Hodgkin Lymphoma Cell Lines Research Articles

The Class I Histone Deacetylase Inhibitor as a Potent Therapeutic Target for Treating Glioblastoma and Mocetinostat as a Novel Inhibitor in the Induction Death of Cancer Cells

Epigenetic abnormality is one of the hallmarks of glioblastoma cancer cells. Histone deacetylase (HDAC) modification has a crucial role in epigenetic abnormality, which results in the initiation and progression of glioblastoma cancer cells. The selective HDAC inhibitors are well-known epigenetic regulators and promising anti-cancer agents that target specific HDAC enzymes and inhibit the proliferation of many cancer cells. Selective HDAC inhibitors isoform provides a high efficacy as chemotherapy in inhibiting cancer confirmation compared to non-selective HDAC inhibitors. Additionally, selective HDAC inhibitors suppress Class -I HDAC1, HDAC2, HDAC3, and HDAC11. HDAC class I inhibitors induce apoptosis, differentiation, autophagic death cells, and reactive oxygen species (ROS)- induced cell death, inhibit cell migration, invasion, and angiogenesis in cancer cells, while the normal cells showed more resistance to HDAC class I inhibitors. Mocetinostat (MGCD0103), a benzamide histone deacetylase, is a potent anti-cancer therapy for the treatment of several cancer cell lines and induction of autophagy. It has been approved by The Food and Drug Administration (FDA) for the treatment of Hodgkin lymphoma (HL) cell lines. MGCD0103 is a synthesized and selective HDAC inhibitor that has vigorous inhibitory activity against Class-I and IV HDAC. MGCD0103 is well tolerated and has favorable pharmacokinetic properties, pharmacodynamic profile, and fast absorption within 1 hour after oral administration, long elimination half-life, and sustained HDAC inhibition. Therefore, MGCD0103 is expected to be a promising anti-cancer drug for treating several types of human cancer cells. Keywords: MGCD0103; Apoptosis; Differentiation; Gliblastoma; HDAC; inhibitors.

Epigenetic Silencing of MTAP in Hodgkin's Lymphoma Renders It Sensitive to a 2 nd Generation PRMT5 Inhibitor

Background: PRMT5 is type II arginine methyltransferase that catalyses symmetric dimethylation of arginine residues (SDMA) and plays an important role in cancer biology. By methylating a number of substrates, PRMT5 can regulate important processes such as DNA repair, RNA splicing, and cellular proliferation. In addition, PRMT5 is overexpressed in various cancer types and has been identified as a candidate for therapeutic intervention through the development of small molecules that inhibit PRMT5 methyltransferase activity. However, first generation PRMT5 inhibitors have shown limited clinical benefit mainly due to development of on-target toxicity, primarily in the bone marrow. AstraZeneca has developed a second generation PRMT5 inhibitor (AZ-PRMT5i), that selectively inhibits PRMT5 in MTAP deficient tumours while sparing MTAP proficient normal cells. MTAP (methylthioadenosine phosphorylase) is a metabolic enzyme involved in methionine salvage pathway. MTAP deficiency results in accumulation of the metabolite methylothioadenosine (MTA) in tumor cells that induces partial inhibition of PRMT5, rendering these tumors sensitive to PRMT5 inhibition. Homozygous deletion of the MTAP gene, that results in the loss of MTAP protein, has been found in approximately 15% of advanced solid tumors. Here, for the first time, we describe epigenetic silencing of the MTAP gene in Hodgkin's Lymphoma (HL) cell lines. This silencing results in the loss of MTAP protein expression thus increasing sensitivity to PRMT5 inhibition. Importantly, MTAP protein loss was also observed in primary cHL samples, opening a novel opportunity for the treatment of HL. Methods: Bioinformatic data mining of the Cancer Cell Line Encyclopedia (CCLE) dataset has been used to overlay levels of expression of MTAP mRNA with MTAP copy number and MTAP promoter DNA methylation. MTAP protein expression and activity of AZ-PRMT5i were assessed in 5 HL cell lines in vitro; efficacy of AZ-PRMT5i and levels of target engagement were tested in vivo in the L540 xenograft model. MTAP expression levels were determined by IHC analysis using 55 primary samples from cHL patients. Results: Using an unbiased analysis of the CCLE dataset, we have identified a subset of cell lines that in the absence of MTAP genetic loss were showing low levels of MTAP mRNA expression, equivalent to levels of MTAP mRNA in MTAP homozygous deleted cell lines. Interestingly, this was predominantly the case in HL cell lines where it was detected in 67% of cases (4 out of 6 cell lines). MTAP methylation profile analysis of HL cell lines revealed elevated MTAP DNA promoter methylation in cell lines with low MTAP mRNA expression. In addition, MTAP protein was not detected by Western Blot. Accordingly, lack of MTAP protein expression resulted in increased sensitivity of these cells to AZ-PRMT5i compared to a HL cell line that expressed MTAP protein. Utilizing RNA-seq for transcriptional profiling of AZ-PRMT5i on a MTAP-deficient HL cell line has identified robust changes in genes that regulate the cell cycle, DNA replication and TP53 pathways thus confirming on target activity. In addition, AZ-PRMT5i demonstrated strong dose dependent efficacy in a MTAP silenced L540 xenograft model where greater than 90% of tumour growth inhibition was detected, with no significant body weight loss. Corresponding dose-dependent inhibition of SDMA is observed in the treated tumours. Finally, IHC analysis of MTAP expression in 55 primary cHL samples has demonstrated that 46 of 55 cHL samples (84%) had absent nuclear MTAP expression. Conclusion: Here we are presenting a novel finding showing MTAP protein expression loss in the majority of tested primary cHL samples, which could provide a collateral vulnerability and novel therapeutic opportunity to 2 nd generation PRMT5 inhibitors as demonstrated in our pre-clinical models.

