell targeted mechanisms, including umbralisib and ublituximab. DisclosuresNormant: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Miskin: TG Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Roue: TG Therapeutics, Inc.: Research Funding.

P26 A case of B-cell lymphoma affecting the eye in a patient with rheumatoid arthritis following a reduction in the frequency of rituximab therapy

P26 A case of B-cell lymphoma affecting the eye in a patient with rheumatoid arthritis following a reduction in the frequency of rituximab therapy

Abstract 1383: A next-generation PI3Kδ/γ inhibitor, ZX-101A inhibiting solid tumor growth by enhancing anti-tumor immunity

Abstract PI3K signaling cascade plays a central role in the regulation of key cellular pathways including growth, proliferation, differentiation, migration and survival. Previous studies suggested that both PI3Kδ and γ activities exert immunosuppression by targeting regulatory T cells (Treg) and myeloid cells. ZX-101A is an inhibitor of PI3K with predominant activity against the PI3Kδ and PI3Kγ isoforms, whose expressions are largely restricted to hematopoietic cells. Dual inhibition of PI3Kδ and PI3Kγ by ZX-101A may potentially restore the immune surveillance to limit tumor growth.The inhibitory effect of ZX-101A on PI3K activity was evaluated by blocking of AKT phosphorylation at T308 and S473 in human B-cell lymphoma DOHH2 cells and stimulated THP1 monocyte cells. ZX-101A significantly decreased the phosphorylation of AKT at S473 site in comparison to DMSO treated DOHH2 cells, but no difference on p-AKT level between the two concentrations of ZX-101A and the three treatment time points. In contrast, ZX-101A blocked the p-AKT on S473 in LPS-stimulated THP-1 cell line in both dose- and time-dependent manner. In vitro cell proliferation assay demonstrated ZX-101A potently inhibited cell proliferation of B-cell and T-cell lymphoma cell lines, but without potent effect on cells (e.g. 4T1 and CT26) derived from solid tumors. In contrast, ZX-101A inhibited tumor growth in CT26 syngeneic mouse model after 21-day treatment with TGI at 76.9% (1mg/kg QD), 79.5% (3mg/kg QD) and 90.0% (12mg/kg), which was attributed to enhanced anti-tumor immunity by ZX-101A treatment. Flow cytometry study on CT 26 tumors demonstrated that ZX-101A decreased Treg and increased M1/M2 macrophage polarization in the tumor microenvironment. Ex vivo T-cell differentiation assay using mouse spleen T cells and M1/M2 polarization assay using human PBMC cells confirmed the effect of ZX-101A on decreasing Treg differentiation and enhancing M1 macrophage function. Similarly, in vivo anti-tumor growth was observed in 4T1 and A20 syngeneic models. In A20 syngeneic model, ZX-101A in combination of anti-PD1 antibody achieved 99.7% TGI after 18 days of treatment, while anti-PD1 antibody alone only had TGI at 11.7%. Safety assessment on the mouse spleen, thymus and colon organs indicated minimal side effects by the treatment of the ZX-101A alone and in combination with anti-PD1. Its absorption, distribution, metabolism, and excretion (ADME) and safety profile, through a series of preclinical in vitro and in vivo studies, supported the ZX-101A clinical development. These results suggested ZX-101A as a potential immunotherapeutic agent to appropriate solid tumor types. Citation Format: Xiaolin Hao, Zhiyuan Peng, Kun Guo, Ying-Ying Li, Deming Kong, Jinfu Yang, Xiaoli (Shelley) Qin. A next-generation PI3Kδ/γ inhibitor, ZX-101A inhibiting solid tumor growth by enhancing anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1383.

