Marginal Zone Lymphoma Cell Research Articles

Abstract PO-028: A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma

Abstract Background. Small molecules targeting the B-cell receptor (BCR) signaling, including PI3K and BTK inhibitors, have represented a step forward for managing lymphoma patients. However, resistance often develops, negatively impacting the benefit of patients exposed to drugs. Thus, developing preclinical tools to identify modalities to overcome this phenomenon is important. The bone marrow is commonly involved in marginal zone lymphoma (MZL) patients, and it might represent a safe environment where lymphoma cells escape therapy. Bone marrow stromal cells (BMSCs) induce proliferation and drug resistance in several tumor entities, mainly through the release of cytokines. To study potential resistance mechanisms to BCR targeting agents, we aimed to develop a 3D co-culture system in which primary BMSCs were used to model the bone marrow tumor microenvironment while exposing an MZL cell line to the PI3K inhibitor copanlisib. Methods. The MZL cell line VL51 was cultured in 3D fibrin gel (10mg/mL), either alone or with primary BMSCs, in 96-well plates using the OT-2 liquid handler (Opentrons) for high-throughput seeding purposes. Copanlisib treatment started on day 3. The effects of the drug were assessed through morphological observations and quantitative measurements of images taken on days 3, 5, and 7 in the presence or absence of BMSCs. On day 7, supernatants were analyzed for 105 secreted factors using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems / Bio-Techne). Results. We observed differences in VL51 growth pattern in the different conditions. Most importantly, the co-culture with BMSCs significantly (p=0.025) decreased VL51 sensitivity to copanlisib. When not treated, VL51 cells formed significantly larger clusters (p<0.001) in the presence of BMSCs. However, upon copanlisib treatment, VL51 cells formed smaller and more distributed clusters comparable in size to those in the mono-culture. Subsequent analyses of the supernatants from the mono- or co-cultures following DMSO or copanlisib treatment identified 14 differentially secreted cytokines among the different conditions. Validation experiments were done using recombinant proteins in 2D cultures for 6 factors. Among these, recombinant IGFBP-3, serpin E1, and PTX-3 significantly decreased VL51 sensitivity to copanlisib in 2D cultures (p=0.05, 0.01, and 0.04, respectively). Validation experiments are ongoing with additional in vitro models and using BTK inhibitors. Conclusions. We created a high-throughput 3D co-culture MZL system and found that BMSCs reduce sensitivity to copanlisib in MZL models. Secretion of IGFBP-3, Serpin E1, and PTX-3 reduced response to copanlisib. This 3D-model will be used to assess resistance mechanisms to clinically relevant compounds and to evaluate alternative therapeutic approaches to improve the treatment of lymphoma patients. Citation Format: Alex Zadro, Alberto Arribas, Maria Vittoria Colombo, Eleonora Cannas, Filippo Spriano, Luciano Cascione, Chiara Arrigoni, Christian Candrian, Matteo Moretti, Francesco Bertoni. A high-throughput bone marrow 3D co-culture system to develop resistance to targeted agents in marginal zone lymphoma [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 PO-028.

Abstract 7140: A new series of flavonoid derivatives with anti-cancer activity includes SDN207, SND210, SND524, and SND562 as potent microtubule-targeting agents

Abstract Background: Plant-derived compounds, including flavonoids, can be potent anti-cancer agents, but low bioavailability, lack of specificity, and complexity in their synthesis can hamper their clinical development. Here, we present seven novel flavonoid derivatives, and test them for their anti-tumor activity in various models derived from hematological cancers and solid tumors. Methods: MTT assessed anti-proliferative activity after 72h of exposure to increasing concentrations of drugs. Tubulin Polymerization Assay was performed with 3-fold dilutions (15-0.02 μM), compared to paclitaxel (1-3 μM) or vinblastine (3 μM) as references. The OncolinesProfiler analysis was used to compare IC50 fingerprints determined on the Oncolines cell line panel to the IC50 fingerprints of 213 pre-profiled reference drugs. Results: SDN207, SND210, SND216, SND462, SND504, SND524, and SND562 were designed and synthesized using a flavonoid polyphenol scaffold selectively modified to markedly increase cytotoxicity towards cancer cells. All seven compounds showed dose-dependent anti-proliferative activity across 15 lymphoma cell lines derived from germinal center B-cell-like (GCB) diffuse large B cell lymphoma (DLBCL) (n=6), activated B cell-like (ABC) DLBCL (n=2), mantle cell lymphoma (n=4), and marginal zone lymphoma (MZL) (n=3). Activity in the nM range was seen with five compounds: SDN207 (median IC50 1.6 nM; 95%CI, 1.3-3.2), SND210 (1.4 nM; 95%CI, 0.9-3.3), SND216 (19.5 nM; 95%CI, 10-38 nM), SND562 (28.9 nM; 95%CI, 15.4-57.7), and SND524 (46.7 nM; 95%CI, 32-133.3). Both other compounds had a median IC50 of 2 μΜ (SND462, 95%CI, 1.1-4.5 μM; SND504, 95%CI, 0.5-3.9 μM). There were no differences based on lymphoma histology. The anti-tumor activity was maintained in MZL cell lines with acquired resistance to BTK and PI3K inhibitors (n=3). Six compounds were also assessed for their anti-tumor activity in models other than lymphomas (SDN207, n=31; SND210, n=20; SND462, n=4; SND504, n=4; SND524, n=9; SND562, n=35), confirming the higher activity for SDN207, SND210, SND562, and SND524. Regarding the mechanism of action, up to now, a tubulin polymerization assay was performed for three compounds. Microtubule depolymerization (SND207), and microtubule polymerization (SND524, SND562) were observed at concentrations as low as 20 nM. The OncolinesProfiler identified the tubulin-targeting agents vinblastine, vincristine, paclitaxel, and docetaxel among the top reference compounds most correlated with SND207 and SND210 (i.e., Pearson correlation > 0.5). Conclusions: Novel compounds developed using plant-derived skeletons have shown in vitro anti-tumor activity in lymphomas, leukemias, and solid tumors, including models of resistance to targeted agents. Microtubule targeting appeared as a possible mechanism of action for at least four of them. Citation Format: Alberto J. Arribas, Eleonora Cannas, Eugenio Gaudio, Paulina Biniecka, Dan Stoicescu, Francesco Bertoni. A new series of flavonoid derivatives with anti-cancer activity includes SDN207, SND210, SND524, and SND562 as potent microtubule-targeting agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7140.