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Truly Refractory Hodgkin Lymphoma Patients Is Highly Effective and Responses Are Mediated By NK Cells

Background In the relapsed/refractory Hodgkin Lymphoma (HL) setting (RHL), the achievement of complete remission (CR) is fundamental in order to proceed with allogeneic stem cell transplantation (HSCT) consolidation. Immune checkpoint inhibitors (CI) have demonstrated clinical activity in patients relapsing after ASCT, although only 20% CR rate is observed. In order to improve the efficacy of CI in RHL, we previously reported a salvage treatment based on early CI therapy following post-autologous stem cell transplantation (ASCT) with the reinfusion of unselected autologous lymphocytes infusions (ALI). The rationale of this approach is based on the achievement of a disease debulking through ASCT conditioning. Early administration of CI (nivolumab) is performed on a minimal disease burden, while reinfusions of ALI reduce post ASCT immune depression eliciting CI efficacy. Aims The aim of this study was to evaluate the efficacy of the procedure in terms of CR rates and overall survival (OS) after an extended follow up. Biological endpoint was to investigate the lymphocyte subpopulations involved in the mechanism of response and investigate the role of NK-cells mediating anti tumor responses in HL. Methods Patient were eligible to this study after failure to respond to first line treatment (i.e. positive PET2 or PET6 scan) and underwent autologous lymphocyte apheresis upon enrollment. All patients received conventional 2nd line chemo followed by 3rd line chemo-immunotherapy with Brentuximab-Vedotin (BV) in non-responding patients. Patient failing to achieve CR after BV were enrolled in the treatment arm and proceeded to ASCT + CI and ALI, whereas patients responding to 2nd- or 3rd treatment received ASCT followed by ALI alone, as a control cohort (figure 1A). NK cell degranulation assay was evaluated by staining for CD107a (LAMP-1) expression on healthy-donors derived NK cells exposed to HL cell lines (L428 and L250), in the presence or absence of monoclonal antibodies targeting inhibitory or activating receptors. Surface and intracellular staining was performed prior to flow cytometry and analysis was performed on the CD3-/CD56+ gated population. Results Twenty-one patients with RHL (median age 32 years; range 18-65) underwent lymphocyte apheresis after failure of 1st line chemo and then proceeded to salvage therapy. Thirteen patients failed to respond to 2nd and 3rd line therapy and thus received early post-transplant CI supported with four ALI. Eight patients responded to 2nd line chemo (n=6) or 3rd line BV (n=2) and received ASCT followed by ALI alone. No adverse events were recorded, specifically, no immune-related toxicity was observed. All patients receiving ALI + CI (treated patients) achieved negative PET scan CR. Eight of them received HSCT consolidation. One patient refusing HSCT after achieving CR in this study relapsed and responded to CI re treatment followed by HSCT. All patients in the treatment arm are alive and disease-free after a median follow-up of 32 months (95% CI 26.4-51.0). Median disease-free survival (DFS) was not reached in treatment arm (Fig 1B). Four of the patients in the control arm relapsed (50%). Three of them responded to 3rd line therapy and proceeded to HSCT. One patient achieved CR with conventional CI treatment but refused HSCT and the other relapsing patient died because of progressive lymphoma after failure to achieve CR with conventional single agent CI. Median DFS in the control arm was 22 months (Fig 1B). NK cells showed higher expansion and a faster maturation in the treatment arm, compared to the control arm (p<0.05). As expected, HL cells lines do not express HLA class I and II, but express activating NK cells receptors (aNKG2D, aNKp30, aNKp46, aDNAM-1). In vitro, the activation of each of those receptors was able to trigger NK cells degranulation. Similarly, PD-L1 and PD-L2 blockade on HL cell lines significantly increased NK cell degranulation (p<0.05). Taken together, those observations support the role of NK cells in this setting. Conclusions Our data show very high anti-tumor activity of ALI + CI for RHL, allowing most patients to proceed to HSCT. RHL patients in the treatment arm had an excellent outcome if compared to the control arm, which included patient who did respond to conventional treatment. Biological data suggests that this approach may accelerate NK cell development/maturation and suggest that NK cells may mediate response to CI in HL.

SGN-35C: A Novel CD30-Directed Antibody-Drug Conjugate for the Treatment of Lymphomas

SGN-35C is an antibody drug conjugate composed of an anti-CD30 antibody conjugated to a camptothecin-derived topoisomerase 1 (TOP1) inhibitor payload. SGN-35C was designed to leverage the antibody backbone from brentuximab vedotin (BV) and the novel mechanism of action of camptothecin-derived ADCs. Here, we evaluated the mechanism of action, cytotoxicity, and safety in in vitro and in vivo models. To characterize the mechanism of action of SGN-35C in vitro and in vivo experiments were performed in CD30-positive anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma (HL) cell lines, and versions of these cell lines that are resistant to BV. SGN-35C binding, internalization, induction of DNA damage, cell cycle analysis, and in vitro cytotoxicity were evaluated. To assess if payload released in CD30-positive cells could kill adjacent CD30-negative cells, an in vitro bystander assay of CD30-positive and CD30-negative co-cultures was developed. The in vivo antitumor activity of SGN-35C was evaluated in animals bearing subcutaneous lymphoma models. SGN-35C antitumor activity was evaluated in a xenograft model of heterogeneous CD30 expression consisting of a mixture of a BV-resistant Karpas-299 cell line mixed with parental Karpas-299 cells to assess bystander activity in vivo. The tolerability of SGN-35C was assessed in a non-human primate model. SGN-35C binding to CD30-positive cells was similar to the unconjugated anti-CD30 antibody and demonstrated internalization into CD30-expressing cells. Treatment of cells with SGN-35C for 24 hours led to an increase of the DNA damage marker phospho-γH2A.X, which induced an increase in the fraction of cells in S-phase, consistent with the mechanism of camptothecin-based TOP1 inhibitors. Cytotoxicity was observed in ALCL and HL cell lines treated with SGN-35C, including an ALCL cell line resistant to BV. In a mixture of CD30-positive and CD30-negative cells, SGN-35C treatment induced cytotoxicity of the CD30-negative cells, demonstrating that SGN-35C can elicit bystander activity. A single dose of SGN-35C led to significant tumor growth inhibition in animals bearing lymphoma xenografts, including a BV-resistant model and a model of heterogeneous CD30 expression, demonstrating that SGN-35C can elicit bystander activity in vivo. SGN-35C demonstrates a tolerability profile in non-human primates that is consistent with camptothecins. A Phase 1, first-in-human study is planned to evaluate the safety and anti-tumor activity of SGN-35C in lymphoid malignancies.