Abstract LB044: Tracking clonal hematopoiesis in patients with classical Hodgkin lymphoma

Abstract Background: Clonal hematopoiesis (CH) is frequent in the elderly and predisposes to blood cancers, mostly of myeloid and T-cell origin. CH incidence and trajectories are not well characterized in classical Hodgkin lymphoma (cHL), a B-cell neoplasm frequent in the young and uniquely featuring rare tumor cells embedded in a supportive microenvironment of hematopoietic origin. Methods: We analyzed for CH 40 cHL cases by subjecting to whole-exome and/or targeted sequencing (mean unique coverage ~150X and ~1000X, respectively) tumor cells microdissected from a tissue biopsy and matched non-neoplastic cells circulating in the blood (n=27 cases) and/or microdissected from the lymph node microenvironment (n=14 cases). Results: 5 cases (12.5%) had blood and/or tissue CH (Table 1), including 3/5 with &amp;gt;70 years and 2/35 &amp;lt;70 years (p-value 0.009). In 3/5 CH cases (aged 30, 45 and 73 years), CH extensively spread through the non-neoplastic tissue microenvironment (32%, 92%, and 60% of cells), being respectively driven by DNMT3AR882H (Case 1), KRASG60D (Case 2) and DNMT3AR882H +TET2Q1274* (Case 5). Notably, in Case 5 CH originated also the tumor cell clone, which was infected by the Epstein-Barr virus and had almost no other somatic mutations exome-wide. This is the first description, in a human B-cell lymphoma clone, of mutant DNMT3AR882, a hotspot in myeloid and T-cell neoplasms. In Case 1, the same mutation determined a massive blood CH (DNMT3AR882H in 94% leukocytes and in 96% B cells), which was surprisingly absent from the tumor cell clone (instead carrying STAT6 and SOCS1 mutations, typical of cHL) and that led, 6 years after therapy for cHL, to a possibly therapy-unrelated NPM1-mutated acute myeloid leukemia (with normal karyotype, wild-type TP53 and PPM1D). Thus, CH trajectories must be carefully disentangled to correctly interpret the histogenesis and pathogenesis of multiple blood cancers arising in patients with CH. Finally, all 3 cases with extensive CH spreading in the cHL tissue progressed after first-line chemotherapy, versus 11/35 (31%; p-value 0.043) evaluable cases with absent or non-extensive tissue CH, who otherwise had similar clinical features (Table 1) and received a no less intense first-line therapy. Conclusion: Our results expand to cHL the spectrum of hematologic malignancies associated with CH, with potential implications for the pathogenesis and prognosis of this cancer. [A. Marra and A. Venanzi are co-first authors of this work.] Clonal Hematopoiesis in patients with classical Hodgkin lymphomaPatients with Clonal Hematopoiesis in a 40-cHL case cohortPt.#Age (years)Time of samplingProgressed after 1st line chemotherapyGene mutationVAF in whole bloodVAF in non-neoplastic tissue microenvironmentVAF in tumor cellsCase 145OnsetYESDNMT3AR882H47%16.4%Not detectedCase 2302nd relapseYESKRASG60DNot available45.9%Not detectedCase 383OnsetNot evaluableCBLG375SNot available2.5%Not detectedCase 481OnsetNOTET2N1487lfs843.2%Not detectedNot detectedCase 5731st relapseYESDNMT3AR882HNot available30%43%TET2Q1274Not available8.4%31.1%Clinico-pathological features of cHL cases stratified by clonal hematopoiesis distribution in cHL tissueN=40 patientsClonal Hematopoiesis in cHL tissueP-valueExtensive (n=3)Absent/non-extensive (n=37)AGE&amp;gt;60 years1 (33%)9 (24%)1&amp;lt;60 years2 (67%)28 (76%)EBV STATUSEBV+1 (33%)8 (22%)0.55EBV-2 (67%)29 (78%)HISTOTYPENodular sclerosis1 (33%)21 (57%)0.58Mixed cellularity2 (67%)12 (32%)Other0 (0%)4 (11%)CLINICAL STAGEEarly ≤IIA1 (33%)13 (37%)1Advanced ≥IIB2 (67%)22 (63%)OUTCOME OF FIRST-LINE CHEMOTHERAPYNo progression0 (0%)24 (69%)0.043Progression3 (100%)11 (31%)Follow-up in months0-6-3564 (median)0.0340-149 (range) Citation Format: Andrea Marra, Alessandra Venanzi, Gianluca Schiavoni, Sara Giulia Milner, Roberto Limongello, Alessia Santi, Valentina Pettirossi, Simona Ultimo, Luisa Tasselli, Alessandra Pucciarini, Sofia Sciabolacci, Maria Paola Martelli, Paolo Sportoletti, Stefano Ascani, Brunangelo Falini, Enrico Tiacci. Tracking clonal hematopoiesis in patients with classical Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB044.

