Cohesin Core Complex Gene Dosage Contributes to Germinal Center Derived Lymphoma Phenotypes and Outcomes

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.

Identification of PLA2G7 as a novel biomarker of diffuse large B cell lymphoma

Background Diffuse large B-cell lymphoma is the most common form of non-Hodgkin lymphoma globally, and patients with relapsed or refractory DLBCL typically experience poor long-term outcomes. Methods Differentially expressed genes associated with DLBCL were identified using two GEO datasets in an effort to detect novel diagnostic or prognostic biomarkers of this cancer type, after which receiver operating characteristic curve analyses were conducted. Genes associated with DLBCL patient prognosis were additionally identified via WCGNA analyses of the TCGA database. The expression of PLA2G7 in DLBCL patient clinical samples was further assessed, and the functional role of this gene in DLBCL was assessed through in vitro and bioinformatics analyses. Results DLBCL-related DEGs were found to be most closely associated with immune responses, cell proliferation, and angiogenesis. WCGNA analyses revealed that PLA2G7 exhibited prognostic value in DLBCL patients, and the upregulation of this gene in DLBCL patient samples was subsequently validated. PLA2G7 was also found to be closely linked to tumor microenvironmental composition such that DLBCL patients expressing higher levels of this gene exhibited high local monocyte and gamma delta T cell levels. In vitro experiments also revealed that knocking down PLA2G7 expression was sufficient to impair the migration and proliferation of DLBCL cells while promoting their apoptotic death. Furthmore, the specific inhibitor of PLA2G7, darapladib, could noticeably restrained the DLBCL cell viability and induced apoptosis. Conclusions PLA2G7 may represent an important diagnostic, prognostic, or therapeutic biomarker in patients with DLBCL.

Karonudib has potent anti-tumor effects in preclinical models of B-cell lymphoma

Chemo-immunotherapy has improved survival in B-cell lymphoma patients, but refractory/relapsed diseases still represent a major challenge, urging for development of new therapeutics. Karonudib (TH1579) was developed to inhibit MTH1, an enzyme preventing oxidized dNTP-incorporation in DNA. MTH1 is highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma, hence confirming a rationale for targeting MTH1. Here, we tested the efficacy of karonudib in vitro and in preclinical B-cell lymphoma models. Using a range of B-cell lymphoma cell lines, karonudib strongly reduced viability at concentrations well tolerated by activated normal B cells. In B-cell lymphoma cells, karonudib increased incorporation of 8-oxo-dGTP into DNA, and prominently induced prometaphase arrest and apoptosis due to failure in spindle assembly. MTH1 knockout cell lines were less sensitive to karonudib-induced apoptosis, but were displaying cell cycle arrest phenotype similar to the wild type cells, indicating a dual inhibitory role of the drug. Karonudib was highly potent as single agent in two different lymphoma xenograft models, including an ABC DLBCL patient derived xenograft, leading to prolonged survival and fully controlled tumor growth. Together, our preclinical findings provide a rationale for further clinical testing of karonudib in B-cell lymphoma.

Safety and efficacy of CAR T cells in a patient with lymphoma and a coexisting autoimmune neuropathy

Key Points CAR T-cell therapy was safe and effective in a DLBCL patient with coexisting autoimmune neuropathy. CD19 CAR T-cell therapy may control refractory autoantibodies and monoclonal gammopathies.