Abstract 534: Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2

Abstract Background: Copanlisib is a PI3K inhibitor with selectivity against PI3Kδ and PI3Kα and is approved for treating patients with follicular lymphoma and marginal zone lymphoma (MZL). Preclinical studies have demonstrated strong synergism of copanlisib and BCL2 inhibitor venetoclax in different lymphoma models, and the combination is under clinical evaluation. Understanding the resistance mechanisms could help the design of improved therapies. Hence, we generated MZL cell lines resistant to the copanlisib/venetoclax combination. Methods: All cells were tested for cell identity and Mycoplasma status. Resistance was confirmed stable after 3-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out by confirming sensitivity to vincristine. Targeted agents already in the clinics were combined with copanlisib/venetoclax. Cells underwent transcriptome and methylation profiling, targeted DNA sequencing, and immunophenotypic and immunoblotting analyses. Results: We developed SSK41-derived models with resistance to copanlisib (n.=1) or venetoclax (n.=1) as single agents and to copanlisib/venetoclax (n.=2) by long IC50 or by upfront IC90 exposure. The single-drug-resistant cells showed only partial resistance to copanlisib/venetoclax. In contrast, the cells that developed exposing cells to copanlisib/venetoclax IC90 exhibited a more substantial decrease in sensitivity to the combination. Copanlisib/venetoclax induced apoptosis and cell cycle arrest in PAR but not in resistant cells. Sensitivity to other PI3K, BCL2, and BTK inhibitors was decreased in all resistant lines. The response to MCL1-i, mTOR-i, and epigenetic agents (decitabine, HDAC, BET, and EZH2 inhibitors) was maintained. Triple combinations of copanlisib/venetoclax with the AURKA inhibitor alisertib, PLK inhibitor rigosertib, or the multikinase inhibitor amcasertib were active in both parental and resistant cells. No acquired mutations were observed via targeted DNA sequencing designed to cover various coding genomic regions known to be recurrently mutated in mature B-cell neoplasms, including BCL2, nor via RNA-Seq for PIK3CA/B/G/D genes. Integration of RNA and methylation profiles showed deregulation of genes involved in type-I IFN, chemokines (CCL25, CXCL13), apoptosis regulation, and JAK-STAT signaling. Resistant cells upregulated CD19 expression compared to parental cells. Immunoblotting showed the inability of copanlisib/venetoclax to block BCR signaling in resistant cells. Conclusions: We created four novel MZL-derived models of secondary resistance to PI3K and BCL2 inhibitors, which will help clarify possible modalities to overcome resistance. Novel potential targets were already identified, such as AURKA or PLK, to be further exploited. Citation Format: Alberto J. Arribas, Luciano Cascione, Eleonora Cannas, Helen Bellerjeau, Federica Fuzio, Aleix Noguera, Andrea Rinaldi, Manel Esteller, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Anastasios Stathis, Francesco Bertoni. Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 534.

Abstract C144: MEN1703/SEL24, a potent PIM inhibitor, demonstrates promising anti-tumour activity in activated B cell like diffuse large B cell lymphoma, mantle cell lymphoma and marginal zone lymphoma cells

Abstract Background. PIM kinases (PIM1, PIM2, PIM3) are serine threonine kinases that function in multiple biological processes including cell cycle regulation, cell survival and proliferation. They also mediate resistance to anti-tumour agents comprising PI3K inhibitors. PIM1 overexpression and mutations are common in diffuse large B cell lymphoma (DLBCL) and PIM1 overexpression has been validated as a rational therapeutic target particularly in ABC and MCD subtypes. PIM1 overexpression is also found in mantle cell lymphoma (MCL). We investigated the pharmacological inhibition of PIM using the small molecule MEN1703/SEL24 in a large panel of lymphoma cell lines. Methods. Anti-proliferative activity was assessed by MTT assay at 72h: MEN1703/SEL24 was tested in 7 ABC-DLBCL, 19 germinal centre-B cell-like (GCB)-DLBCL, nine MCL, six marginal zone lymphoma (MZL), one primary mediastinal, four MZL ibrutinib-and PI3K-resistant models and one canine lymphoma. Cell cycle and apoptosis assays were done by flow cytometry. RNA-Seq was performed on two ABC- and two GCB-DLBCL cell lines treated with MEN1703/SEL24 or DMSO for 4, 8, 12h, followed by limma and functional annotation analysis with gene set enrichment analysis (GSEA). Results. MEN1703/SEL24 showed anti-proliferative activity in all lymphoma histotypes tested (IC50 median = 593 nM; range = 30 - 10,193 nM). MCL (IC50 median = 108 nM; range = 53 - 412 nM) and ABC-DLBCL (IC50median = 994 nM; range = 30 - 1756 nM) were the most sensitive histotypes; among them OCI-Ly-10 (30 nM) and OCI-Ly-3 (142 nM) had mutated PIM1. GCB-DLBCLs comprised the least sensitive cell lines (IC50 median = 961 nM; range = 252 - 10,193 nM) accounting for 8/11 cell lines with IC50 in the upper percentile. More importantly, MEN1703/SEL24 was active in MZL cell lines with acquired resistance to idelalisib, copanlisib and ibrutinib (IC50 median = 367 nM; range = 273 - 410 nM). MEN1703/SEL24 induced apoptosis in 6/6 cell lines and minor G1 arrest in 1/6 cell lines. In DLBCL cell lines more sensitive to MEN1703/SEL24 treatment, RNA-Seq and GSEA revealed differential modulation of immune, MYC and E2F targets. Conclusions. MEN1703/SEL24 shows robust preclinical activity in B cell lymphomas of different histotypes. RNA-Seq indicates that MEN1703/SEL24 modulates the transcriptome of highly responsive DLBCL cell lines differently from poorly responsive cells, providing novel clues to mechanisms involved in sensitivity to PIM inhibitors and supporting a potential for clinical development in this indication. Citation Format: Afua A. Mensah, Alessandro Ghiringhelli, Giulio Sartori, Filippo Spriano, Chiara Tarantelli, Luciano Cascione, Andrea Rinaldi, Alberto A. Arribas, Daniela Bellarosa, Monica Binaschi, Francesco Bertoni. MEN1703/SEL24, a potent PIM inhibitor, demonstrates promising anti-tumour activity in activated B cell like diffuse large B cell lymphoma, mantle cell lymphoma and marginal zone lymphoma cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C144.