Fascin Inhibition As a Novel Therapeeutic Strategy in Diffuse Large B-Cell Lymphoma

Fascin is an actin bundling protein used to form cellular projections (e.g. filipodia) involved in cell motility and invasion. Cancer cells often upregulate fascin, and in many cancer types this is associated with poor prognosis, likely due to increased invasive and metastatic potential. We have developed fascin inhibitors that prevent actin bundling and block breast cancer metastasis in mice. Here we explored the potential role of fascin inhibition in lymphoma. We have shown that fascin is upregulated in 44% of canine B-cell (n=62) and 57% of T-cell lymphoma cases (n=28), and that increased fascin expression is correlated with worse overall survival (p=0.03). B-cell lymphoma cases had a median survival time of 388 days while that of T-cell cases was 263 days. We have further shown that a fascin inhibitor, NP-G2-044, kills human and canine diffuse large B-cell lymphoma (DLBCL) cells in vitro, and in vivo in mice bearing canine DLBCL xenografts, with GI50s ranging from 5 to 200uM. Animal toxicology studies (performed by Charles River Laboratories) show no hematologic or other toxicity in dogs and rats at concentrations up to 1,000 mg/kg/day for 28 days; these doses are much higher than the doses needed to achieve concentrations that kill lymphoma in vivo . Therefore, we are planning clinical studies in pet dogs with lymphoma to establish proof of principle prior to a human clinical trial. Collectively, our results demonstrate that fascin is a potential new therapeutic target in DLBCL. DisclosuresHuang:Novita: Equity Ownership, Patents & Royalties: Cornell University and Novita Pharmaceutical has patient on the fascin described here. .

A Single Gene-Mediated Perturbation of the Human Genome Leads to Malignant Transformation in a Murine Transgenic Model

We hypothesized that induction or activation of a gene that directly affects DNA integrity can provide a tool for ongoing genomic changes in a cell and may eventually lead to oncogenesis. In the human genome, apurinic/apyrimidinic (AP) sites are created by number of external and internal factors including exposure to chemicals, UV radiation, DNA damaging agents, reactive oxygen species (ROS), DNA glycosylases and/or spontaneous hydrolysis by other mechanisms. AP sites are pre-mutagenic lesions that can also affect normal DNA replication. An evolutionarily conserved mechanism “base excision repair (BER)” has been established to identify and repair such sites. As part of BER, AP nucleases cleave the phosphodiester backbone at 5' to the AP site, prior to its repair. Although APEX 1 and 2 are the nucleases required for this repair, their hyperactivity has been connected with increased DNA breaks. We have previously observed that over expression of either APEX 1 or APEX 2 leads to malignant transformation with number of genomic changes including copy number alterations, single nucleotide variants (SNVs) and structural changes.In this study, we investigated if transgenic overexpression of APEX2 in B cells in mice will cause tumorogenesis. We specifically overexpressed APEX2 in B cells by generating Tg,CD19Cre+/-,APEX2+/- mouse lines and used Tg,CD19Cre+/- mouse (siblings of trail group) as control. The expression of APEX2 was detected by immunohistochemical (IHC) staining, which confirmed specific expression of APEX2 in lymph nodes. We next evaluated impact of APEX2 expression on B cell genome. We isolated B220+ B cells from spleen of 8 weeks old mice, and identified both increased single strand break (SSB) and double strand breaks (DSB). After 45 weeks of age and at variable times thereafter, 40% (23/58) of Tg,CD19Cre+/-,APEX2+/- mice showed enlargement of the lymph nodes and spleen. Histology showed effacement of normal tissue architecture by a nodular or diffuse population of atypical lymphoid cells in interfollicular areas. A distinct pattern of B220+BCL6+CD5- B-cell lymphomas were identified after IHC staining, which resemble human diffuse large B-cell lymphoma (DLBCL). To investigate the changes at genomic level, we performed whole genome sequencing of tumor tissues from 3 mice as well as heart tissues (with lowest APEX2 expression) from the same mice to be used as controls. From single nucleotide varients (SNVs) calling, we observed average of 24,388 SNVs in the tumor with acquisition of new copy number changes and significant number of structural variants including average of 43 translocations, 51 deletions, 21 duplications and 24 inversion. Indel frequency was variable with average of 348. We observed mutational hotspots on chr6, chr12 and chr16. Variant allele frequency (VAF) data shows clonality of chr11 related mutations. We observed most frequent T>G, and then T>C mutations in all 3 samples. Interestingly, the mutational signature analysis showed signature 17 accounting for an average of 60% of mutations followed by signature 9. According to the mutational signature profile of different diseases, both signature 9 and 17 are observed in human B cell lymphoma.To understand the genesis of the malignant transformation, we scarified Tg,CD19Cre+/-,APEX2+/- mice and control Tg,CD19Cre+/- mouse at 25 weeks after birth to see the pre-malignant changes in lymphoid organs. We observed enlarged and increased follicle numbers in lymph nodes and in spleen in Tg,CD19Cre+/-,APEX2+/- mice compared to control. A single cell sequencing is underway to detect clonal changes at an early stage.Our results demonstrate a single DNA-repair related gene responsible for inducing genomic instability with malignant transformation providing a model to study oncogenesis as well as possible preventative measures. DisclosuresAnderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Modeling Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia in Transgenic Mice Using Human MYD88L256P Mutated Protein

Lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) is a B-cell disorder resulting from the accumulation of cloncal lymphoplasmacytic cells in the bone marrow (BM) along with excessive secretion of immunoglobulin M paraprotein. Despite recent advances in treatment LPL/WM remains incurable. Generating a refined LPL/WM mouse model that would facilitate studying this disease as well as enable stringent in vivo validation of potential therapeutic approaches has been a long-sought goal for the filed. Interestingly, among the most common mutations identified by recent high-throughput studies there was a single gain of function point mutation in the MYD88 gene, with a predicted non-synonymous amino acid (L265P) change, found in over 91% of patients. Therefore, we hypothesized that generation of transgenic mice overexpressing MYD88L256Pcould recapitulate the human setting of LPL/WM.To overcome problems inherently related to embryonic lethality, we generated MYD88WT and MYD88L256P conditional transgenic mice expressing human proteins. To remove a transcriptional stop cassette and activate MYD88 expression in vivo, we mated the conditional mice with mice expressing Cre recombinase under the control of Activation-Induced Cytidine Deaminase (AID) gene promoter to generate AIDCre/-;MYD88WT/- and AIDCre/-;MYD88L256P/- double-mutant mice. Recombinase activity driven by the AID gene promoter caused removal of the stop cassette and expression of MYD88 in germinal center (GC) and post-GC B cells, the appropriate B-cell compartment from which LPL/WM clones are thought to originate in humans. Young (up to 30 weeks of age) AIDCre/-;MYD88WT/- and AIDCre/-;MYD88L256P/- mice where indistinguishable from AIDCre/- mice as assessed by external physical examination. Moreover, obvious histologic and IHC differences in spleens, lymph nodes, and bone marrows between the mutant genotypes and the AIDCre/- control mice were not found. No clonal bands for the IgH gene, nor M-spikes were detected in Southern blots and serum protein electrophoresis, respectively. However, after 30 weeks of age, and at variable times thereafter, some of the AIDCre/-; MYD88L256P/- aging cohort (n=27) mice developed skin rash and excoriation as well as loss of hair in the submandibular areas. A fraction of AIDCre/-; MYD88L256P/- animals that weren't sacrificed because of the severe dermatitis had enlarged lymph nodes and appeared sick. To assess whether MYD88 transgenic mice develop B-cell lymphomas or major pathologic alterations consistent with the diagnosis of LPL/WM, animals were euthanized at different ages and lymphoid organs were rigorously examined. In spleens and lymph nodes of 8 of the AIDCre/-; MYD88L256P/- animals, a nodular lymphoid infiltrate composed predominantly of small atypical cells varyingly infiltrated with CD138+ plasma cells was observed. Lymphoid cells infiltrated other organs including the BM. Enhanced expression of MYD88 and activation of NF-κB activity within atypical lymphoid nodules in diseased transgenic mice was documented by IHC staining for MYD88 and p65. However, no clonal bands of the IgH gene were detected in Southern blots, suggesting that overexpression of MYD88L256P/- mutant protein in activated B-cells gave rise to a polyclonal lymphoproliferative disorder. Consistently, serum protein electrophoresis analysis showed diffuse, polyclonal increase in γ-globulins. Moreover, stains of peripheral blood smears revealed rouleaux formation suggesting increased concentrations of plasma proteins. In addition, one of AIDCre/-; MYD88L256P/- animals developed a more aggressive monoclonal disease resembling human diffuse large B-cell lymphoma. However, large atypical lymphoid cells in this mouse lacked expression of MYD88 and nuclear p65. All AIDCre/- controls and AIDCre/-; MYD88WT/- mice lacked described phenotypes and were histologically unremarkable.Taken together, these results demonstrate that although MYD88L256P/- alone might not be sufficient to induce a malignant transformation in the mature B-cell compartment, it gave rise to a low-grade mature non-clonal B-cell lymphoproliferative disorder with some of the characteristic futures of LPL/WM. Therefore, the MYD88L256P/- mutation might be necessary but not sufficient for LPL/WM development and must be present with concomitant “second hit” genetic alterations for a neoplasm to develop. DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding.

