cHL Cell Lines Research Articles

A first-in-class inhibitor of HSP110 to potentiate XPO1-targeted therapy in primary mediastinal B-cell lymphoma and classical Hodgkin lymphoma

BackgroundPrimary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) are distinct hematological malignancies of B-cell origin that share many biological, molecular, and clinical characteristics. In particular, the JAK/STAT signaling pathway is a driver of tumor development due to multiple recurrent mutations, particularly in STAT6. Furthermore, the XPO1 gene that encodes exportin 1 (XPO1) shows a frequent point mutation (E571K) resulting in an altered export of hundreds of cargo proteins, which may impact the success of future therapies in PMBL and cHL. Therefore, targeted therapies have been envisioned for these signaling pathways and mutations.MethodsTo identify novel molecular targets that could overcome the treatment resistance that occurs in PMBL and cHL patients, we have explored the efficacy of a first-in-class HSP110 inhibitor (iHSP110-33) alone and in combination with selinexor, a XPO1 specific inhibitor, both in vitro and in vivo.ResultsWe show that iHSP110-33 decreased the survival of several PMBL and cHL cell lines and the size of tumor xenografts. We demonstrate that HSP110 is a cargo of XPO1wt as well as of XPO1E571K. Using immunoprecipitation, proximity ligation, thermophoresis and kinase assays, we showed that HSP110 directly interacts with STAT6 and favors its phosphorylation. The combination of iHSP110-33 and selinexor induces a synergistic reduction of STAT6 phosphorylation and of lymphoma cell growth in vitro and in vivo. In biopsies from PMBL patients, we show a correlation between HSP110 and STAT6 phosphorylation levels.ConclusionsThese findings suggest that HSP110 could be proposed as a novel target in PMBL and cHL therapy.

Protein kinase CK2α is overexpressed in classical hodgkin lymphoma, regulates key signaling pathways, PD-L1 and may represent a new target for therapy.

In classical Hodgkin lymphoma (cHL), the survival of neoplastic cells is mediated by the activation of NF-κB, JAK/STAT and PI3K/Akt signaling pathways. CK2 is a highly conserved serine/threonine kinase, consisting of two catalytic (α) and two regulatory (β) subunits, which is involved in several cellular processes and both subunits were found overexpressed in solid tumors and hematologic malignancies. Biochemical analyses and in vitro assays showed an impaired expression of CK2 subunits in cHL, with CK2α being overexpressed and a decreased expression of CK2β compared to normal B lymphocytes. Mechanistically, CK2β was found to be ubiquitinated in all HL cell lines and consequently degraded by the proteasome pathway. Furthermore, at basal condition STAT3, NF-kB and AKT are phosphorylated in CK2-related targets, resulting in constitutive pathways activation. The inhibition of CK2 with CX-4945/silmitasertib triggered the de-phosphorylation of NF-κB-S529, STAT3-S727, AKT-S129 and -S473, leading to cHL cell lines apoptosis. Moreover, CX-4945/silmitasertib was able to decrease the expression of the immuno-checkpoint CD274/PD-L1 but not of CD30, and to synergize with monomethyl auristatin E (MMAE), the microtubule inhibitor of brentuximab vedotin. Our data point out a pivotal role of CK2 in the survival and the activation of key signaling pathways in cHL. The skewed expression between CK2α and CK2β has never been reported in other lymphomas and might be specific for cHL. The effects of CK2 inhibition on PD-L1 expression and the synergistic combination of CX-4945/silmitasertib with MMAE pinpoints CK2 as a high-impact target for the development of new therapies for cHL.

In Doxorubicin-Adapted Hodgkin Lymphoma Cells, Acquiring Multidrug Resistance and Improved Immunosuppressive Abilities, Doxorubicin Activity Was Enhanced by Chloroquine and GW4869.