ERBB4-Mediated Signaling Is a Mediator of Resistance to PI3K and BTK Inhibitors in B-cell Lymphoid Neoplasms.

BTK and PI3K inhibitors are among the drugs approved for the treatment of patients with lymphoid neoplasms. Although active, their ability to lead to long-lasting complete remission is rather limited, especially in the lymphoma setting. This indicates that tumor cells often develop resistance to the drugs. We started from a marginal zone lymphoma cell line, Karpas-1718, kept under prolonged exposure to the PI3Kδ inhibitor idelalisib until acquisition of resistance, or with no drug. Cells underwent transcriptome, miRNA and methylation profiling, whole-exome sequencing, and pharmacologic screening, which led to the identification of the overexpression of ERBB4 and its ligands HBEGF and NRG2 in the resistant cells. Cellular and genetic experiments demonstrated the involvement of this axis in blocking the antitumor activity of various BTK/PI3K inhibitors, currently used in the clinical setting. Addition of recombinant HBEGF induced resistance to BTK/PI3K inhibitors in parental cells and in additional lymphoma models. Combination with the ERBB inhibitor lapatinib was beneficial in resistant cells and in other lymphoma models already expressing the identified resistance factors. An epigenetic reprogramming sustained the expression of the resistance-related factors, and pretreatment with demethylating agents or EZH2 inhibitors overcame the resistance. Resistance factors were also shown to be expressed in clinical specimens. In conclusion, we showed that the overexpression of ERBB4 and its ligands represents a novel mechanism of resistance for lymphoma cells to bypass the antitumor activity of BTK and PI3K inhibitors and that targeted pharmacologic interventions can restore sensitivity to the small molecules.

Aggressive Lymphoplasmacytic Neoplasm With an Unusual In-frame Deletion of MYD88 Associated With TRAF3 and TP53 Mutations and Complex Karyotype.

Lymphoplasmacytic lymphoma often needs to be differentiated from other B-cell lymphomas with plasmacytic differentiation, especially marginal zone cell lymphoma. Molecular detection of MYD88 p.L265P hotspot mutation supports the diagnosis of lymphoplasmacytic lymphoma since it is seen in about 90% of such lymphoma, which is much higher than other B-cell lymphomas. MYD88 p.L265P is a gain-of-function mutation that enhances the activity of the NF-κB signaling pathway and therefore drives lymphomagenesis. Other mutations in MYD88 are rarely reported. This study aims to report an unusual MYD88 in-frame deletion in an aggressive lymphoplasmacytic neoplasm. This is an IgM-positive, CD5- and CD10-negative mature B-cell lymphoma with prominent plasmacytic differentiation and aggressive features. The clinical and pathologic findings were most consistent with lymphoplasmacytic lymphoma. Next-generation sequencing identified an unusual MYD88 in-frame deletion in the absence of the hotpot p.L265P mutation. Other concurrent pathogenic mutations also include truncating mutations of TRAF3, which is a negative regulator of the NF-κB signaling pathway, and a missense mutation of TP53. Karyotype analysis showed complex karyotypes, including chromosome 6q deletion. By searching literature and online cancer databases, we identified only 8 other mature B-cell lymphomas with MYD88 in-frame deletions, but none of them was diagnosed with lymphoplasmacytic lymphoma. Recognizing such in-frame deletions is necessary to help understand the mutational spectrum of MYD88 in B-cell lymphomas. It remains to be further investigated whether such MYD88 in-frame deletions are also overrepresented in lymphoplasmacytic lymphoma among other B-cell lymphomas.