Classic Hodgkin Lymphomas Display Neuronal-Glial Lineage Transdifferentiation

The molecular drivers of classic Hodgkin lymphoma (HL) underlying its pathologic and clinical distinctions from non-Hodgkin lymphoma (NHL) have been challenging to uncover. The study of HL has been impeded by the rarity of the Hodgkin-Reed-Sternberg (HRS) cells within a dense tumor microenvironment (TME). Previously, we developed a FACS approach to isolate the HRS cells and profiled their whole exomes and genomes (Reichel et al 2015; Maura et al 2023). These studies did not reveal an HRS-specific mutational signature, but rather mutations that overlap with those of NHL, such as those in SOCS1 and HIST1H1E. These data suggested that non-genetic factors such as the underlying cell state may cooperate with somatic mutations to induce HL. However, we previously lacked the ability to co-map somatic mutations and cell states within the same cancer cells. We thus developed Genotyping of Transcriptomes (GoT; Nam et al 2019) that links single-cell RNA-seq (scRNA-seq) and somatic mutations within the same thousands of individual cells. We applied GoT to primary HL samples (n = 10 HL; 68291 cells, Fig. 1A) after FACS-isolation of HRS cells. We analytically integrated the cells across the samples based on gene expression and identified the expected cell types including B-cells, T-cells, monocytes and HRS cells (Fig. 1B). As expected, the HRS cells displayed low expression of B-cell antigens ( CD19, CD79A) and high levels of TNFRSF8 (CD30), CCL17, CCR7 and PD-L1 (Fig. 1C). GoT data revealed that mutations were restricted to the HRS cells (Fig. 1D). Copy number variation (CNV) analysis further confirmed that CNVs were specific to the HRS cells (Fig. 1E). As scRNA-seq has unveiled marked intratumoral cell state heterogeneity across cancers, we posited that the HRS cells may display heterogeneous cell states. Indeed, we identified distinct cell states (Fig. 1F), including cycling HRS and metabolically-active HRS cells upregulating the translation machinery ( NACA, RACK1). We also observed a cell state with elevated inflammatory signals ( TNF, JUN, FOS, CCL22). One group demonstrated a partially preserved B cell program with high CD19 and CD79A. Unexpectedly, we also identified an HRS state with features of neuronal-glial (NG) transdifferentiation, including genes involved in synapse formation ( NRG2, NRXN3). Consistently, genes upregulated in the NG state were enriched in nervous system and neuronal development pathways (Fig. 1G). We further assessed NG gene expressions across normal cell types and confirmed their specificity to NG lineage cells (Fig. 1H). These findings were consistent with dendritic processes of HRS cells highlighted by CD15 and CD30 (Fig. 1I, red arrows). Areas of CD15/CD30 staining devoid of nucleus (Fig. 1I, blue arrows) corroborated the presence of dendritic processes. These results suggested that the NG differentiation may be a key defining feature of HRS cells, whereby dendritic processes facilitate a tight control of the TME. Consistently, we identified strong HRS-immune complexes that persisted through tissue processing and sorting (Fig. 1F). To determine the degree of plasticity or heritability of the HRS cell states, we integrated VDJ sequencing with GoT (GoT-VDJ), whereby somatic hypermutations in the VDJ served as endogenous barcodes. Phylogenetic reconstructions revealed that HRS cell states were distributed across clades (Fig. 1J), unveiling a plastic interchange of HRS cell states. Consistently, GoT showed that mutant cells were present across the cell states (Fig. 1K), indicating that the cell states were decoupled from the genetic identities. We therefore postulated that the NG program may be encoded epigenetically. We performed single nuclei ATAC-seq on HRS-enriched HL samples (n = 5 samples; Fig. 1L). Compared to admixed B cells, the HRS cells displayed enhanced accessibility for binding motifs of the NG lineage transcription factors (TF), e.g. OLIG1/2 and SOX family (Fig. 1M), revealing the TF networks that govern the lineage infidelity program. We further identified that the NG program is cell intrinsic and at least partially stable through in vitro passaging, as 4 of 5 HL cell lines exhibited the highest NG gene expression compared to NHL cell lines (Fig. 1N). Altogether, these single-cell multi-omics studies indicated that an epigenetically encoded NG program may cooperate with lymphoma driving mutations to drive the development of HL, paving novel avenues of therapy.