Preliminary Results of an Ongoing Phase 1 Dose Escalation Study of the Novel Anti-CD74 Antibody Drug Conjugate (ADC), STRO-001, in Patients with B-Cell Non-Hodgkin Lymphoma

Background: CD74 is highly expressed on B cell malignancies, including non-Hodgkin's lymphoma (NHL). STRO-001, a novel CD74-targeting ADC was generated using cell-free protein synthesis and site-specific conjugation platform technologies. STRO-001 contains a potent maytansinoid warhead conjugated to two specific sites (drug-antibody ratio of 2) using a stable non-cleavable linker. This first-in-human Phase 1, open-label, multicenter, dose escalation study was designed to evaluate the safety, tolerability, and preliminary anti-tumor activity of STRO-001 in adults with B-cell malignancies (NHL and multiple myeloma). Herein we report preliminary results from the B-cell NHL cohort. Methods: Patients with advanced, relapsed/refractory NHL are eligible for enrollment. STRO-001 is administered as a 60-minute IV infusion. STRO-001 was initially administered on Days 1 and 15 of a 28-day cycle. Starting at 0.91 mg/kg, STRO-001 was administered on Day 1 of a 3-week cycle. Treatment is administered until disease progression or unacceptable toxicity. The study employed a modified 3+3 design with an accelerated dose titration (N=1 per cohort until set specified AEs are observed) for initial dosing cohorts. Results: 18 patients with NHL have been treated at 9 dose levels: .05, .075, .15, .27, .43, .65, .91, 1.27 and 1.78 mg/kg. NHL subtypes include: 6 diffuse large B-cell lymphoma (DLBCL), 5 follicular lymphoma (FL), 2 mantle cell lymphoma (MCL), 2 marginal zone lymphoma, 1 Burkitt's lymphoma, 1 composite DLBCL/FL and 1 composite DLBCL/CLL. Median age is 64.5 (range 21-82). Median ECOG performance status is 1 (range 0-2). Median number of prior therapies is 4 (range 1-12). Three patients received prior CAR-T therapy. Median number of STRO-001 doses administered is 2 (range 1-12). 17 patients have completed at least one cycle of STRO-001 and are evaluable for safety and toxicity for dose escalation recommendation. One patient at the 1.78 mg/kg dose level is currently completing Cycle 1 and not yet evaluable for DLT assessment. Most AEs are grade 1 or 2 (90%) with the most common grade 1-2 TEAEs of chills, fatigue, nausea, anemia, headache, pyrexia, infusion reaction, decreased appetite, and abdominal pain occurring in ≥ 20% of patients. There was one DLT in the NHL cohort, a grade 3 thromboembolic event at the 0.91 mg/kg dose level. 16 patients are evaluable for response. The preliminary clinical benefit/disease control rate for all patients is 25% (4/16) including 1 patient with complete response (CR) 2 with partial response (PR) and 1 with stable disease (Table). One patient with DLBCL treated at .075 mg/kg achieved a CR after 2 cycles (4 doses) and progressed after 12 doses (on study 24 weeks). A DLBCL patient treated at 0.65 mg/kg achieved a PR at Cycle 3 and progressed after 8 doses (on study 15 weeks). A DLBCL patient treated at 1.27 mg/kg who achieved a PR has received 10 cycles and remains on study after 27 weeks. Preliminary PK analysis of ADC shows exposure increased (Cmax from 0.39 to 19 