Single Cell RNA-Seq of Extranodal Marginal Zone Lymphoma (MZL) Identifies Site-Specific and Shared Features in Both B and T Cell Components

Background. Extranodal MZL of MALT type (MALT lymphomas) represents 5-8% of all human B-cell lymphomas can occur at any anatomical site. We and others have previously highlighted the genetic, biological and clinical peculiarities of this tumor, differences due in part to the diverse primary sites of disease. Almost by definition, the cellular composition of MALT lymphomas can be very heterogenous, encompassing small B cells resembling MZ cells, monocytoid cells, and small lymphocytes, in addition to scattered immunoblasts, centroblast-like cells and plasma cells. This issue and the possible presence of anatomical site-specific nonmalignant cell biopsies make single cell (sc) RNA-Seq ideal for studying MALT lymphomas. scRNA-Seq has the potential to provide detailed information on the transcriptome of non-neoplastic cells and give insight on the heterogeneity of the lymphoma cell population. Methods. Cells were analyzed with the 10× Chromium Chip and the Single Cell 3′ Reagent Kit v3. Libraries were run on an Illumina Nextseq 500. A minimum of 50'000 reads per cell were sequenced. FASTQ files were mapped to human reference genome (HG38) using the cellranger pipeline. Data went through quality control and downstream analysis in R (v3.6.1) using Seurat (v3.0). Cell clusters were generated with the FindNeighbors/FindClusters functions and cells annotated with a mixed approach considering known biomarkers and unsupervised computational methods (celldex, SingleR). Non-linear dimensional reduction (UMAP) was used to visualize clustering results and Monocle 2.0 for pseudo-time analysis. Results. ScRNA-seq was performed on pulmonary (n=2), gastric and parotid (n=1, each one) MZLs. Publicly available data for a second parotid case were included in the analyses. A total of ~19K cells passed QC with a median of 1,368 detected genes per cell. Each specimen contained mostly B cells (median 76%; range 49-85%), followed by T cells (median 21%; 12-47%), plasma cells (median 2%; 1.6-2.7%), epithelial and myeloid cells (median <=1%). Each sample contained both neoplastic B cells (median percentage 49%; range 8-79%), defined as those bearing a monoclonality for κ (n.=4) or λ (n.=1), and normal B cells (median 26%; 6-41%). The latter mainly comprised naïve and memory B cells. When we merged the five cases together, neoplastic B cells had highly patient-specific profiles, whereas nonmalignant B cells and plasma cells shared similar gene expression profiles across different cases. CD4+T cells also showed patient specific-signatures, while CD8+ cells co-clustered, indicating a more homogenous phenotype across patients. Transcripts expressed across MZL cells were enriched for IRF4 and BCL6 targets, genes higher in splenic MZL vs normal spleen, genes expressed in memory B cells, plasma cells, ABC-DLBCL with high STAT3, and in OxPhos DLBCL. Examples of genes expressed in MZL cells vs normal B cells were PAX5 and CXCR4 (pulmonary MZLs), TNFRSF1B and KRAS in parotid MZL, PIK3CD in gastric MZL. SPIBand SOX5, among other genes, showed high expression levels in malignant cells from all individuals compared to the other cells. MZL cells showed high inter- and intra-tumoral heterogeneous expression of potential therapeutic targets (for example, MS4A1/CD20, CD19, BCL2, MCL1). Pseudo-temporal analysis identified a main trajectory that ordered naïve B cells and healthy memory B cells to plasma cells, while the differentiation toward malignant B cells was inferred as a distinct branch. Analysis of immune-related ligand-receptor (L-R) pair interactions showed that most of the B cell subclusters provided costimulatory signals to CD4+ or CD8+ T cells via CD22-PTPRC interactions. Another suggestive costimulatory interaction occurred through CD70-CD27, involving malignant B cells, CD4+, CD8+ cells and also Tregs. Regarding coinhibitory pairs, the CD86-CTLA4 axis was frequently identified between CD4+/Tregs subclusters and malignant B cells but not between non-malignant B and T cells. Conclusions. scRNA-Seq highlighted peculiar inter- and intra-tumoral behaviors of B and T cells in MALT lymphomas. The results indicate that although each anatomical site can have a different molecular portrait, common gene expression programs are found in malignant cell populations of MALT as well as in cells of the tumor microenvironment. Acknowledgments: supported by Barletta foundation and EASI-Genomics.

PI3K and BTK Inhibition Induces the Upregulation of CD19 and Increases Sensitivity to CAR T Cells in a Model of Marginal Zone Lymphoma (MZL)

Background. Marginal zone lymphomas (MZLs) are characterized by an indolent course with median survival of over 10 years. Unfortunately, relapses/progressions are common and might heavily impact patients' survival. Chimeric antigen receptor (CAR) T cells revolutionized the treatment of several relapsing/refractory B cell malignancies. However, CAR T cells success in MZL is still limited (Jacobson et al, Lancet Oncol 2022) and none of the currently available products has been approved so far for this indication. Little is known about the reasons behind the limited efficacy of CAR T cells against MZL. Given the importance of CD19-antigen density in CAR T cell killing of B-cell malignancies, in this work we investigated the contribution of CD19 levels in sensitivity of MZL in vitro models to CAR T cells. We previously presented MZL-derived models with secondary resistance to BTK/PI3K inhibitors, also observing increased CD19 levels in some of the resistant cells (Arribas et al, ASH2019, ENA2019, Haematologica 2022). Based on these data, we hypothesized that the CD19 modulation induced by exposure to BTK/PI3K inhibitors might be a strategy to increase CAR T cell efficacy in MZL. Here, we tested this hypothesis using MZL models, parental or chronically exposed to BTK/PI3K inhibitors and with acquired resistance to the small molecules. Methods. CD19 expression was determined at protein level by flow cytometry. CD19-specific CAR T with CD28 co-stimulatory domain were produced from healthy donors using retroviral vectors. Transduction efficacy was evaluated by protein L staining. MZL cells were co-cultured with the CAR T cells at an E:T ratio of 1:1 after adjusting for transduction efficacy. Viability was measured by flow cytometry at 48h. CAR T cell-mediated cytotoxicity was calculated as percentage of cell death compared to MZL cells cultured alone. Longitudinal cell growth was measured by bioluminescence in mCherry/Luciferase transduced cells after normalization to day 0. Results. As we previously reported, CD19 expression levels are very heterogenous both across MZL clinical specimens (n.=41; Bonfiglio et al, Blood 2022; Figure 1A) and cell lines (n.=6; Hicks et al, Haematologica 2019; Figure 1B). We selected models expressing the highest (Karpas-1718) and the lowest (VL51) CD19 levels and tested their sensitivity to CAR T cell killing. CD19-specific CAR T cells displayed a robust cytotoxic activity against Karpas-1718 (median 74%, range 61-88%) while no CAR T cell-specific killing was observed against VL51 (Figure 1C). The results were in agreement with the CD19 levels, higher in Karpas-1718 and lower in VL51. PI3K or BTK inhibition led to CD19 upregulation on VL51 surface (Figure 1D). Compared to the non-treated (NT), co-incubation of parental VL51 with CAR T cells or control untransduced T (UT) cells did not inhibit tumor cell growth (Figure 1E, left panel). Conversely, CAR T cells, but not control UT cells, led to 43% growth inhibition at day 4 after co-incubation with idelalisib-treated VL51 (Figure 1E, middle panel) and completely inhibited ibrutinib-treated VL51 growth (Figure 1E, right panel). Conclusions. CD19 modulation by PI3K/BTK inhibition increased the sensitivity to CAR T cells in a CD19low MZL model. These data support exploring alternative modalities of CAR T cell treatment for MZL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Resistance to PI3Kδ inhibitors in marginal zone lymphoma can be reverted by targeting the IL-6/PDGFRA axis.