Proliferation-promoting roles of linear and circular PVT1 are independent of their ability to bind miRNAs in B-cell lymphoma

Plasmacytoma Variant Translocation 1 (PVT1) is a long non-coding RNA located at 8q24.21 immediately downstream of MYC. Both the linear and circular PVT1 transcripts contribute to cancer pathogenesis by binding microRNAs. However, little is known about their roles in B-cell lymphoma. Here we studied their expression patterns, role in growth, and ability to bind miRNAs in B-cell lymphoma. Linear PVT1 transcripts were downregulated in B-cell cell lymphoma lines compared to germinal center B cells, while circPVT1 levels were increased. Two Hodgkin lymphoma cell lines had a homozygous deletion including the 5′ region of the PVT1 locus, resulting in a complete lack of circPVT1 and 5′ linear PVT1 transcripts. Inhibition of both linear and circular PVT1 decreased growth of Burkitt lymphoma, while the effects on Hodgkin lymphoma and diffuse large B cell lymphoma were less pronounced. Overexpression of circPVT1 promoted growth of B-cell lymphoma lacking or having low endogenous circPVT1 levels. Contrary to other types of cancer, linear and circular PVT1 transcripts did not interact with miRNAs in B-cell lymphoma. Overall, we showed an opposite expression pattern of linear and circular PVT1 in B-cell lymphoma. Their effect on growth was independent of their ability to bind miRNAs.

Establishment of the lymphoid ETS-code reveals deregulated ETS genes in Hodgkin lymphoma.

The human family of ETS transcription factors numbers 28 genes which control multiple aspects of development, notably the differentiation of blood and immune cells. Otherwise, aberrant expression of ETS genes is reportedly involved in forming leukemia and lymphoma. Here, we comprehensively mapped ETS gene activities in early hematopoiesis, lymphopoiesis and all mature types of lymphocytes using public datasets. We have termed the generated gene expression pattern lymphoid ETS-code. This code enabled identification of deregulated ETS genes in patients with lymphoid malignancies, revealing 12 aberrantly expressed members in Hodgkin lymphoma (HL). For one of these, ETS gene ETV3, expression in stem and progenitor cells in addition to that in developing and mature T-cells was mapped together with downregulation in B-cell differentiation. In contrast, subsets of HL patients aberrantly overexpressed ETV3, indicating oncogenic activity in this B-cell malignancy. Analysis of ETV3-overexpressing HL cell line SUP-HD1 demonstrated genomic duplication of the ETV3 locus at 1q23, GATA3 as mutual activator, and suppressed BMP-signalling as mutual downstream effect. Additional examination of the neighboring ETS genes ETS1 and FLI1 revealed physiological activities in B-cell development and aberrant downregulation in HL patient subsets. SUP-HD1 showed genomic loss on chromosome 11, del(11)(q22q25), targeting both ETS1 and FLI1, underlying their downregulation. Furthermore, in the same cell line we identified PBX1-mediated overexpression of RIOK2 which inhibited ETS1 and activated JAK2 expression. Collectively, we codified normal ETS gene activities in lymphopoiesis and identified oncogenic ETS members in HL.

Identification of two unannotated miRNAs in classic Hodgkin lymphoma cell lines.

MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1.

Hodgkin Lymphoma Cell Lines and Tissues Express mGluR5: A Potential Link to Ophelia Syndrome and Paraneoplastic Neurological Disease.

Ophelia syndrome is characterized by the coincidence of severe neuropsychiatric symptoms, classical Hodgkin lymphoma, and the presence of antibodies to the metabotropic glutamate 5 receptor (mGluR5). Little is known about the pathogenetic link between these symptoms and the role that anti-mGluR5-antibodies play. We investigated lymphoma tissue from patients with Ophelia syndrome and with isolated classical Hodgkin lymphoma by quantitative immunocytochemistry for mGluR5-expression. Further, we studied the L-1236, L-428, L-540, SUP-HD1, KM-H2, and HDLM-2 classical Hodgkin lymphoma cell lines by FACS and Western blot for mGluR5-expression, and by transcriptome analysis. mGluR5 surface expression differed significantly in terms of receptor density, distribution pattern, and percentage of positive cells. The highest expression levels were found in the L-1236 line. RNA-sequencing revealed more than 800 genes that were higher expressed in the L-1236 line in comparison to the other classical Hodgkin lymphoma cell lines. High mGluR5-expression was associated with upregulation of PI3K/AKT and MAPK pathways and of downstream targets (e.g., EGR1) known to be involved in classical Hodgkin lymphoma progression. Finally, mGluR5 expression was increased in the classical Hodgkin lymphoma-tissue of our Ophelia syndrome patient in contrast to five classical Hodgkin lymphoma-patients without autoimmune encephalitis. Given the association of encephalitis and classical Hodgkin lymphoma in Ophelia syndrome, it is possible that mGluR5-expression in classical Hodgkin lymphoma cells not only drives tumor progression but also triggers anti-mGluR5 encephalitis even before classical Hodgkin lymphoma becomes manifest.