Classical Hodgkin lymphoma (cHL) is a highly curable disease (70-80%), even though long-term toxicities, drug resistance, and predicting clinical responses to therapy are major challenges in cHL treatment. To solve these problems, we characterized two cHL cell lines with acquired resistance to doxorubicin, KM-H2dx and HDLM-2dx (HRSdx), generated from KM-H2 and HDLM-2 cells, respectively. HRSdx cells developed cross-resistance to vinblastine, bendamustin, cisplatin, dacarbazine, gemcitabine, brentuximab vedotin (BV), and γ-radiation. Both HDLM-2 and HDLM-2dx cells had intrinsic resistance to BV but not to the drug MMAE. HDLM-2dx acquired cross-resistance to caelyx. HRSdx cells had in common decreased CD71, CD80, CD54, cyt-ROS, HLA-DR, DDR1, and CD44; increased Bcl-2, CD58, COX2, CD26, CCR5, and invasive capability; increased CCL5, TARC, PGE2, and TGF-β; and the capability of hijacking monocytes. In HRSdx cells less sensitive to DNA damage and oxidative stress, the efflux drug transporters MDR1 and MRP1 were not up-regulated, and doxorubicin accumulated in the cytoplasm rather than in the nucleus. Both the autophagy inhibitor chloroquine and extracellular vesicle (EV) release inhibitor GW4869 enhanced doxorubicin activity and counteracted doxorubicin resistance. In conclusion, this study identifies common modulated antigens in HRSdx cells, the associated cross-resistance patterns, and new potential therapeutic options to enhance doxorubicin activity and overcome resistance.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: We previously performed global miRNA expression profiling of cHL vs. non-Hodgkin lymphoma (NHL) cell lines and sorted germinal centre B-cells (GCB). In the group of 79 deregulated miRNAs (p < 0.05) we became particularly interested in the upregulated miR-23a-3p, a potential oncomiR, predicted to regulate the expression of TNFAIP3 and the downregulated miR-148a-3p, a potential tumor suppressor miRNA, predicted to regulate the expression of IL15. Therefore, we aimed to decipher the role of miR-23a-3p and miR-148a-3p in cHL pathogenesis. Methods: Expression analyses were performed with TaqMan Advanced miRNA Assays versus two miRNA controls. Laser capture microdissection was used for primary HRS collection. 1,000 (miRNA expression) and 2 × 200 (DNA methylation) HRS cells were collected per case. Bisulfite pyrosequencing assay was designed using the PyroMark Software and sequencing performed using the PyroMark Q24 sequencer. MiRNA-mRNAs interactions were validated with Dual-Glo Luciferase Assay using respective miRNA mimics. For overexpression pre-miRNAs were cloned into the pCDH-CMV-MCS-EF1α-GreenPuro vector and transductions were performed in triplicate. Cell proliferation was measured using CCK-8 in four replications in three independent reactions by the GloMax®96 Microplate reader. Results: We confirmed the overexpression of miR-23a-3p (p < 0.001) and downregulation of miR-148a-3p (p < 0.014) in microdissected HRS cells (n = 10) vs. GCB cells (n = 10). Moreover, we observed inverse correlation of miR-148a-3p expression with DNA methylation level of an adjacent CpG island (r = −0.72, p < 0.01; cHL n = 7, NHL n = 10). Elevated DNA methylation of this CpG island was detected also in 2/6 cHL primary cases. Next, we validated interactions between miR-23a-3p and miR-148a-3p and their target genes. A reduction by 34% (p < 0.001) and 40% (p < 0.01) of the luciferase signal was observed in consequence of the interaction of miR-23a-3p mimic—TNFAIP3 3’UTR and miR-148a-3p mimic—IL15 3’UTR, respectively. Moreover, the abundance of TNFAIP3 protein was reduced after overexpression of the miR-23a (coexpressed with miR-27a) in L428 (p = 0.0018) and GCB6-16 (p = 0.0083) cell lines, compared to empty vector transductions. Whereas overexpression of miR-148a in three cHL cell lines caused a 32% decrease (p < 0.05) in proliferation in the KM-H2 cell line compared to empty vector transductions. The research was funded by: This research was funded by the National Science Center, Poland grants: 2014/14/M/NZ2/00529 to M.G. and the 2019/32/T/NZ5/00441 Etiuda scholarship to JP. SH is supported by the German Research Foundation (DFG, HA6145/3-1). MG, ADz-K and NR received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952304. Keywords: Hodgkin lymphoma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through activation of the adaptor protein STING. The latter, in turn, activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The expression patterns of STING in Hodgkin and Reed-Sternberg (HRS) cells and the potential role of cGAS-STING pathway in anti-tumor immune responses in classical Hodgkin lymphoma (cHL) remain unknown to date. Methods: In this prospective study, STING expression was immunohistochemically analysed in a cohort of 52 previously untreated patients with cHL and available tissue for flow cytometry (FC) analysis and histology as well as available peripheral blood samples for FC. An arbitrary 10% cutoff was used for positivity in HRS. The in vitro system included 6 cHL cell lines (MDAV, L1236, L428, L540, HDLM2, KMH2). Gene (mRNA level) and protein expression/activation of cGAS-STING components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot, respectively. The cHL cell lines were treated with a STING agonist or were subjected to silencing of STING gene using transient transfection with specific STING siRNA construct. The gene expression of cGAS-STING-associated IFNs and cytokines, including IFN-β, CXCL10, IFN-γ, STING, and a control gene (GAPDH), was analysed with RT-qPCR. Results: STING was positive in HRS cells of cHL in 22 of 52 (42%) patients. STING positivity in HRS significantly correlated with lower numbers of STING+ T-lymphocytes (p < 0.0001) and CD3+ T-lymphocytes (p = 0.04) but higher numbers of CD20+ B-cells assessed by FC in the tissues. In peripheral blood samples assessed by FC at presentation, STING expression by HRS correlated with higher numbers of CD3+ T-cells (p = 0.005) but lower numbers of NK-cells (p = 0.02). STING expression did not correlate with clinical features at presentation, including age, gender, Ann Arbor stage, B-symptoms, tumor burden, bulky disease, anemia, or other hematologic values. STING expression at the mRNA and protein level was substantially higher in L1236, L428 and HDLM2 compared to other cHL cell lines. Treatment with STING agonists stimulated gene expression of IFN-β and/or CXCL10 at a variable level indicating functional cGAS-STING pathway in HRS. Knocking down STING gene resulted in dramatic increase in CXCL10 gene expression in cHL cells. Conclusions: STING is expressed by HRS in a subset of cHL and significantly correlates with the lymphocytic populations in the tumor microenvironment and peripheral blood. The cGAS-STING pathway is functional in HRS suggesting that STING agonists may have therapeutic implications in patients with cHL. Keywords: Diagnostic and Prognostic Biomarkers, Hodgkin lymphoma, Microenvironment No conflicts of interests pertinent to the abstract.

