KM-H2 Cells Research Articles

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.

Integrating network pharmacology and experimental studies for uncovering the molecular mechanisms of Dioscorea bulbifera L. in the treatment of thyroid cancer

IntroductionThyroid cancer (TC), a common endocrine malignant tumor with a higher incidence in females than in males, is a serious threat to human life and health. Although current clinical treatments can alleviate this disease, the recurrence rate remains high. Tuber Dioscoreae Bulbiferae, also called Huang-Yao-Zi (HYZ), has remarkable curative properties, few side effects and is used for the treatment of sore throat, goiter, hemoptysis, and other diseases in traditional Chinese medicine (TCM). Existing clinical studies have found that HYZ can improve the clinical symptoms of TC patients and reduce tumor volume, while in vitro studies have found that HYZ can induce the death of thyroid cancer cells. However, the mechanism of HYZ in the treatment of TC is still unclear. MethodsIn this study, based on network pharmacology and bioinformatics, the target and molecular mechanism of HYZ in the treatment of TC were preliminarily explored. The results suggest that the antithyroid cancer effect of HYZ may be related to the PI3K-Akt and focal adhesion pathways. Then, a TC cell model was established to further explore the detail molecular mechanisms. ResultsFortunately, HYZ induced apoptosis in KMH-2 cells and regulated the expression of apoptosis-related proteins and genes. At the same time, HYZ can also significantly inhibit the migration and invasion of TC cells. Further studies showed that the pharmacological activities of HYZ were related to the regulation of the PI3K-Akt and focal adhesion pathways. ConclusionOur study provides a reference for further animal or clinical studies investigating the effectiveness and molecular mechanisms of HYZ against thyroid cancer.

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.

Heparanase Expression Is Associated With Cancer Stem Cell Features and Radioresistance in Hodgkin's Lymphoma Cells.

Heparanase (HPSE) is relevant to therapy resistance in many malignancies yet is largely unstudied in Hodgkin's lymphoma. Here, we investigated links between HPSE, cancer stem cell (CSC) features and radioresistance in KM-H2 and L428 Hodgkin's lymphoma cells. Firstly, HPSE expression in unsorted and sorted CSCs was assessed. Post-irradiation, HPSE and CSC-related gene expression changes were then quantified. Clonogenic ability was investigated with and without artificial changes in HPSE expression pre and post irradiation. HPSE was highly expressed in L428 but barely present in KM-H2 cells. HPSE was overexpressed in sorted L428 CSCs. Irradiation induced HPSE and expression of CSC markers. High HPSE-expressing L428 cells showed higher clonogenic ability than low HPSE-expressing KM-H2 cells after irradiation. Down-regulation of HPSE in L428 cells reduced their clonogenic capability post-radiation, whilst overexpression of HPSE in KM-H2 cells increased colony formation. HPSE expression is associated with CSC features and contributes to radioresistance in Hodgkin's lymphoma cells.

RNA Sequencing Reveals Mechanisms Underlying Modulation of Hodgkin Lymphoma Cells and Tumor Microenvironment By the PI3K δ/γ Inhibitor, RP6530