IL-6 as an Inflammatory Marker in Hodgkin Lymphoma

Hodgkin lymphoma (HL) is a lymphoid tumor that develops from embryological or post-germinal center B-cells. The disease was originally recognized in 1832 by Thomas Hodgkin. Globally, 0.4% of clinically diagnosed cancers in 2018 were caused by HL. Regardless of age, ethnicity, or location, the prevalence of HL varies. It's a particular kind of malignant tumor in which the cancerous Hodgkin and Reed-Sternberg (HRS) cells are surrounded by a significant cellular infiltration of immune cells (lymphocytes, macrophages, eosinophils, mast cells, plasma cells, and collagen cells), which also contribute to the microenvironment of the tumor. Numerous chemotherapy drugs and low-dose field radiation have been found to significantly reduce illness mortality [1].
 High levels of cytokines that stimulate the immune system, such as interleukin (IL)-6, tumor necrosis factor (TNF), and eotaxin are produced by developing tumor cells and/or by reactive immune cells associated with tumors. These markers are anticipated to promote B-cell activation and related high-risk DNA-modifying activities, which will lead to the growth of B-cell lymphoma. IL-6 is a critical cytokine that stimulates the growth of hematological and solid tumors and is synthesized by a wide range of cell types, including fibroblasts, ECs, epithelial cells, and monocytes as hematological tumor lines. Through the activation of JAK, it stimulates the transcription factors STAT. In NHL, lower progression-free survival and overall survival rates have been linked to higher blood IL-6 levels, and IL-6 production in HL cell lines has been demonstrated. Numerous investigations revealed that IL-6 expression correlated with early onset vascular development events, indicating that it may be involved in angiogenesis [2].
 Interleukin-6 (IL-6) is thought to play a crucial pathobiological function in classical HL among the biologic variables like cytokines. According to reports, adult patients with HL who have elevated serum interleukin-6 (IL-6) have better response rates, longer survival times, and B symptoms. TH-17 response may be elicited by HRS cells that express IL-6, but TH-2 response may be elicited by HRS cells that do not express IL-6. T-lymphocytes are known to be attracted by IL-6, and depending on how it interacts with other cytokines like TGF-beta, the lymphocytes may induce a TH-2 or TH-17 response. A significant cancer hallmark known as immune evasion can be brought on in cancer patients by the TH-2 response [3].

Replacing Procarbazine with Dacarbazine in Escalated Beacopp Dramatically Reduces the Post Treatment Haematopoietic Stem and Progenitor Cell Mutational Burden in Hodgkin Lymphoma Patients with No Apparent Loss of Clinical Efficacy

Introduction When treating Hodgkin lymphoma (HL), it is common UK practice to modify escalated BEACOPP (eBPP) by removing oral procarbazine and replacing it with intravenous dacarbazine (250mg/m2 D2-3) to reduce haematopoietic stem cell and gonadal toxicity. A similar replacement of procarbazine in COPP (to COPDac) has reduced gonadal toxicity and conferred a comparable long-term event-free survival to children (EuroNet-PHL-C1 trial; Mauz-Körholz et al Lancet Oncol 2021). Our own retrospective study has shown that adults treated with first-line escalated BEACOPDac (eBPDac) have a reduced blood transfusion requirement and earlier return of menstrual periods compared with real-world eBPP patients (pts) with no loss of clinical efficacy compared to HD18 eBPP pts (Santarsieri et al Blood 2021). To better understand the toxicity differences, we performed this study to compare the haematopoietic stem and progenitor cell (HSPC) mutational burden in pts who have received procarbazine and dacarbazine containing regimens. Chemotherapeutic agents are known to damage DNA and this mutational process likely causes many of the long-term chemotherapy toxicities. Little is known about the mutational signatures and mutation burdens caused by HL therapies, although a novel mutational signature (SBS25) has recently been described in two HL cell lines (Petljak et al Cell 2019). Methods We whole genome sequenced 6-8 single-cell derived HSPC colonies from each of 12 advanced stage HL pts (n = 91; mean sequencing depth 26X). The pts were all in remission > 6 months and had been previously treated with either eBPDac (6 cycles (n=2); 4 cycles (n=2)), eBPP (6 cycles (n=3); 5 cycles (n=1); 4 cycles (n=1)) or ABVD (n=3; all 6 cycles). Single HSPC somatic mutation burdens and mutation spectra from these chemotherapy exposed individuals were compared to those from our previously published normal cohort (n = 110; mean sequencing depth 24X) (Mitchell et al Nature 2022). In addition we have updated the outcomes of our retrospective multi-centre study of pts treated with eBPDac including additional pts and longer follow-up of the entire cohort. Mutational analysis The somatic single nucleotide variant mutation burden in normal adult HSPCs ranges from approximately 400 aged 20yr to 1500 aged 80yr; an average of 18 mutations accumulate with every year (Figure 1A). The HSPCs from ABVD and eBPDac-treated pts had similar minor excess somatic mutation burdens compared to aged-matched normal HSPCs of 183 (CI95%= 110-256) and 291 (CI95%= 242-340) mutations respectively. In contrast, the HSPCs from eBPP-treated pts had a striking and significantly increased excess mutation burden of 1153 (CI95%= 937-1369). Of the eBPP pts, the single patient who received 4 cycles had the lowest number of excess mutations, but noting the inter-patient variation, we cannot yet conclude that eBPP cycle number correlates with the number of mutations. Descriptive analysis of the mutational profiles (Figure 1B) revealed that every patient who received procarbazine had a clear SBS25-like mutational signature, characterised by T>A base substitutions, demonstrating for the first time that this DNA mutational signature is directly attributable to procarbazine. Clinical data analysis The clinical data presented last year have been updated with more pts and longer follow-up (n=288; median 2.3 yr). With this high-risk group of advanced stage pts, our data suggest that the eBPDac pts have reduced red cell transfusion requirements, reduced hospitalisation, improved menstrual period recovery and improved sperm count preservation compared with eBPP pts. The eBPDac 24-month PFS is similar to HD18 3-yr PFS (94.9% vs 92.3%) and appears superior to RATHL 5-yr PFS (81.4%). The eBPDac 24-month OS estimate is 99%. Conclusions We have shown that dacarbazine-containing regimens (ABVD and eBPDac) confer a lower mutation burden to HSPCs compared with procarbazine-containing eBPP and that procarbazine is the agent responsible for the SBS25 mutational signature. This work provides a model example of how genomic analysis could be used to inform therapeutic decision making in cancer. Our retrospective analysis provides strong supportive evidence that eBPDac is a highly efficacious HL treatment and noting the clear benefits in terms of HSPC genomic health, we would encourage clinicians offering eBPP to HL pts to consider replacing procarbazine with dacarbazine. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phase I Trial of Brentuximab Vedotin Plus Cyclosporine in Relapsed/Refractory Hodgkin Lymphoma