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.

Modulation of the CGAS-Sting Anti-Tumor Immune Response Pathway in an NF-KB-Dependent or -Independent Manner As a Potential Therapeutic Target in Classical Hodgkin Lymphoma (cHL)

Introduction: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through production of the second messenger cGAMP and subsequently activation of the adaptor protein STING. The latter activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The potential role of cGAS-STING pathway in anti-tumor immune responses in classical Hodgkin lymphoma (cHL) remains unknown to date. In this study, we hypothesized that NF-kB pathway, a critical player in cHL oncogenesis, may regulate anti-tumor immune responses through modulation of the cGAS-STING pathway activity and gene expression of the relevant interferons and chemokines. Methods: STING expression was immunohistochemically analyzed in a pilot study group of 32 untreated patients with cHL and available tissue from diagnostic lymph node biopsies using a previously validated monoclonal antibody and standard methods. In addition, control lymphoid tissue (2 reactive lymph nodes, 2 tonsils) were used. At least 500 neoplastic Hodgkin & Reed-Sternberg (HRS) cells were counted and an arbitrary 10% cutoff was used for positivity. The in vitro system included 6 cHL cell lines (MDA-V, L-1236, L-428, L-540, HDLM-2, KM-H2). Gene expression (mRNA level) and protein expression and activation (phosphorylation) of cGAS-STING pathway components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot analysis, respectively. The cHL cell lines were treated with a STING agonist and TBK1/IKK inhibitor (Amlexanox) alone or in combination with other agents. Silencing of STING and NF-kB genes was performed in all cHL cell lines using transient transfection (Nucleofector system, Amaxa, Lonza) with specific STING and NF-kB (RELA) siRNA constructs. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of IFN-β, CXCL10, IFN-γ, and a control gene (GAPDH), with RT-qPCR. Results: In reactive lymphoid tissue, STING expression was restricted to dendritic cells and a subset of T-lymphocytes with a cytoplasmic staining pattern, which served as internal positive controls in all immunostainings. Using an arbitrary 10% cutoff, STING was positive in the neoplastic HRS cells of cHL in 20 of 32 (63%) patients with a predominant cytoplasmic pattern. STING expression at the mRNA and protein level was substantially higher in L-1236, L-428 and HDLM-2 compared to other cHL cell lines. Treatment with STING agonist alone stimulated gene expression of IFN-β and/or CXCL10 at a variable level depending on the cell line indicating functional cGAS-STING anti-tumor immune response pathway in cHL. Knocking down STING gene resulted in dramatic increase in CXCL10 gene expression in cHL cells. Silencing of NF-kB (RELA) resulted in activation of cGAS-STING pathway as shown by IRF3 activation (phosphorylation) and increased IRF3 protein levels in L-428 cells, which was associated with increased CXCL10 mRNA levels. Treatment with Amlexanox resulted in downregulation of IFN-β and/or CXCL10 gene expression in cHL cell lines. Conclusion: The cGAS-STING pathway is activated and, at least in part, regulated by the oncogenic NF-kB pathway. STING agonists and Amlexanox modulate gene expression of type 1 IFNs in cHL with direct therapeutic implications. A large cohort of cHL patients is currently being analyzed for the expression and prognostic significance of STING and the final results will be available at the time of the meeting.

P053: The cGAS-STING anti-tumor immune response pathway as a potential therapeutic target in classical Hodgkin Lymphoma (cHL)

Background: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through production of the second messenger cGAMP and subsequently activation of the adaptor protein STING. The latter activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The potential role of cGAS-STING pathway in anti-tumor immune responses in cHL remains unknown to date. Methods: STING expression was immunohistochemically analysed in a pilot study group of 32 untreated patients with cHL and available tissue. The in vitro system included 6 cHL cell lines (MDA-V, L1236, L428, L540, HDLM2, KMH2). Gene expression (RNA level) and protein expression and activation (phosphorylation) of cGAS-STING pathway components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot, respectively. The cHL cell lines were treated with a STING agonist and TBK1/IKK inhibitor (Amlexanox) alone or in combination with other agents. Silencing of STING gene was performed using transient transfection with specific STING siRNA. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of IFN-β, CXCL10, IFN-γ, and a control gene (GAPDH) with RT-qPCR. Results: Using an arbitrary 10% cutoff, STING was positive in the neoplastic Hodgkin & Reed-Sternberg (HRS) cells of cHL in 20 of 32 (63%) patients with a membranous and cytoplasmic pattern. STING expression at the mRNA and protein level was substantially higher in L1236, L428 and HDLM2 compared to other cHL cell lines. Treatment with STING agonist alone stimulated gene expression of IFN-β and/or CXCL10 at a variable level depending on the cell line indicating functional cGAS-STING anti-tumor immune response pathway in cHL. Knocking down STING gene resulted in decreased CXCL10 and type 1 IFN gene expression by cHL cells. Amlexanox treatment also resulted in downregulation of IFN-β or CXCL10 gene expression in vitro. Conclusion: STING agonists and Amlexanox modulate gene expression of type 1 IFNs in cHL with direct therapeutic implications. A large cohort of cHL patients is currently being analysed for the expression and prognostic significance of STING, and the final results will be available at the time of ISHL 2022.