INTRODUCTION: Aberrant activation of the phosphoinositide 3-kinase (PI3K) pathway plays a central role in Hodgkin Lymphoma (HL) pathogenesis. The pleiotropic activities of the δ and γ isoforms of PI3K in regulating the interactions of Hodgkin/Reed-Sternberg (HRS) cells with the tumor microenvironment suggest thatPI3K inhibitors might exert a therapeutic potential in the treatment of HL. The dual PI3K δ/γ inhibitor RP6530 was proven to affect HL tumor cells and to exert immunosuppressive activities. However, a genome-wide search for molecular mediators of RP6530 is largely unexplored in HL. The present study was therefore aimed at evaluating the anti-lymphoma functions and the underlying genes affected by RP6530 in HL cell lines.METHODS: We identified differentially expressed genes (DEGs) in two HL cell lines (L-540 and KM-H2) treated with RP6530 for 6 and 24 h by next generation RNA sequencing (RNA-Seq). The expression level of some of these genes was verified in HL cell lines using real time-PCR. Ingenuity Pathway Analysis (IPA) software was used to assign the DEGs to distinct canonical pathways and to predict the biological functions of the transcriptional changes. Gene silencing experiments were used to investigate and validate potential predictive biomarkers of response to RP6530 treatment. Additionally, serum levels of Thymus- and activation-regulated chemokine (TARC/CCL17) were analyzed by commercially available sandwich enzyme-linked immunosorbent assay (ELISA) in RP6530-treated HL patients enrolled in a phase 1 study.RESULTS: RNA-Seq analysis following treatment of L-540 cells for 6 h caused an upregulation of 1544 and downregulation of 839 genes. After a 24 h treatment period, 625 genes remained upregulated while the number of genes that were downregulated increased to 1362 (FDR ≤ 0.1). In KM-H2 cells, treatment with RP6530 for 6 or 24 h upregulated 137 and 980 genes while downregulating 139 and 833 genes, respectively (FDR ≤ 0.1). We further classified modulated genes according to their canonical pathways using the IPA suite. The most significantly upregulated pathways detected in both L-540 and KM-H2 cell lines included apoptosis, p53, death receptor signaling and G1/S cell cycle checkpoint regulation. In contrast, PI3K/AKT, ERK/MAPK, JAK/Stat, CD40, IL-4, IL-6, IL-10 and iNOS signaling were downregulated. We also identified 118 downregulated genes shared between the two cell lines that were associated with glycolysis, HIF1, chemokine and cytokine signaling. Upon RP6530 incubation, RNA-seq analysis showed a strikingly reduced expression of Pyruvate kinase muscle isozyme M2 (PKM2), which is a limiting glycolytic enzyme that catalyzes the final step in glycolysis. Here we show that lactic acid produced by tumor cells, as a by-product of glycolysis, has a critical function in inducing the M2-like polarization of tumor-associated macrophages (TAMs). Furthermore, we demonstrated that this effect was mediated by PKM2 and was inhibited after RP6530 treatment.We also found that the lactate-induced expression of M2 markers by macrophages, i.e., CCL-17 and CCL-22, were markedly inhibited both by RP6530 [-30% and -50% (mean of HL cells), respectively] and PKM2 silencing [-50% and -30% (mean of HL cells), respectively]. However, RP6530 increased the expression of M1 markers, switching the activation of macrophages from an immunosuppressive M2-like phenotype to an inflammatory M1-like state. PKM2 pathway therefore appears to be a critical determinant of RP6530-mediated inhibition of M2 macrophage polarization. Finally, RP6530 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines and angiogenic factors (including, TARC/CCL17, CCL22, CCL5, IL-7, IL-13, and VEGF) known to be secreted by HRS cells to promote a pro-tumoral microenvironment. As the chemokine TARC/CCL17 is considered a biomarker of disease activity in HL patients, we observed a severe reduction of serum TARC/CCL17 levels in RP6530-treated HL patients that achieved CR or PR, but not in PD patients. Collectively, these findings suggest that RP6530 affects a mechanism of communication between HL tumor cells and tumor microenvironment.CONCLUSIONS: Results demonstrate that RP6530 can affect both HL tumor cells and HL tumor microenvironment, suggesting that a novel therapeutic opportunity may be achievable for the treatment of HL patients. DisclosuresViswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Santoro:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees. Carlo-Stella:Boehringer Ingelheim: Consultancy, Honoraria; Rhizen Pharmaceuticals: Research Funding; ADC Therapeutics: Research Funding.

Investigating the Thioredoxin and Glutathione Systems' Response in Lymphoma Cells after Treatment with [Au(d2pype)2]CL.