M7583 Is a Highly Selective and Potent Second Generation BTK Inhibitor for Treatment of B-Cell Malignancies

▪The advent of ibrutinib has represented a new era in the treatment of B-cell malignancies, with remarkable clinical efficacy shown in previously difficult to treat lymphomas. Now, second generation Bruton's tyrosine kinase (BTK) inhibitors are in development with more refined pharmacologic profiles that maintain the potent anti-tumor effects of ibrutinib, but that improve upon overall tolerability and circumvent ibrutinib's inhibitory effect on antibody-dependent cell-mediated cytotoxicity (ADCC).M7583 is a potent, orally administered, highly selective, adenosine triphosphate (ATP)-competitive, second generation, irreversible inhibitor of BTK. Kinase specificity has been determined in vitro using the Merck Millipore Kinase Profiler panel. Only 3 kinases (Blk, BMX, and Txk) had IC50s within a 10-fold range of BTK (BTK IC50 = 18.5 nM; Blk IC50 = 77 nM; BMX IC50= 5 nM; Txk IC50 = 62 nM). Target inhibition was confirmed with a concentration dependent decrease in BTK auto-phosphorylation detected by western blot in the Ramos cell line (human Burkitt's B-cell lymphoma). To examine the impact of kinase selectivity on the overall efficacy of M7583, its preclinical activity was compared with that of ibrutinib in a series of in vitro studies. M7583 inhibited the growth of diffuse large B-cell lymphoma (DLBCL) cell lines and primary chronic lymphocytic leukemia (CLL) blasts. In vivo , treatment with M7583 inhibited tumor growth in mantle cell lymphoma cell line-derived xenografts and patient-derived xenograft (PDX) models of DLBCL with the activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) subtype being the most sensitive to M7583; consistent with the findings for other BTK inhibitors. To examine the potential for inhibition of ADCC by therapeutic antibodies, M7583 and ibrutinib were combined with rituximab or an ADCC incompetent version of rituximab in ADCC cell lysis experiments. At clinically relevant concentrations, ibrutinib inhibited the ADCC mechanism of rituximab, whereas M7583 did not. Collectively, these findings show the potency and selectivity of M7583 for BTK, as well as its efficacy in models of B-cell malignancies. The safety and efficacy of M7583 is currently being investigated in Phase I/II trials in patients with B-cell malignancies. DisclosuresGoodstal:EMD Serono: Employment. Ma:EMD Serono: Employment. Lin:EMD Serono: Employment. Crandall:EMD Serono: Employment. Crowley:EMD Serono: Employment. Bender:EMD Serono: Employment. Iadevaia:EMD Serono: Employment. Clark:EMD Serono: Employment.