T-cell-derived Hodgkin lymphoma has motility characteristics intermediate between Hodgkin and anaplastic large cell lymphoma.

Classic Hodgkin lymphoma (cHL) is usually characterized by a low tumour cell content, derived from crippled germinal centre B cells. Rare cases have been described in which the tumour cells show clonal T‐cell receptor rearrangements. From a clinicopathological perspective, it is unclear if these cases should be classified as cHL or anaplastic large T‐cell lymphoma (ALCL). Since we recently observed differences in the motility of ALCL and cHL tumour cells, here, we aimed to obtain a better understanding of T‐cell‐derived cHL by investigating their global proteomic profiles and their motility. In a proteomics analysis, when only motility‐associated proteins were regarded, T‐cell‐derived cHL cell lines showed the highest similarity to ALK− ALCL cell lines. In contrast, T‐cell‐derived cHL cell lines presented a very low overall motility, similar to that observed in conventional cHL. Whereas all ALCL cell lines, as well as T‐cell‐derived cHL, predominantly presented an amoeboid migration pattern with uropod at the rear, conventional cHL never presented with uropods. The migration of ALCL cell lines was strongly impaired upon application of different inhibitors. This effect was less pronounced in cHL cell lines and almost invisible in T‐cell‐derived cHL. In summary, our cell line‐derived data suggest that based on proteomics and migration behaviour, T‐cell‐derived cHL is a neoplasm that shares features with both cHL and ALCL and is not an ALCL with low tumour cell content. Complementary clinical studies on this lymphoma are warranted.

Loss of function mutations of BCOR in classical Hodgkin lymphoma

BCOR is a component of a variant Polycomb repressive complex 1 (PRC1.1). PRC1 and PRC2 complexes together constitute a major gene regulatory system critical for appropriate cellular differentiation. The gene is upregulated in germinal center (GC) B cells and mutated in a number of hematologic malignancies. We report BCOR inactivating alterations in 4/7 classic Hodgkin lymphoma (cHL) cell lines, subclonal somatic mutations in Hodgkin and Reed–Sternberg (HRS) cells of 4/10 cHL cases, and deletions in HRS cells of 7/17 primary cHL cases. In mice, conditional loss of Bcor driven by AID-Cre in GC B cells resulted in gene expression changes of 46 genes (>2-fold) including upregulated Lef1 that encodes a transcription factor responsible for establishing T-cell identity and Il9r (interleukin-9 receptor), an important member of the cytokine network in cHL. Our findings suggest a role for BCOR loss in cHL pathogenesis and GC-B cell homeostasis.

The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

The NLPHL Tumor Microenvironment Is Markedly Enriched in the Tigit and PD-1 Signalling Axes Compared to Classical Hodgkin Lymphoma