µg/mL) and (AUC0-tlast from 0.6 to 71 h*µg/mL) as dose increased from 0.05 to 0.91 mg/kg. Summary/Conclusion: STRO-001 is the first ADC generated with novel cell-free protein synthesis technology and site-specific conjugation to be tested in the clinic. STRO-001 has been well-tolerated. No ocular or neuropathy toxicity signals have been observed and the MTD has not been reached. Preliminary anti-tumor activity has been observed in this heavily pre-treated patient population, including two DLBCL patients who had previously progressed after a CAR-T (Table). The study continues to enroll patients in dose escalation. Next planned dose levels are 2.5 mg/kg and 3.5 mg/kg. This study is registered with clinicaltrials.gov identifier NCT03424603. Table Disclosures Shah: Verastim: Consultancy; Kite Pharma: Consultancy, Honoraria; Lily: Consultancy, Honoraria; Cell Vault: Research Funding; TG Therapeutics: Consultancy; Miltenyi Biotec: Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Incyte: Consultancy. Popplewell:Pfizer: Research Funding; Novartis: Research Funding; Roche: Research Funding. Andreadis:Gilead/Kite: Consultancy; Merck: Research Funding; Incyte: Consultancy; Karyopharm: Honoraria; Jazz Pharmaceuticals: Honoraria; Genentech: Consultancy, Current equity holder in publicly-traded company; BMS/Celgene/Juno: Honoraria, Research Funding; Novartis: Research Funding. Melear:AstraZeneca: Speakers Bureau; Janssen: Speakers Bureau. Spira:Cardiff Oncology: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Merck: Consultancy; BMS: Consultancy; Incyte: Consultancy; Janssen: Consultancy; ADCT: Research Funding. Manda:AbbVie: Other: Investigator in AbbVie-sponsored clinical trials. Burke:Roche: Consultancy; AbbVie: Consultancy; Bayer: Consultancy; Astra Zeneca: Consultancy; Verastem: Consultancy; Morphosys: Consultancy; Adaptive: Consultancy; Epizyme: Consultancy; Kura: Consultancy; Celgene: Consultancy; Adaptive Biotechnologies: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Consultancy; Seattle Genetics: Speakers Bureau. Sharman:TG Therapeutics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Acerta: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; BeiGene: Research Funding. Krishnan:Sanofi: Consultancy; Sutro: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Takeda: Speakers Bureau; BMS/Celgene: Consultancy, Other: Stock BMS, Speakers Bureau; Janssen: Consultancy; Regeneron: Consultancy; Z Predicta: Membership on an entity's Board of Directors or advisory committees. Shah:BMS, Janssen, Bluebird Bio, Sutro Biopharma, Teneobio, Poseida, Nektar: Research Funding; GSK, Amgen, Indapta Therapeutics, Sanofi, BMS, CareDx, Kite, Karyopharm: Consultancy. Kuriakose:Sutro Biopharma: Current Employment. Berman:Sutro Biopharma: Current Employment. Matheny:Sutro Biopharma: Current Employment. Leonard:Miltenyi: Consultancy; BMS/Celgene: Consultancy; Regeneron: Consultancy; Karyopharm: Consultancy; GenMab: Consultancy; Sutro: Consultancy; Roche/Genentech: Consultancy; Epizyme: Consultancy; Bayer: Consultancy; Gilead/Kite: Consultancy; ADC Therapeutics: Consultancy; MEI Pharma: Consultancy; AstraZeneca: Consultancy. Molina:Sutro Biopharma: Current Employment.