Introduction Treatment of relapsed/refractory (R/R) Hodgkin lymphoma (HL) following failure of brentuximab vedotin (BV) and PD-1 blockade is a major unmet need. Resistance to BV is associated with multidrug resistance gene 1 (MDR1) overexpression. There are pre-clinical data showing that MDR1 inhibitors such as cyclosporine (CsA) or verapamil (VRP) are synergistic with BV in BV-resistant HL cell lines. Preliminary results of a phase I study evaluating the safety and efficacy of combination of BV with CsA and VRP in R/R HL were previously reported. Here we report the final results of the completed study. Methods This is a single center, open-label phase I investigator-initiated study. Patients (pts) with HL whose disease had relapsed or was refractory to ≥1 prior lines of therapy were enrolled. The dose finding portion followed a 3+3 design with 4 planned dose levels (DL). BV was given at 1.2 mg/kg (DL1) or 1.8 mg/kg (DL2-4) intravenously every 3 weeks. Planned dose of CsA was 5 mg/kg (DL1-2) or 7.5 mg/kg (DL3-4) orally twice daily on days 1-5. Planned dose of VRP was 120 mg orally four times daily on days 1-5 (DL4). Each cycle was 3 weeks. Once the maximum tolerated dose (MTD) was determined, BV-refractory pts were enrolled in an expansion cohort at the MTD. Primary objective was to determine the MTD and safety of the combination. Secondary objectives were overall response rate (ORR), complete response (CR) rate, response duration (DOR), overall survival (OS), and progression-free survival (PFS). Results Twenty-nine pts were enrolled onto the study, 14 in the dose-finding portion and 15 in the dose expansion BV-refractory cohort. With respect to baseline characteristics, 38% of patients were female, 69% had advanced stage disease, 45% had extranodal disease, and 24% had B symptoms. Median age was 36 (20-69) and pts had a median of 5 (3-12) prior lines of therapy. All had prior BV (only 2 BV-sensitive) and 27 had prior PD-1 blockade. Four pts were treated on DL1, 22 on DL2, and 3 on DL3. MTD was DL2. DLTs observed were: DL1 10-day CsA (n=1) grade (Gr) 3 hyperbilirubinemia, abdominal pain, and hypertension, DL2 (n=1) Gr3 abdominal pain and Gr3-4 neutropenia, DL3 (n=2) Gr3 bone pain/constipation/Gr4 lymphopenia (n=1) and Gr3 hyperglycemia (n=1). Median duration of treatment was 3 (1-16) cycles. Most frequent adverse events (AEs) were nausea (90%), hypertension (90%), anemia (86%), fatigue (76%), neutropenia (76%), and leukopenia (76%). All pts had Gr3+ AEs with most frequent being neutropenia (62%). Reasons for treatment discontinuation included death (n=4), disease progression (n=10), toxicity (n=2), patient refusal (n=8), loss to follow-up (n=2), MD decision (n=1), proceeding to HCT (n=1), and study completion (n=1). Treatment-related death occurred in 3 pts (pneumonitis at DL1, respiratory failure and hypotension at DL2). The ORR/CR was 62%/24%. The median DOR and PFS was 5 months and median OS was not reached. Discussion The combination of BV and CsA was effective in BV-refractory R/R HL but was also associated with toxicity, including treatment-related deaths that led to early termination of the clinical trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Navitoclax Most Promising BH3 Mimetic for Combination Therapy in Hodgkin Lymphoma.

The treatment of young patients with Hodgkin lymphoma (HL) is often successful but a significant proportion of patients suffers from late toxicity. In the current era there are new opportunities for less toxic and more targeted treatment options. In this respect, the anti-apoptotic pathway is an attractive target since Hodgkin tumor cells abundantly express components of this pathway. We measured the effect of BH3 mimetics that interfere with anti-apoptotic proteins in cell lines, also in combination with the standard of care chemotherapeutic doxorubicin and the recently discovered preclinically active tamoxifen. Several anti-apoptotic BCL-2 family proteins were expressed in each case (n = 84) and in HL cell lines (n = 5). Cell lines were checked for sensitivity to BH3 mimetics by BH3 profiling and metabolic assays and monotherapy was only partially successful. Doxorubicin was synergistic with a BCL-XL inhibitor and BCL2/XL/W inhibitor navitoclax. Tamoxifen that targets the estrogen receptor β present in the mitochondria of the cell lines, could induce cell death, and was synergistic with several BH3 mimetics including/as well as navitoclax. In conclusion, targeting the anti-apoptotic pathway by the triple inhibitor navitoclax in combination with doxorubicin or tamoxifen is a promising treatment strategy in HL.