Dissecting the Adaptive Response to Arginine Deprivation in Hodgkin Lymphoma

Background In Hodgkin Lymphoma (HL), neoplastic cells orchestrate an inflammatory microenvironment leading to sterile inflammation, T-cell anergy, and immune deficiency. Our group showed that in HL patients the aminoacid degrading enzyme Arginase-1 is increased, associated with poor outcome, and leads to arginine (Arg) deprivation. However, how the reduction of Arg in the extracellular milieu of the tumor microenvironment can contribute to neoplastic cell fitness is largely unknown.Aims To detect the adaptive response (via evaluation of global transcriptome and metabolome changes) in human HL cell lines exposed to Arg deprivation.Methods To better understand the impact of extra-cellular Arg1 deprivation on the metabolome of human cHL cells, four human cHL cell lines (L428, L540, HDMYZ and KM-H2) were individually cultured with customized complete media or lacking or Arg (R0), supplemented with 10% dialyzed fetal bovine serum, in six independent experiments. After 48 hours of culture, the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc and transcriptome profiling by Illumina platform. Following normalization to DNA concentration, log transformation, and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups.Results While Arg deprivation did not affect cell viability but delayed cell cycle due to arrest in G2 phase in all tested cell lines, the effect of Arg deficiency on the cellular metabolome depended largely on the cell type examined with L428 and KMH2 cells having significantly changed metabolomes. Pyruvate was significantly higher in the KMH2 cells deprived of Arg compared to controls. Conversely, lactate was significantly lower, with increased levels of long-chain saturated fatty acids and long-chain polyunsaturated fatty acids (PUFAs). Taken together the metabolomics changes suggested that specific-amino acid deficiency can lead to an increase in free fatty acids synthases to preserve cytoplasmatic and mitochondrial membrane dynamics. Consistent with a metabolic rewiring to maintain mitochondrial integrity (the pyruvate is an important intermediary in the conversion of carbohydrates into fatty acids), the adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione in KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine).Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements in KMH2 and HDMYZ cell lines, involving upregulation of genes required for the unfolded protein response (UPR, including XBP1, EIF2S1, EIF4A2, EIF4A3, ATF3, ATF4, DDIT4, EDEM1, GADD45B, SQSTM1, HMOX), NF-kB response to TNF (including RAF1, TNF, LIF, NKBIA, SGK1, BIRC3, ICAM1, BCL6, IL6, RELA, CDKN1A), p53 pathway and networks (including CDKN2B, STOM, TRAF4, RRAD, SESN1, FOXO3, SERPINB5, JAG2) and proteosome degradation (HSPA4, PSMD11, PSMD13, PSMD2, PSMA5, PSMA7, PSMC4), with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation.All lines tested showed down-regulation of CCNI2, LCROL, MKI67, NCAPG, PEX10 and UFSP2, suggesting that early response to arginine deprivation includes modulation of UFMylation pathway, the most recently discovered post-translational protein modification system, whose biological function is largely unknown.Conclusions The removal of Arg from L428 and KMH2 resulted in changes in the specific-amino acid-related metabolites. The adaptive response to Arg-depleted environment increases oxidative stress and promotes a shift in glucose use in the attempt to preserve mitochondrial function. The cell-cycle arrest in G2, the increase of pyruvate availability and the upregulation of proteasome function via upregulation of the UFMylation pathway suggest the dependency of HL cell lines on mitochondrial function integrity. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. DisclosuresMartinelli: Stemline Therapeutics: Consultancy; Roche: Consultancy; Astellas: Consultancy, Speakers Bureau; Daichii Sankyo: Consultancy; Pfizer: Consultancy, Speakers Bureau; Incyte: Consultancy; Abbvie: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy. Di Raimondo: Pfizer: Honoraria; Jazz Pharmaceutical: Honoraria; Amgen: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Deregulated miRNAs Contribute to Silencing of B-Cell Specific Transcription Factors and Activation of NF-κB in Classical Hodgkin Lymphoma.

Simple SummaryThe role of transcriptionally deregulated miRNAs (microRNAs) in classical Hodgkin lymphoma (cHL) is still not fully understood. To address this issue, we have performed global miRNA expression profiling of commonly used cHL cell lines and we present a complete cHL miRNome (microRNome). Within this group, we identify miRNAs recurrently deregulated in cHL cell lines, and compare them to non-Hodgkin lymphoma cell lines and sorted normal CD77+ germinal centre B-cells. Moreover, we show that several of the recurrently overexpressed miRNAs in cHL cell lines, and also primary microdissected HRS (Hodgkin and Reed-Sternberg) cells, target known B-cell-related transcription factors and NF-κB inhibitors. These findings provide evidence that deregulated miRNAs contribute to the loss of B-cell phenotype and NF-κB activation observed in this lymphoma.A hallmark of classical Hodgkin lymphoma (cHL) is the attenuation of B-cell transcription factors leading to global transcriptional reprogramming. The role of miRNAs (microRNAs) involved in this process is poorly studied. Therefore, we performed global miRNA expression profiling using RNA-seq on commonly used cHL cell lines, non-Hodgkin lymphoma cell lines and sorted normal CD77+ germinal centre B-cells as controls and characterized the cHL miRNome (microRNome). Among the 298 miRNAs expressed in cHL, 56 were significantly overexpressed and 23 downregulated (p < 0.05) compared to the controls. Moreover, we identified five miRNAs (hsa-miR-9-5p, hsa-miR-24-3p, hsa-miR-196a-5p, hsa-miR-21-5p, hsa-miR-155-5p) as especially important in the pathogenesis of this lymphoma. Target genes of the overexpressed miRNAs in cHL were significantly enriched (p < 0.05) in gene ontologies related to transcription factor activity. Therefore, we further focused on selected interactions with the SPI1 and ELF1 transcription factors attenuated in cHL and the NF-ĸB inhibitor TNFAIP3. We confirmed the interactions between hsa-miR-27a-5p:SPI1, hsa-miR-330-3p:ELF-1, hsa-miR-450b-5p:ELF-1 and hsa-miR-23a-3p:TNFAIP3, which suggest that overexpression of these miRNAs contributes to silencing of the respective genes. Moreover, by analyzing microdissected HRS cells, we demonstrated that these miRNAs are also overexpressed in primary tumor cells. Therefore, these miRNAs play a role in silencing the B-cell phenotype in cHL.

Usefulness of BATF3 Immunohistochemistry in Diagnosing Classical Hodgkin Lymphoma.