Immunocluster: A Computational Tool to Explore the Immune Profile and Cellular Heterogeneity of Hematological Diseases Using Liquid and Imaging Mass, and Flow Cytometry Data

Introduction Our understanding of 'immunome" and its role in the pathogenesis and clinical outcomes of hematological diseases has significantly improved in recent years, which highlights the importance of multidimensional cytometry techniques in investigating immune response both in clinical and research settings. Liquid mass cytometry (LMC), imaging mass cytometry (IMC) and flow cytometry (FC) are powerful techniques for immunophenotyping, biomarker discovery, and patients' immune-monitoring. These techniques provide the ability to profile over 40 markers per cell, resulting in large amounts of data which could be challenging to analyze and interpret. Therefore, we developed ImmunoCluster, an easy-to-use open-source computational pipeline, to explore high dimensionality single-cell cytometry datasets. Case studies Here we describe three examples of the implementation of the ImmunoCluster tool, which is a self-contained R package (accessed via GitHub: https://github.com/kordastilab/ImmunoCluster). Previously published LMC data from 15 leukemia patients 30 and 90 days after bone marrow transplantation (BMT) were used to test ImmunoCluster's ability to reproduce results. Post-BMT 3/15 patients suffered acute graft versus host disease (GvHD). For IMC data a lymph node section from a diffuse large B-cell lymphoma (DLBCL) patient. Finally, FC data from BM of 7 healthy donors (HDs) taken during hip surgery. The pipeline provides tools to allow researchers to follow a workflow which guides them through experimental design, data analyses and interpretation, to publishable graphics identifying differences in phenotype and abundance of cells between conditions (Figure 1A). ImmunoCluster comprises of three core computational stages: Stage 1. Data import and quality control In the experimental design stage, the high dimensional dataset is imported into ImmunoCluster with an associated metadata file, this included timepoints, and response to treatment. All data was stored within a SingleCellExperiment (SCE) object, a data container in which you can store/retrieve information such as metadata and UMAP/tSNE coordinates (Figure 1B). Initial exploratory visualization of the data, e.g. Multidimensional scaling (MDS) plots, and heatmaps showing marker expression for each patient were created and metadata used for annotation. Stage 2. Dimensionality reduction and unsupervised clustering We used three dimensionality reduction tools: MDS, uniform manifold approximation and projection (UMAP), and t-Distributed Stochastic Neighbor Embedding (tSNE). Two clustering algorithms: an ensemble clustering method of FlowSOM and Consensus clustering; and PhenoGraph. The aim of these algorithms were to assign all cells to clusters corresponding to true cell types. Stage 3. Annotation and differential testing Tools exploring cluster marker expression via projection onto UMAP/tSNE, or heatmaps, aided the identification of cell types and phenotypically distinct clusters. Metadata were used to annotate figures, allowing for visualization of the distribution of cell islands between different conditions/timepoints. Tools such as median marker expression, hierarchical clustered heatmaps, and box plots of cell cluster abundance were applied. Statistically significant differences between conditions were identified. Results We successfully replicated the findings from Hartmann et al., 24 cell populations were identified (Figure 2A-B). Significant differences between memory B-cells (FDR p=4.38 x 10-3), naïve B-cells (FDR p=1.35 x 10-2), and naïve CD4+ T-cells (FDR p=3.47 x 10-2) were identified between the GvHD and none (Figure 2C). From the FC HD CD4+ BM population data we were able to identify regulatory T cells (Tregs), including subpopulations, Treg A and Treg B (as low as 0.7% (0.1-2.0) and 0.9% (0.2-2.4), respectively). A marker expression ranking tool was applied to the DLBCL patient IMC data. We identified the majority of Ki-67 high population were proliferating tumor cells (84%), and the Ki-67 low population consisted of a heterogeneous collection of immune cell populations (Figure 3). These case studies show that ImmunoCluster could help clinicians and researchers with varying experience in computational biology to drive their projects from experimental design, wet lab/clinical trial, through to the data analysis process and visualization. Disclosures McLornan: JAZZ PHARMA: Honoraria, Speakers Bureau; NOVARTIS: Honoraria, Speakers Bureau; CELGENE: Honoraria, Speakers Bureau. Harrison:Gilead Sciences: Honoraria, Speakers Bureau; Incyte Corporation: Speakers Bureau; Janssen: Speakers Bureau; Sierra Oncology: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau; AOP Orphan Pharmaceuticals: Honoraria; Shire: Honoraria, Speakers Bureau; Promedior: Honoraria; Roche: Honoraria; Celgene: Honoraria, Research Funding, Speakers Bureau; CTI Biopharma Corp: Honoraria, Speakers Bureau. Kordasti:Celgene: Research Funding; Novartis: Research Funding; Alexion: Honoraria.

Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.

Ongoing, first-in-human, phase I dose escalation study of the investigational CD47-blocker TTI-622 in patients with advanced relapsed or refractory lymphoma.