PI3Kδ inhibitors are active in patients with lymphoid neoplasms and a first series of them have been approved for the treatment of multiple types of B-cell lymphoid tumors, including marginal zone lymphoma (MZL). The identification of the mechanisms underlying either primary or secondary resistance is fundamental to optimize the use of novel drugs. Here we present a model of secondary resistance to PI3Kδ inhibitors obtained by prolonged exposure of a splenic MZL cell line to idelalisib. The VL51 cell line was kept under continuous exposure to idelalisib. The study included detailed characterization of the model, pharmacological screens, silencing experiments, and validation experiments on multiple cell lines and on clinical specimens. VL51 developed resistance to idelalisib, copanlisib, duvelisib, and umbralisib. An integrative analysis of transcriptome and methylation data highlighted an enrichment of upregulated transcripts and low-methylated promoters in resistant cells, including IL-6/STAT3- and PDGFRA-related genes and surface CD19 expression, alongside the repression of the let-7 family of miRNA, and miR-125, miR-130, miR-193 and miR-20. The IL-6R blocking antibody tocilizumab, the STAT3 inhibitor stattic, the LIN28 inhibitor LIN1632, the PDGFR inhibitor masitinib and the anti-CD19 antibody drug conjugate loncastuximab tesirine were active compounds in the resistant cells as single agents and/or in combination with PI3Kδ inhibition. Findings were validated on additional in vitro lymphoma models and on clinical specimens. A novel model of resistance obtained from splenic MZL allowed the identification of therapeutic approaches able to improve the antitumor activity of PI3Kδ inhibitors in B-cell lymphoid tumors.

Immunohistochemical algorithms and gene expression profiling in primary cutaneous B-cell lymphoma

Objectiveto assess whether immunohistochemical (IHC) algorithms used to classify the cell of origin (COO) of nodal Diffuse Large B-cell lymphoma (nDLBCL) in Germinal Center type (GCB) and non-GCB subtypes may be applied to Primary Cutaneous B-cell lymphoma (PCBCL) too, and which of these algorithms performs better on PCBCL. DesignRetrospective case control study. SettingPathology Department of the University Hospital “San Giovanni di Dio e Ruggi d′Aragona” Salerno, Italy. ParticipantsFourteen PCBCL, including Primary Cutaneous follicle centre lymphoma (PCFCL) and primary cutaneous diffuse large B-cell lymphoma, Leg type (PCDLBCL-LT) and 14 nDLBCL were evaluated for 7-year period (January 2011 to December 2017). Primary cutaneous marginal zone cell lymphoma (PCMZL) cases were not included in the present study. InterventionEvaluation of immunohistochemical CD10, BCL6, MUM1/IRF4, BCL2, MYC and Ki-67 expression and classification according to three different algorithms. Gene expression profiling (GEP) was performed on the same series using Lymph2Cx assay (Nanostring). The data obtained were compared and analysed. ResultsAll the IHC algorithms showed 13 GCB and 15 non-GCB. GEP showed 12 GCB, 12 activated B cell–type and 4 unclassified. ConclusionsThe PCBCL were classifiable as GCB and non-GCB like the nDLBCL as IHC algorithms were concordant to GEP and produced the same results.

Abstract P073: Pharmacological inhibition of IRAK4 with CA-4948 is beneficial in marginal zone lymphoma models with secondary resistance to PI3K and BTK inhibitors