It is well recognized that the AP-1 transcription factor BATF3 is constitutively expressed in Hodgkin/Reed-Sternberg (HRS) cells, but its potential as a diagnostic marker for classical Hodgkin lymphoma (cHL) has not yet been addressed. In this study, we performed immunohistochemistry and analyzed the BATF3 expression in lymphoma cells on 218 lymphoma samples belonging to 14 different lymphoma entities. We observed varying degrees of BATF3 expression in nearly half of the cases (n = 100) with BATF3 expression being a constitutive feature of cHL (n = 53) and anaplastic large cell lymphoma (ALCL). By scoring BATF3 expression (BATF3-score) we observed constitutively high BATF3-scores in cHL and ALCL and low to moderate BATF3-scores in all other entities examined. Western blot analysis confirmed BATF3 protein expression in cell lysates from cHL cell lines (n = 7). Thus, BATF3 can be considered a useful IHC marker for the diagnosis of cHL as it is highly sensitive and sufficiently specific when analyzed by BATF3-scoring.

Apoptotic regulator BCL-2 blockade as a potential therapy in classical Hodgkin Lymphoma

The challenge in classical Hodgkin Lymphoma (cHL) management is the 30–40% of refractory/relapsed cases. AimsThe aim of this work was to determine whether NIK and BCL-2 could be useful as prognosis biomarkers in cHL. In addition, we evaluated BCL-2 as a directed-therapy in cHL cell lines using venetoclax. Main methodsWe evaluated NIK and BCL-2 expression in 112 untreated cHL patients' lymph-node biopsies by immunohistochemistry. cHL cell lines were treated with venetoclax alone or combined with vincristine or doxorubicin. Cell viability, metabolic activity and cell death were analyzed by trypan-blue exclusion method, MTS assay and FDA/IP staining respectively. Key findingsNo correlation between NIK or BCL-2 expression and the majority of the clinical parameters was found. Patients with ≥60% BCL-2+ HRS-cells had a shorter disease-free survival (DFS) and overall survival (OS) (p = 0.002, p = 0.02 respectively). A decision tree analysis, in a 30 patients subgroup, showed that patients with <60% NIK+ HRS-cells but with ≥60% BCL-2+ HRS-cells had a worse outcome in terms of DFS and OS. These parameters performed better as prognosis indicators as compared to the diagnosis bone marrow status. Human cHL cell lines U-H01, KM-H2, L1236, SUPHD1, L540 showed sensitivity to venetoclax. The co-treatment effect of venetoclax and vincristine or doxorubicin on cell viability was diverse depending on the cell line evaluated. SignificanceBCL-2 should be considered as a prognosis biomarker as well as a potential new therapeutic target in cHL. We report for the first time the cytotoxic effect of venetoclax in human cHL cell lines.

The PTP1B mutant PTP1B∆2-4 is a positive regulator of the JAK/STAT signalling pathway in Hodgkin lymphoma.

The neoplastic Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) depend on chronic activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathways to maintain survival and proliferation. Accumulating reports highlight the importance of the inactivation or reduced expression of negative JAK/STAT regulators such as the protein-tyrosine phosphatase 1B (PTP1B/PTPN1) in this process. Various PTPN1 mRNA variants as well as truncated PTP1B proteins were identified in cHL cell lines and primary cHL tumour samples. These PTPN1 mRNA variants lack either one or several exon sequences and therefore render these PTP1B variants catalytically inactive. Here, we show that one of these mutants, PTP1B∆2–4, is not only a catalytically inactive variant, but also augmented the IL-4-induced JAK/STAT activity similar to the recently reported PTP1B∆6 splice variant. Moreover, while PTP1B∆6 diminished the activity and protein levels of PTP1BWT, PTP1BWT remained unaffected by PTP1B∆2–4, arguing for different molecular mechanisms of JAK/STAT modulation by PTP1B∆6 and PTP1B∆2–4. Collectively, these data indicate that PTPN1 variants missing one or more exon sequences originated either from alternative splicing or from gene mutation, create PTP1B gain-of-function variants with oncogenic potential by augmenting JAK/STAT signalling in cHL.

Trabectedin overcomes doxorubicin-resistance, counteracts tumor-immunosuppressive reprogramming of monocytes and decreases xenograft growth in Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) tumor cells are surrounded by a protective tumor microenvironment (TME). Trabectedin, an anticancer drug targeting both tumor cells and TME, demonstrated a potent antitumor activity against Hodgkin Reed Sternberg (HRS) cells. It was cytotoxic against cHL cell lines, including the doxorubicin-resistant clones, with subnanomolar IC50 values, and inhibited clonogenic growth and heterospheroid cell viability. It induced necroptosis, caused DNA damage, G2/M cell cycle arrest, and increased reactive oxygen species production. It reduced HRS cell secretion of CCL5, M-CSF, IL-6, IL-13 and TARC, and inhibited migration. Conditioned medium from trabectedin-treated HRS cells was less chemoattractive toward monocytes, mesenchymal stromal cells and lymphocytes, and less effective in educating monocytes to become immunosuppressive macrophages. These monocytes expressed lower levels of indoleamine 2,3-dioxygenase-1, CD206 and PD-L1, secreted lower amounts of IL-10, TARC, and TGF-β, and were less able to inhibit the growth of activated lymphocytes. In vivo, trabectedin inhibited by >75% the growth of cHL murine xenografts with minimal weight loss; tumors of trabectedin-treated mice had fewer TAMs and less angiogenesis. Altogether, this study offers a preclinical rationale for trabectedin use as a new drug candidate in relapsed/refractory cHL patients.