The noncatalytic regions of the tyrosine kinase Tnk1 are important for activity and substrate specificity

Human Tnk1 (thirty-eight negative kinase 1) is a member of the Ack family of nonreceptor tyrosine kinases. Tnk1 contains a sterile alpha motif, a tyrosine kinase catalytic domain, an SH3 (Src homology 3) domain, and a large C-terminal region that contains a ubiquitin association domain. However, specific physiological roles for Tnk1 have not been characterized in depth. Here, we expressed and purified Tnk1 from Sf9 insect cells and established an invitro assay system using a peptide substrate derived from the Wiskott-Aldrich Syndrome Protein (WASP). By Tnk1 expression in mammalian cells, we found that the N-terminal SAM domain is important for self-association and kinase activity. We also studied a fusion protein, originally discovered in a Hodgkin's Lymphoma cell line, that contains an unrelated sequence from the C17ORF61 gene fused to the C-terminus of Tnk1. Cells expressing the fusion protein showed increased tyrosine phosphorylation of cellular substrates relative to cells expressing WT Tnk1. A truncated Tnk1 construct (residues 1-465) also showed enhanced phosphorylation, indicating that the C17ORF61 sequence was dispensable for the effect. Additionally, invitro kinase assays with the WASP peptide substrate showed no increase in intrinsic Tnk1 activity in C-terminally truncated constructs, suggesting that the truncations did not simply remove an autoinhibitory element. Fluorescence microscopy experiments demonstrated that the C-terminus of Tnk1 plays an important role in the subcellular localization of the kinase. Taken together, our data suggest that the noncatalytic regions of Tnk1 play important roles in governing activity and substrate phosphorylation.

P029: Are Reed-Sternberg cells stuck in mitosis by short nucleoplasmic bridges as a consequence of centromeric instability?

Background: Multinucleated giant tumor cells are frequently observed in lymph nodes of untreated lymphoma patients. Malignant Hodgkin lymphoma (HL) cells i. e. small Hodgkin and large bi- or multinucleated Reed-Sternberg (HRS) cells are characterized by genomic instability and downregulation of numerous key regulators of e.g. cell cycle, spindle apparatus, cytokinesis and cell lineage differentiation. So far, the formation of HRS cells remains obscure. We have demonstrated previously that telomere instability is involved in the proliferation of small cells into large binucleated cells. Here, we have used a collection of HL cell lines to analyze the successive steps involved in the transformation of the mononucleated into the multinucleated RS-like cells. Materials and Methods: Seven HL cell lines and 30 HL lymph nodes were used. Cytokinesis-block micronucleus assay and telomere and centromere staining followed by the M-FISH technique were employed to analyze chromosomal instability. DNA repair pathways were investigated. Results: We demonstrate that telomere dysfunction in HL cell lines is associated with the formation of dicentric chromosomes with both centromeres in close proximity, but also with a high rate of formation of micronuclei. Two morphologically different types of nucleoplasmic bridges (NPBs) were observed: (1) Long NPBs related to telomere dysfunction and chromosome fusions, and (2) short NPBs related to centromere breakpoints with configurations of cells looking like “stuck” together as binucleated cells. Short NPBs and binucleated cells were seen in the cultures of all HL cell lines. However, each HL cell line was characterized by an individual signature of the proportion of long and short NPBs, which correlated with centrosome amplification and formation of stable binucleated cells. Similar mechanisms were identified in HL patient lymph nodes prior to treatment, thus underscoring the biological significance of our findings in cell cultures. Transcriptome analysis confirmed the variations in the DNA repair pathways regarding the rate of large cells among the different HL cell lines. Conclusion: The formation of dicentric chromosomes related to centromere instability, and short NPBs were associated with the emergence of binucleated cells through different mechanisms. Our findings open a novel route to understanding the transition from mononucleated cells to multinucleated cells in HL and in other B-cell lymphomas.

P046: Preclinical evaluation of novel repurposed drug combinations in Hodgkin lymphoma

Although the outcome of patients with Hodgkin lymphoma (HL) is comparably high, it worsens rapidly in relapsed or refractory disease. In the pursuit of improving cancer treatment and finding new therapy options, repurposing drugs has evolved as a promising strategy. This project takes a systematic look at the direct antitumor effect of 12 non-oncological, repurposed drugs in HL that survived initial screening in a broad panel of cell lines. Additionally, we consider their immunological effects and aim to decipher strategies on how to potentially use them in conjunction with immunotherapy. Luciferase toxicity assays in different concentrations were performed with four HL cell lines, narrowing down the list of 12 to the five most toxic agents which were then tested again in three different combinations. The most effective combination was then evaluated in mouse models. To evaluate the influence of the screening survivor drugs on immune response, assays for T-cell activation, macrophage polarization and phagocytosis were conducted. Five compounds, Albendazole (Alb), Auranofin (Aur), Disulfiram (Dis), Fluvastatin (Flu) and Propranolol (Pro) showed markedly elevated in-vitro toxicity. The three combinations Alb+Aur+Dis+Pro, Aur+Dis+Flu+Pro and a combination of all 5 drugs showed synergistic cytotoxicity in HL cell lines. Latter combination clearly reduced tumor growth in mouse models. Except for Doxycycline, which markedly repressed T-cell activation, none of the other tested 11 compounds showed negative effects on either T-cell activation, or macrophage polarization and phagocytosis. On the contrary, Eprosartan (Epro), Metformin (Met), Clofibrate (Clof) and Ornidazole (Orni) increased antibody dependent cellular phagocytosis (ADCP) in in-vitro assays. This project shows that combinations of repurposed, non-oncological drugs at safe doses have a direct cytotoxic effect against HL and a potentially beneficial impact on key immune response mechanisms. Taken together, our results justify further evaluation of combinations of these clinically known drugs with chemotherapy or checkpoint blockade in cases of HL without other treatment options.