Abstract Background. Marginal zone lymphoma (MZL) is a heterogeneous B-cell malignancy for which no standard treatment exists. B-cell receptor (BCR) signaling dysregulation is a hallmark of MZL and can mimic antigen dependent BCR activation. However, single treatment with BCR signaling inhibitors shows limited complete response rate and relapsing patients. IRAK4 is a protein kinase downstream the Toll-like receptor signaling. CA-4948 is the first-in-class inhibitor of IRAK4 and it is in phase 1/2 studies for patients with relapsed/refractory clinical studies with favorable activity in lymphomas and myeloid neoplasms. Here, we assessed IRAK4 inhibition with CA-4948 against a panel of MZL cell lines in which we have developed secondary resistance to inhibitors of PI3K (idelalisib, copanlisib) or BTK (ibrutinib). Methods. Models with secondary resistance were developed by continuous exposing cell lines derived from splenic MZL (Karpas 1718 and VL51) to high doses of drugs for long period of times. Resistant and parental lines were exposed to increasing doses of CA-4948, alone or in combination with compounds they are resistant to. Cell viability was tested by MTT assay (72h treatment). Synergy of combinations was evaluated according to the Chou-Talalay combination index (CI), as well as potency and efficacy, estimated according to the MuSyC algorithm. Results. The parental Karpas1718, bearing a MYD88 L265P mutation, had an IC50 of 3.29 µM for CA-4948 as single agent, while the parental VL51 and all resistant models presented IC50 values in the range of 19-35 µM. IRAK4 inhibition with CA-4948 had strong benefit when combined to downstream BCR inhibitors. In resistant cells, CA-4948 was strongly synergistic with idelalisib, ibrutinib and, at lesser extent, with copanlisib. CA-4948 in combination with ibrutinib was strongly synergistic especially in the VL51 ibrutinib resistant model compared to the parental one. CA-4948 (from 1 to 5 µM) recovered sensitivity to ibrutinib at IC50 values close to the parental condition. Similarly, CA-4948 in combination with idelalisib was also synergistic and increased sensitivity to idelalisib in idelalisib resistant VL51. Less benefit was seen adding CA-4948 to copanlisib: the combination was synergistic in VL51 parental cell, but with only weak efficacy. In the Karpas1718, CA-4948 was synergistic either in idelalisib resistant or in parental, with improved benefit in the latter, in which synergistic effect was observed when combined with both idelalisib and ibrutinib. The synergistic activity of CA-4948 was mainly due to improved efficacy rather than potency of the combinatorial partners. Conclusions. Our results in MZL cell lines show that the IRAK4 inhibitor CA-4948 is synergistic with small molecules targeting BCR signaling, especially with idelalisib and ibrutinib. Data also suggest that concentrations of CA-4948 comparable to those achieved in patients might overcome or reduce secondary resistance to the tested tyrosine kinase inhibitors. Citation Format: Francesca Guidetti, Alberto J. Arribas, Filippo Spriano, Laura Barnabei, Reinhard von Roemeling, Elizabeth Martinez, Emanuele Zucca, Francesco Bertoni. Pharmacological inhibition of IRAK4 with CA-4948 is beneficial in marginal zone lymphoma models with secondary resistance to PI3K and BTK inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P073.

842P Secreted factors determine resistance to idelalisib in splenic marginal zone lymphoma (MZL) models

PI3Kδ has a central role in the BCR signaling in hematological malignancies. Idelalisib was the first-in-class PI3Kδ inhibitor, and several novel compounds are undergoing clinical investigation. To identify modalities to overcome the resistance developed to these agents, we have developed idelalisib-resistant models derived from MZL cell lines.

Improved Primary Staging of Marginal-Zone Lymphoma by Addition of CXCR4-Directed PET/CT.

PET/CT with 18F-FDG is an integral component in the primary staging of most lymphomas. However, its utility is limited in marginal-zone lymphoma (MZL) because of inconsistent 18F-FDG avidity. One diagnostic alternative could be the targeting of C-X-C motif chemokine receptor 4 (CXCR4), shown to be expressed by MZL cells. This study investigated the value of adding CXCR4-directed 68Ga-pentixafor PET/CT to conventional staging. Methods: Twenty-two newly diagnosed MZL patients were staged conventionally and with CXCR4 PET/CT. Lesions identified exclusively by CXCR4 PET/CT were biopsied as the standard of reference and compared with imaging results. The impact of CXCR4-directed imaging on staging results and treatment protocol was assessed. Results: CXCR4 PET/CT correctly identified all patients with viable MZL and was superior to conventional staging (P < 0.001). CXCR4-directed imaging results were validated by confirmation of MZL in 16 of 18 PET-guided biopsy samples. Inclusion of CXCR4 PET/CT in primary staging significantly impacted staging results in almost half of patients and treatment protocols in a third (upstaging, n = 7; downstaging, n = 3; treatment change, n = 8; P < 0.03). Conclusion: CXCR4 PET/CT is a suitable tool in primary staging of MZL and holds the potential to improve existing diagnostic algorithms.

Expression of germinal center cell markers by extranodal marginal zone lymphomas of MALT type within colonized follicles, a diagnostic pitfall with follicular lymphoma

We reviewed 341 consecutive cases of marginal zone lymphoma (MZL) (47) or follicular lymphoma (FL) (294) of which 7 were difficult to distinguish due to perceived coexpression of BCL6 and BCL2 by tumor cells in follicular foci. This stimulated us to develop dual BCL6/BCL2 immunohistochemistry, allowing us to assess coexpression among individual cells. Dual staining confirmed coexpression in 6 of 7 cases, all extranodal MZL (ENMZL) based on overall features and representing 13% of MZL in this series. These findings confirm that MZL cells have plasticity regarding protein expression within the germinal center (GC) microenvironment, an important diagnostic pitfall. Intriguingly, in all MZL expressing BCL6, non-neoplastic GC B cells within colonized follicles showed diminished or absent CD10 expression but preserved BCL6 and high ki67. This finding suggests plasticity of CD10 expression in non-neoplastic GC B cells in the context of colonization by MZL, possibly related to NF-kB dysregulation.