Targeting DNA Repair to Overcome Drug Resistance in Hodgkin Lymphoma

Classical Hodgkin lymphoma (cHL) is characterized by rare tumor-initiating Hodgkin and Reed-Sternberg cells (HRS) surrounded by a microenvironment with a reactional immune cells infiltrate. cHL accounts for 15% to 25% of all lymphomas. This neoplasm is curable in the majority of cases with chemotherapy including ABVD (Doxorubicin, Bleomycin, Vinblastine, Dacarbazine) or BEACOPP (Bleomycin, etoposide, Doxorubicin, cyclophosphamide, Vincristine, procarbazine and Prednisone) and/or radiation. However, 15-20% of high-risk patients ultimately relapse and high dose chemotherapy in combination with autologous stem-cell transplantation is successful in only half of the patients with relapsed/refractory cHL (Eichenaueret al., Eur. Soc. Med. Oncol,2018). HRS cells are characterized by genetic instability, abnormal DNA damage response and repair that may play a role in drug resistance in high-risk cHL (Reichelet al., Blood,2015, Monroyet al. Mol. Carcinog,2011). In this work, we hypothesized that inhibiting DNA repair mechanisms using small molecules might represent a promising strategy to overcome drug resistance to genotoxic agents in cHL, such as cyclophosphamide and topoisomerase II inhibitors (O'Connor,Mol. Cell, 2015; Shaheenet al., Blood 2011). We characterized the drug-response of 4 cHL cell lines to DNA repair inhibitors including PJ34 (PARP inhibitor), NU7441 (DNAPK inhibitor), KU55933 (ATM inhibitor), PF477736 (CHK1 inhibitor), AZD6738 (ATR inhibitor), AZD1775 (Wee1 inhibitor), MP-470 (Rad51 inhibitor) and genotoxic agents used in standard chemotherapy (cyclophosphamide, doxorubicin and etoposide). We showed that DNA repair inhibitors targeting ATR, CHK1 or Wee1 significantly induced apoptosis of cHL cell lines (AnnexinV staining, p&amp;lt;0.05), significant inhibition of proliferation (BrdU incorporation). ATR inhibitor induces a significant accumulation of cHL cells in G0/G1 phase (p&amp;lt;0.05) whereas CHK1 and Wee1 inhibitors induced a significant accumulation in G2/M phase (p&amp;lt;0.01). Moreover, ATR, CHK1 and Wee1 inhibitors induced double strand breaks monitored by 53BP1 foci formation, γH2A.X staining (p&amp;lt;0.01) and chromosomic instability characterized by a higher frequency of micronucleation (p&amp;lt;0.05) and nucleoplasmic bridges formation (p&amp;lt;0.01). Since DNA repair pathways play a role in drug resistance, we sought to identify new synthetic lethal and synergistic combinations associating IC20 DNA repair targeted treatments with conventional genotoxic agents in cHL. Applying a standard threshold of 2 SDs below the IC50 of the genotoxic agent alone (Srivaset al., Mol. Cell, 2016), a total of two synthetic lethal combinations in cHL cells have been identified: CHK1 inhibitor (PF477736) combined with a Wee1 inhibitor (AZD1775) (IC50 reduced from 5.00 µM to 1.75 µM for L-428 and from 4.81 µM to 1.86 µM for KMH2) and cyclophosphamide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 3.26 µM to 1.12 µM for L-428 and from 1.27µM to 0.10µM for KMH2). In addition, we identified 3 synergistic/additive combinations (combination index &amp;lt; 1): doxorubicin combined with a DNAPK inhibitor (NU7441) (IC50 reduced from 44 nM to 22 nM for L-428 and from 27 nM to 8 nM for KMH2), cyclophosphamide combined with CHK1 inhibitor (PF477736) (IC50 reduced from 2.9 µM to 2.4 µM for L-428 and from 1.2µM to 0.9µM for KMH2) and etoposide combined with an ATR inhibitor (AZD6738) (IC50 reduced from 420 nM to 195 nM for KMH2). The treatment of Hodgkin's lymphoma is successful for a majority of patients. However, patients with advanced-stage or high-risk disease are only cured in ∼70% of cases. Alternative treatment options for the subsets of patients for whom first- or second-line therapies fail are needed. These results open new perspectives to improve the treatment of relasped/refractory cHL patients and provide new strategies to overcome drug resistance. Disclosures Moreaux: Diag2Tec:Consultancy.