Combined Targeting of the Proteasome and Bcl-2 Sensitizes Human B-Cell Lymphoma and Multiple Myeloma to Cyclophosphamide in In Vitro and In Vivo Models of These Diseases.

The balance of apoptotic proteins is a critical mechanism used by the cell to maintain the mitochondrial transmembrane potential, which plays a critical role in governing apoptosis. Bortezomib (B) is a proteasome inhibitor that selectively inhibits the 26S proteasome, leading to an increase in many intracellular proteins, including proteins that lead to inhibition of NF-kB, as well as proapoptotic proteins like bik and Bax. Oblimersen (Obl) is a 18 mer oligonucleotide results in decreased transcription of the antiapoptotic protein bcl-2. We hypothesized that the mitochondrial membrane potential could be influenced by the co-administration of these agents to favor aopotosis. We report results of pre-clinical in vitro and in vivo experiments using these agents in combination in various lymphoma (NHL) & myeloma (MM) models. NHL & MM cell lines were exposed to Obl in the presence of cationic lipid and incubated for 48 hours. DNA synthesis & cell growth arrest was quantified by 3H-thymidine incorporation assays at 72 hrs, & revealed a concentration dependent cell killing. Induction of apoptosis in the presence or absence of different concentrations of B was detected by multiparameter flow cytometric analysis. Data reveals addition of bortezomib, even at relatively low concentrations (10 nM), additively improved the effects of Obl in all the cell lines studied. A SCID beige model of aggressive large B-cell lymphoma was used by injecting 1x10e7 cells (SKI-DLCL-1 or RL, a cell line over expressing bcl-2 through the 14:18 translocation) as a subcutaneous flank tumor. In over 6 in vivo experiments, the combination of B (0.5 mg/kg days 3 & 7) + Obl (3 mg/kg days 1,3,5, &7) consistently resulted in about 50% inhibition of growth of both SKI-DLCL-1 & RL compared to that in the control. Surprisingly, results were evident even using a low dose schedule of bortezomib (about one quarter of the MTD), suggesting the possibility of a small sensitizing dose of this drug. In all experiments, growth delay seen with the doublet exceeded the results noted with any singlet (B or Obl alone). Integration of cyclophosphamide at a dose of 50 mg/kg produced marked tumor shrinkage that appeared synergistic in several circumstances. Single agent CTX produced excellent growth delay (similar to any doublet), but in all these cases, tumor re-growth was evident by 21–25 days post tumor transplant. CTX dose & schedule was also important. Same day ‘lump and dump' of all drugs failed to effect cured, while CTX preceding B consistently produced best results, with cures found only in this triplet cohort. Results suggest that optimal scheduling of agents requires a ‘lead in' exposure of Obl, followed by CTX preceding all doses of bortezomib. These studies support the efficacy of this regimen.

Tumor-targeted nanoparticles improve the therapeutic index of BCL2 and MCL1 dual inhibition

Cancer and normal cells use multiple antiapoptotic BCL2 proteins to prevent cell death. Therapeutic targeting of multiple BCL2 family proteins enhances tumor killing but is also associated with increased systemic toxicity. Here, we demonstrate that the dual targeting of MCL1 and BCL2 proteins using the small molecules S63845 and venetoclax induces durable remissions in mice that harbor human diffuse large B-cell lymphoma (DLBCL) tumors but is accompanied by hematologic toxicity and weight loss. To mitigate these toxicities, we encapsulated S63845 or venetoclax into nanoparticles that target P-selectin, which is enriched in tumor endothelial cells. In vivo and ex vivo imaging demonstrated preferential targeting of the nanoparticles to lymphoma tumors over vital organs. Mass spectrometry analyses after administration of nanoparticle drugs confirmed tumor enrichment of the drug while reducing plasma levels. Furthermore, nanoparticle encapsulation allowed 3.5- to 6.5-fold reduction in drug dose, induced sustained remissions, and minimized toxicity. Our results support the development of nanoparticles to deliver BH3 mimetic combinations in lymphoma and in general for toxic drugs in cancer therapy.