Abstract PO-46: Mechanisms of resistance to the PI3K inhibitor copanlisib in marginal zone lymphoma

Abstract Background: PI3Kδ is expressed in B cells and has a central role in the B-cell receptor signaling. Copanlisib is a highly selective PI3Kδ and PI3Kα inhibitor, and it is currently under clinical development in indolent lymphomas including marginal zone lymphoma (MZL). Copanlisib is Food and Drug Administration (FDA) approved for the treatment of patients with relapsed or refractory follicular lymphoma. Nevertheless, a subset of patients can eventually relapse due to acquired resistance. A better understanding of resistance mechanisms could help to design improved therapies; hence, we generated MZL cell lines resistant to copanlisib. Materials and Methods: Cells were kept on copanlisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 3 weeks of drug-free culture. Multidrug resistance phenotype was ruled out by confirming sensitivity to vincristine. Cells underwent transcriptome profiling (RNA-Seq) and immunophenotypic analysis. Results: The RES models were obtained from VL51 cell line with over 50-fold times higher IC50s than PAR counterparts. Of note, the copanlisib-resistant lines showed decreased sensitivity to other PI3K inhibitors such as duvelisib (50-fold) and idelalisib (5-fold) and to the BTK inhibitor ibrutinib (15-fold), suggesting that the mechanism observed here might drive resistance to other downstream B-cell receptor inhibitors. Gene expression profiles of RES showed the upregulation of cytokine signaling (IL1A, IL1B, CXCR4), NFkB (LTA, TNF), MAPK (RASGRP4, RASGRP2), and JAK-STAT (STAT3, JAK3) signaling pathways and negative regulators of apoptosis (CD44, JUN). Conversely, repressed genes in RES were involved in cell adhesion (ITGA4, ITGB1), antigen presentation (HLAs), and IFN response (PARP12, GBP6). Consistent with the overexpression of antiapoptotic signaling genes, RES cells exhibited also resistance to the BCL2-inhibitor venetoclax, either as a single as in combination with copanlisib. Flow cytometry confirmed the CXCR4 upregulation and the downregulation of CD49d (ITGA4), paired with reduced CD20 and CD81 surface expression. In accordance, addition of a CXCR4 inhibitor overcame resistance to copanlisib. Conclusions: We created a model of secondary resistance to the PI3K inhibitor copanlisib, derived from an MZL cell line. This model will help in clarifying mechanisms of resistance to the drug and to evaluate alternative therapeutic approaches. Indeed, we already identified novel potential targets, such as IL1 and CXCR4, that might be exploited in overcoming resistance to copanlisib and are worthy of further investigation. Citation Format: Alberto J. Arribas, Sara Napoli, Luciano Cascione, Eugenio Gaudio, Roberta Bordone-Pittau, Marilia Barreca, Giulio Sartori, Chiara Tarantelli, Filippo Spriano, Andrea Rinaldi, Anastasios Stathis, Georg Stussi, Davide Rossi, Emanuele Zucca, Francesco Bertoni. Mechanisms of resistance to the PI3K inhibitor copanlisib in marginal zone lymphoma [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 PO-46.

149 Poster - Secondary resistance to the PI3K inhibitor copanlisib in marginal zone lymphoma

Background: PI3K kinase has a prominent role in the B-cell receptor signaling. Copanlisib, a pan-PI3K inhibitor with predominant selectivity to PI3Kα and PI3Kδ, is Food and Drug Administration (FDA) approved for the treatment of patients with relapsed or refractory follicular lymphoma, and it is currently under clinical development in other indolent lymphomas including marginal zone lymphoma (MZL). However some patients might eventually relapse because of acquired resistance and so a better understanding of resistance mechanisms is needed. Thus we generated MZL cell lines resistant to copanlisib which could help to design improved therapies.

IBCL-338: The Incidence of Composite Lymphomas: One Center Experience (Russia)

Context: Composite lymphomas (CL) can occur simultaneously or sequentially and are of a particular interest in hematology. Objective: Approaches to the diagnosis and treatment of CL are complex with a full range of modern laboratory methods. Design: Analysis of cases of CL with histological and molecular features of these cases. Setting: Study setting can be used in hematological inpatient and outpatient departments. Patients: There were 37 cases of CL (M:F=2:1, median age 64 (38-83 years)). Interventions: All patients underwent a full range of diagnostic procedures, including histological, immunophenotypic, cytogenetic and molecular tests. Main outcomes measures: Study the combination types of CL. Results: Metachronous CL were observed in 17 cases; synchronous CL in 20 cases. Metachronous CL cases: the most common type of the second lymphoma was diffuse B-large cell lymphoma (DBLCL) with 58.8% and Hodgkin's lymphoma (HL) with 29.4%. In 30% of cases, DBLCL developed after HL therapy, and in 30% - after therapy of angioimmunoblastic lymphoma. The median time to development of the second lymphoproliferative disease (LD) was 6 years (range 7 mo - 40 yrs). Synchronous CL were observed in 55% of cases; the most common were the combination of chronic lymphocytic leukemia (CLL) with other LD, hairy cell leukemia (HCL) 54.5%, large granular lymphocytic leukemia, marginal zone lymphoma (MZL), follicular lymphoma, multiple myeloma, and fungal mycosis. Combination of HL with other LD was observed in 25% of cases (HL+MZL represent 60% of cases). It is not clear whether HL in this combination represents a clonal transformation from MZL cells or it is a secondary tumor that develops de novo after MZL treatment. Since common IGVH gene rearrangements and somatic mutation status could be detected for both types of cells; this may indicate that MZL and HL cell clones may originate from a single B–cell of the follicular germinal center. Conclusions: Careful examination is needed in atypical clinical course of lymphomas to exclude the second LD, including clonally related. CL are excellent models for studying the complex process of tumor cells evolution during the course of the disease. Treatment of CL is a non-trivial task and aims to control both diseases.