An Integrative Unbiased Global Analysis of the Metabolic Adaptive Response to Tryptophan Deprivation in Classical Hodgkin Lymphoma

Background Tryptophan (trp) is an essential aminoacid, required for de novo NAD+ synthesis. Our previous work showed that the microenvironment in classical Hodgkin Lymphoma is characterized by dysfunctional neutrophils and myeloid derived suppressor cells that produce a trp-degrading enzyme indoelamine deoxygenase (IDO-1), lowering the amount of trp. Aims To detect metabolomics changes in human cHL cell lines exposed in vitro to tryptophan deprivation, an amino acid involved in immune dysregulation and generation of anergic and tolerogenic T- cells. Methods In order to better understand the impact of extra-cellular IDO1 increase on the metabolome of human cHL cells, three human cHL cell lines (L428, L540 and KM-H2) were individually cultured with customized complete media or media lacking tryptophan (W0), +10% dyalised fetal bovum serum, in six independent experiments. After 48 hours of culture the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc. Following normalization to DNA concentration, log transformation and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups (Table 1). Results The lack of tryptophan in media had a profound effect on the cell metabolome in 2 cell lines, KMH2 and L428 cells, while L540 cell line was pretty resilient (Table 1). In all cell lines, the removal of tryptophan from the media resulted in significantly lower levels of tryptophan. Kynurenine, the metabolic product of IDO-1 action on tryptophan, was lower in all cells, but did not reach significance in the L540 cells, whereas it trended lower in L428 and was significantly lower in the KMH2 compared to controls. Indolelactate, another major tryptophan metabolite was also significantly lower in the L428 and KMH2 cells lines compared to controls. Glucose uptake and aerobic glycolysis are frequently upregulated in tumor cells to support energy needs and provide biosynthetic precursors (e.g. pentose phosphate pathway intermediates for nucleotide synthesis). Known as the Warburg effect, this process of reliance on glucose for energy results in high levels of lactate production. We found that trp deficiency lead to lower levels of the hexose diphosphates (fructose 1,6-diphosphate/glucose 1,6-diphosphate/myo-inositol diphosphates isobar) and dihydroxyacetone phosphate, suggesting the revert of Warburg effect due to reduced bio-energetic requirements for proliferation. In line with this observation, culture in trp deficient media resulted in increased levels of long chain saturated fatty acids and long chain polyunsaturated fatty acids (PUFAs), suggesting that specific-amino acid deficiency leads to an increase in uptake of free fatty acids from the media, to preserve membrane dynamics. Since prolonged trp deprivation (up to 10 days) delayed cell cycle length without affecting proliferation or changes in intracellular amount of NAD+, we investigated changes in mitochondrial membranes network to explain these findings. Trp deprivation induced the rearrangement of the mitochondrial network at 48 hours, with more fission than fusion, as suggested by increased expression of Fis1 and Drp1 and reduced expression of Tfam and Opa1, without affecting significantly mitochondrial mass and depolarization. This adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the L540 and KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine). Conclusions The removal of trp from L428 and KMH2 resulted in changes in the specific-amino acid related metabolites. The adaptive response to trp-depleted microenvironment can revert the Warburg effect, promoting a shift in the glucose use in the futile attempt to preserve mitochondrial function, and increase oxidative stress. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. Disclosures Puglisi: Amgen:Honoraria.Di Raimondo:Takeda:Consultancy, Honoraria;GILEAD, Incyte:Research Funding;Amgen, Takeda, Novartis:Honoraria;Celgene:Consultancy, Honoraria;Janssen:Consultancy, Honoraria;GSK:Consultancy, Honoraria;Amgen:Consultancy, Honoraria.Romano:Takeda:Honoraria;Novartis:Honoraria.

The Tumor Suppressive mir-148a Is Epigenetically Inactivated in Classical Hodgkin Lymphoma.

DNA methylation was shown previously to be a crucial mechanism responsible for transcriptional deregulation in the pathogenesis of classical Hodgkin lymphoma (cHL). To identify epigenetically inactivated miRNAs in cHL, we have analyzed the set of miRNAs downregulated in cHL cell lines using bisulfite pyrosequencing. We focused on miRNAs with promoter regions located within or <1000 bp from a CpG island. Most promising candidate miRNAs were further studied in primary Hodgkin and Reed-Sternberg (HRS) cells obtained by laser capture microdissection. Last, to evaluate the function of identified miRNAs, we performed a luciferase reporter assay to confirm miRNA: mRNA interactions and therefore established cHL cell lines with stable overexpression of selected miRNAs for proliferation tests. We found a significant reverse correlation between DNA methylation and expression levels of mir-339-3p, mir-148a-3p, mir-148a-5p and mir-193a-5 demonstrating epigenetic regulation of these miRNAs in cHL cell lines. Moreover, we demonstrated direct interaction between miR-148a-3p and IL15 and HOMER1 transcripts as well as between mir-148a-5p and SUB1 and SERPINH1 transcripts. Furthermore, mir-148a overexpression resulted in reduced cell proliferation in the KM-H2 cell line. In summary, we report that mir-148a is a novel tumor suppressor inactivated in cHL and that epigenetic silencing of miRNAs is a common phenomenon in cHL.