3030 Background: CD47 is an immune checkpoint that binds signal regulatory protein alpha (SIRPα) and delivers a "do not eat" signal to suppress macrophage phagocytosis. Cancer cells frequently overexpress CD47 to escape immune surveillance. TTI-622 is a fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG4. TTI-622 acts as a decoy receptor, preventing CD47 from delivering its inhibitory signal and enabling macrophage activation and anti-cancer activity via pro-phagocytic signals present on cancer cells. Unlike many CD47-blocking antibodies, TTI-622 does not bind to human erythrocytes and thus may not cause anemia in patients. Methods: In phase 1A, patients with advanced relapsed or refractory lymphoma received IV TTI-622 once per week with dose increased based on traditional 3+3 escalation. Dosing was on a mg/kg basis with the third and subsequent weekly doses approximately 2-fold higher than the first 2 doses (e.g., 0.05, 0.05, and 0.1 mg/kg for weeks 1, 2 and 3). Blood samples were obtained for PK analysis and assessment of CD47 receptor occupancy (RO) on peripheral T cells. Results: At data cut-off, 19 patients (11 M, 8 F) of median age 62 years (range, 24-86) with the following lymphomas: DLBCL 10; HL 6; and TCL, MCL and FL, 1 each, with a median of 3 prior therapies (range, 1-8) were enrolled. No DLTs have been observed in 5 dose levels (0.05 to 4.0 mg/kg). Grade ≥3 related neutropenia occurred in 2 patients; other related AEs occurring in 2 patients each included abdominal pain, fatigue, and nausea; no patients experienced a related SAE. Acute, post-dose platelet decreases occurred transiently and generally were Grade 1- 2; no related Grade ≥3 thrombocytopenia or anemia AEs have been observed. Preliminary PK data indicate a dose-proportional increase in exposure and a T1/2 of approximately 4-5 days following repeat infusions (Week 6). Preliminary biomarker data reveal approximately 60% RO at the end of the first infusion of 2 mg/kg and more sustained 24-hour RO at 1 and 2 mg/kg vs ≤ 0.8 mg/kg. To date, 1 patient with stage 4 non-GCB DLBCL (5 prior therapies) initially achieved PR by Wk 8 and CR by Wk 36, with response ongoing. Conclusions: TTI-622 is well tolerated at doses up to 4 mg/kg per week. Preliminary data indicate dose-dependent increases in PK exposure and target engagement with 1 DLBCL patient having achieved a durable, ongoing CR. Dose escalation is ongoing and additional safety, PK, biomarker and response data will be available at the time of meeting presentation. Clinical trial information: NCT03530683 .

Venetoclax response is enhanced by selective inhibitor of nuclear export compounds in hematologic malignancies

The selective inhibitor of nuclear export (SINE) compounds selinexor (KPT-330) and eltanexor (KPT-8602) are from a novel class of small molecules that target exportin-1 (XPO1 [CRM1]), an essential nucleo-cytoplasmic transport protein responsible for the nuclear export of major tumor suppressor proteins and growth regulators such as p53, p21, and p27. XPO1 also affects the translation of messenger RNAs for critical oncogenes, including MYC, BCL2, MCL1, and BCL6, by blocking the export of the translation initiation factor eIF4E. Early trials with venetoclax (ABT-199), a potent, selective inhibitor of BCL2, have revealed responses across a variety of hematologic malignancies. However, many tumors are not responsive to venetoclax. We used models of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) to determine in vitro and in vivo responses to treatment with venetoclax and SINE compounds combined. Cotreatment with venetoclax and SINE compounds demonstrated loss of viability in multiple cell lines. Further in vitro analyses showed that this enhanced cell death was the result of an increase in apoptosis that led to a loss of clonogenicity in methylcellulose assays, coinciding with activation of p53 and loss of MCL1. Treatment with SINE compounds and venetoclax combined led to a reduction in tumor growth in both AML and DLBCL xenografts. Immunohistochemical analysis of tissue sections revealed that the reduction in tumor cells was partly the result of an induction of apoptosis. The enhanced effects of this combination were validated in primary AML and DLBCL patient cells. Our studies reveal synergy with SINE compounds and venetoclax in aggressive hematologic malignancies and provide a rationale for pursuing this approach in a clinical trial.