Copanlisib synergizes with conventional and targeted agents including venetoclax in B- and T-cell lymphoma models

AbstractCopanlisib is a pan–class I phosphoinositide 3-kinase (PI3K) inhibitor with preferred activity toward PI3Kα and PI3Kδ. Despite the clear overall clinical benefit, the number of patients achieving complete remissions with the single agent is relatively low, a problem shared by the vast majority of targeted agents. Here, we searched for novel copanlisib-based combinations. Copanlisib was tested as a single agent, in combination with an additional 17 drugs in 26 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and T-cell lymphomas. In vivo experiments, transcriptome analyses, and immunoblotting experiments were also performed. Copanlisib as a single agent showed in vitro dose-dependent antitumor activity in the vast majority of the models. Combination screening identified several compounds that synergized with copanlisib. The strongest combination was with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax. The benefit of the combination over single agents was also validated in an MZL xenograft model and in MCL primary cells, and was due to increased induction of apoptosis, an effect likely sustained by the reduction of the antiapoptotic proteins myeloid cell leukemia 1 (MCL1) and BCL-XL, observed in MCL and MZL cell lines, respectively. These data supported the rationale for the design of the Swiss Group for Clinical Cancer Research (SAKK) 66/18 phase 1 study currently exploring the combination of copanlisib and venetoclax in relapsed/refractory lymphomas.

Clinical Outcomes of Obinutuzumab Therapy across Non-Hodgkin Lymphoma Subtypes

Clinical Outcomes of Obinutuzumab Therapy across Non-Hodgkin Lymphoma Subtypes

COPANLISIB SYNERGIES WITH CONVENTIONAL AND TARGETED AGENTS INCLUDING VENETOCLAX IN PRECLINICAL MODELS OF B‐ AND T‐CELL LYMPHOMAS

Introduction: Copanlisib has shown activity in lymphomas, but the number of patients achieving complete remission is relatively low, a problem shared by many targeted agents. We performed a pharmacological screening to identify active combinations. Methods: We tested copanlisib as single agent and in combination with other molecules in cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and T cell lymphomas (T-NHLs). Synergism was evaluated with Chou-Talalay combination index, as previously done (Tarantelli et al, CCR 2018). Results: Copanlisib showed an in vitro dose-dependent antitumor activity in the vast majority of the models with a median IC50 of 75 nM (95% C.I., 21-160 nM). The drug was active both in B- (n = 17) and T-NHLs (n = 9), but the latter were less sensitive (285 vs 22 nM, P = 0.0002). The in vitro activity was confirmed using the human JeKo-1 MCL xenograft model. We evaluated 16 combination partners, including rituximab (B-NHL), crizotinib and brentuximab vedotin (T-NHLs), lenalidomide, bendamustine, agents targeting AKT1/2, BCL2, BET, BTK, CDKs (pan, CDK4/6, CDK9), HDAC, JAK1/2, MALT1, and the proteasome. In B-NHLs, copanlisib/venetoclax were the most beneficial (14 synergism, 2 additive), and the 4thbest in T-NHLs (5 synergism, 1 additive). The combination led to increased apoptosis, as shown by a dose-dependent increased PARP cleavage in 3 MZL, and 1 MCL cell lines and by a reduction of cell viability in 2 MCL primary cells. In MZL (SSK41) and MCL (Jeko1) 24h exposure to copanlisib determined a dose-dependent down-regulation of pAKT and anti-apoptotic proteins (BCL-XL in the MZL and of MCL1 in the MCL). These changes were maintained in cells exposed to the combination, but not after venetoclax alone. An in vivo experiment using the SSK41 MZL cell line validated the beneficial effect of this combination and also of copanlisib/lenalidomide (in vitro beneficial in 12/17 B- and 4/9 T-NHL). In T-NHLs, benefit was observed in all ALCL-ALK+ with copanlisib/crizotinib (3/4 synergisms). All but one of the non ALCL-ALK+ cell lines achieved a synergism with copanlisib/ruxolitinib. Copanlisib/brentuximab vedotin was synergistic in the non ALCL-ALK+ and Sezary Syndrome cell lines. Transcriptome analysis identified the PIM1 and PIM2 kinases and the anti-apoptotic gene MCL1 as up-regulated in the MZL cell line HAIR-M exposed to copanlisib (5nM, for 4, 8 and 12h). Thus, we tested combinations with the PIM inhibitor AZD1208 and the MCL1 inhibitor MIK665 (S63845) in 3 MCL and 3 MZL cell lines. Both combinations were synergistic in the HAIR-M and beneficial in additional 5/5 and 3/5 cell lines, respectively. Conclusions: Our study identified a series of active copanlisib-containing combinations and provided the rational for the design of the new SAKK 66/18 phase I study exploring copanlisib plus venetoclax in relapsed/refractory B-cell lymphomas. Keywords: marginal zone lymphoma (MZL); PI3K/AKT/mTOR; venetoclax. Disclosures: Lange, M: Employment Leadership Position: Bayer AG. Jourdan, T: Employment Leadership Position: Bayer AG. Berthold, M: Employment Leadership Position: Bayer AG. Rossi, D: Consultant Advisory Role: Gilead, AbbVie, Janssen, AstraZeneca; Honoraria: Gilead, AbbVie Janssen, Roche; Research Funding: Gilead, AbbVie, Janssen. Stathis, A: Research Funding: Bayer, Merck, Pfizer, Novartis, Roche; Other Remuneration: travel grant from AbbVie. Liu, N: Employment Leadership Position: Bayer AG. Zucca, E: Consultant Advisory Role: Roche, Mei Pharma, Astra Zeneca; Research Funding: Roche, Janssen; Other Remuneration: travel grants from Abbvie and Gilead; expert statements provided to Gilead. Politz, O: Employment Leadership Position: Bayer AG. Bertoni, F: Research Funding: Acerta, Bayer AG; Other Remuneration: travel grants from Astra Zeneca, PIQUR Therapeutics AG.