P054: The Kinase CK2 is deregulated and targetable in classical Hodgkin Lymphoma

Introduction: CK2 is a pleiotropic kinase consisting of 2 catalytic (a) and 2 regulatory (b) subunits, usually overexpressed, that sustains cancer signaling cascades through the activation of NF-kB, PI3K and STAT pathways. Considering that NF-kB, STAT and PI3K pathways are key players in Hodgkin lymphoma (HL), it is likely that CK2 might play a role in the pathogenesis of this disease. Methods: Experiments were performed and replicated at least for five times employing 4 HL cell lines (L-428, L-540, KM-H2 and HDLM-1) cultured in RPMI. CK2a, CK2b, RelA-Serine (S)529, RelA, PARP, AKT-S473, AKT, STAT3-S727, STAT3, tubulin, ubiquitin expression levels were evaluated by western blot analysis (WB). Silmitasertib and bortezomib were used as CK2a and proteasome inhibitors. Apoptosis was assessed by Annexin V/Propidium iodide assay and PARP cleavage by WB. Immunohistochemistry (IHC) for CK2a and CK2b was performed on formalin fixed paraffin embedded sections of lymph-nodes from patients with HL, indolent and aggressive NHL. Chou-Talalay methods was used to calculate the combination index (C.I.) of two drugs. Results: By WB and IF we found that all the 4 HL cell lines expressed higher levels of CK2a, but not CK2b, as compared to normal B lymphocytes (p<0.001). By IHC on 35 patients’ lymph-nodes, we confirmed that CK2a but not CK2b was highly expressed in Reed-Sternberg cells. In addition, patients with lower levels of CK2b display a better progression-free survival (p<0.05). The unbalance between a and b subunits was not observed in low or high-grade NHL (p<0.001). Furthermore, we observed that mRNA levels of both CSNK2A and CSNK2B were similar to normal B lymphocytes, and that proteasome inhibition with bortezomib caused the upregulation of CK2b. AKT, RelA and STAT3 were constitutively phosphorylated in HL at their activatory S-residue (S473, S529, and S727 respectively). Treatment of HL cell lines with silmitasertib caused down-regulation of these phospho-serine proteins, time and dose-dependent apoptosis (p<0.0001). In addition, we found that silmitasertib did not modulate surface CD30 and had a synergistic anti-apoptotic effect in combination with monomethyl auristatin E (C.I.<1) Conclusions: We demonstrated that CK2a is overexpressed, active, induced key pro-survival signals in HL, and its inhibition trigger apoptosis. CK2b is likely downregulated due to proteasome degradation. These preliminary data suggest that CK2 might be a new therapeutic target in HL.

Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL.

Distinct Gene Expression Patterns of Calcium Channels and Related Signaling Pathways Discovered in Lymphomas.

Cell surface calcium (Ca2+) channels permit Ca2+ ion influx, with Ca2+ taking part in cellular functions such as proliferation, survival, and activation. The expression of voltage-dependent Ca2+ (CaV) channels may modulate the growth of hematologic cancers. Profile analysis of Ca2+ channels, with a focus on the Ca2+ release-activated Ca2+ (CRAC) and L-type CaV channels, was performed on RNA sequencing data from lymphoma cell lines and samples derived from patients with diffuse large B cell lymphoma (DLBCL). CaV1.2 expression was found to be elevated in classical Hodgkin lymphoma (CHL) cell lines when compared to other B cell lymphoma cell lines. In contrast, CHL exhibited reduced expression of ORAI2 and STIM2. In our differential expression analysis comparing activated B cell-like DLBCL (ABC-DLBCL) and germinal centre B cell-like DLBCL (GCB-DLBCL) patient samples, ABC-DLBCL revealed stronger expression of CaV1.3, whereas CaV1.1, CaV1.2, and CaV1.4 showed greater expression levels in GCB-DLBCL. Interestingly, no differences in ORAI/STIM expression were noted in the patient samples. As Ca2+ is known to bind to calmodulin, leading to calcineurin activation and the passage of nuclear factor of activated T cells (NFAT) to the cell nucleus, pathways for calcineurin, calmodulin, NFAT, and Ca2+ signaling were also analyzed by gene set enrichment analysis. The NFAT and Ca2+ signaling pathways were found to be upregulated in the CHL cell lines relative to other B cell lymphoma cell lines. Furthermore, the calmodulin and Ca2+ signaling pathways were shown to be downregulated in the ABC-DLBCL patient samples. The findings of this study suggest that L-type CaV channels and Ca2+-related pathways could serve as differentiating components for biologic therapies in targeted lymphoma treatments.

Focal structural variants revealed by whole genome sequencing disrupt the histone demethylase KDM4C in B-cell lymphomas.

Histone methylation-modifiers, such as EZH2 and KMT2D, are recurrently altered in B-cell lymphomas. To comprehensively describe the landscape of alterations affecting genes encoding histone methylation-modifiers in lymphomagenesis we investigated whole genome and transcriptome data of 186 mature B-cell lymphomas sequenced in the ICGC MMML-Seq project. Besides confirming common alterations of KMT2D (47% of cases), EZH2 (17%), SETD1B (5%), PRDM9 (4%), KMT2C (4%), and SETD2 (4%), also identified by prior exome or RNA-sequencing studies, we here found recurrent alterations to KDM4C in chromosome 9p24, encoding a histone demethylase. Focal structural variation was the main mechanism of KDM4C alterations, and was independent from 9p24 amplification. We also identified KDM4C alterations in lymphoma cell lines including a focal homozygous deletion in a classical Hodgkin lymphoma cell line. By integrating RNA-sequencing and genome sequencing data we predict that KDM4C structural variants result in loss-offunction. By functional reconstitution studies in cell lines, we provide evidence that KDM4C can act as a tumor suppressor. Thus, we show that identification of structural variants in whole genome sequencing data adds to the comprehensive description of the mutational landscape of lymphomas and, moreover, establish KDM4C as a putative tumor suppressive gene recurrently altered in subsets of B-cell derived lymphomas.