Lymphoma is a blood cancer comprising various subtypes. Although effective therapies are available, some patients fail to respond to treatment and can suffer from side effects. Antioxidant systems, especially the thioredoxin (Trx) and glutathione (GSH) systems, are known to enhance cancer cell survival, with thioredoxin reductase (TrxR) recently reported as a potential anticancer target. Since the GSH system can compensate for some Trx system functions, we investigated its response in three lymphoma cell lines after inhibiting TrxR activity with [Au(d2pype)2]Cl, a known TrxR inhibitor. [Au(d2pype)2]Cl increased intracellular reactive oxygen species (ROS) levels and induced caspase-3 activity leading to cell apoptosis through inhibiting both TrxR and glutathione peroxidase (Gpx) activity. Expression of the tumour suppresser gene TXNIP increased, while GPX1 and GPX4 expression, which are related to poor prognosis of lymphoma patients, decreased. Unlike SUDHL2 and SUDHL4 cells, which exhibited a decreased GSH/GSSG ratio after treatment, in KMH2 cells the ratio remained unchanged, while glutathione reductase and glutaredoxin expression increased. Since KMH2 cells were less sensitive to treatment with [Au(d2pype)2]Cl, the GSH system may play a role in protecting cells from apoptosis after TrxR inhibition. Overall, our study demonstrates that inhibition of TrxR represents a valid therapeutic approach for lymphoma.

Melittin Increases Cisplatin Sensitivity and Kills KM-H2 and L-428 Hodgkin Lymphoma Cells.

Hodgkin lymphoma (HL) is neoplasia with high cure rates. However, not all patients can be cured with the current treatment. Chemo-resistance of tumor cells is one factor involved in treatment failure. In addition to its pore-forming activity on lipid bilayer membranes, the toxin melittin from bee venom is an inhibitor of several cancer-related signaling pathways. Moreover, melittin analogs have been shown to inhibit the activity of ATP binding cassette (ABC) transporters which are known to play important roles in the chemo-resistance of tumor cells. Therefore, we tested the toxicity of melittin for HL cell lines KM-H2 and L-428 and whether melittin can increase the chemo-sensitivity of cisplatin-resistant HL cells. We found high toxicity of melittin for KM-H2 and L-428 cells. In co-cultures with normal blood cells, melittin preferentially killed KM-H2 and L-428 cells. In addition, we observed increased cisplatin sensitivity of chemo-resistant L-428 cells after treatment with melittin. ABC transporter activity was not reduced after treatment with melittin. Our data suggest that melittin or melittin analogs might be promising agents for the future development of treatment strategies for HL patients with resistant disease.

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.

Luteolin Induces Cytotoxicity in Mix Cellularity Classical Hodgkin's Lymphoma via Caspase Activated-cell Death.

We investigated the effects of luteolin (LUT) on classical Hodgkin's lymphoma (cHL), since such studies in malignant lymphomas are lacking. Effect of LUT on cell growth was assessed with water-soluble tetrazolium 1 (WST-1) cell proliferation assay and automated hemocytometry on trypan blue-exclusion assay. Cell death was investigated with acridine orange/ethidium bromide live-dead assay, propidium iodide (PI) flow cytometry, and Annexin-V-PI microscopy. Caspase activation was studied using CellEvent Caspase-3/7 Green detection reagent. High resolution immunofluorescence microscopy was used to detect cleaved-PARP-1. LUT induced a dose-dependent decrease in the growth of KMH2 and L428 cells, cellular models of mix-cellularity (MC) and nodular sclerosis (NS) cHL, respectively. However, LUT induced cell death only in KMH2, at a higher concentration, and this was associated with caspase activation and cleaved PARP-1. LUT induces cytotoxicity in the MC-cHL cellular model KMH2 via caspase activation.

CASK, the Soluble Glomerular Permeability Factor, Is Secreted by Macrophages in Patients With Recurrent Focal and Segmental Glomerulo-Sclerosis.

Introduction: Focal and segmental glomerulosclerosis (FSGS) is a frequent form of glomerulonephritis that may be caused by a soluble permeability factor and regulated by the immune system. We previously described a soluble form of calcium/calmodulin-dependent serine/threonine kinase (CASK) acting as a permeability factor in patients with recurrent FSGS (rFSGS). Here, we aimed to identify the immune cells associated with CASK secretion in patients with rFSGS.Methods: FACS, western blotting and immunoprecipitation were performed to detect CASK in peripheral blood mononuclear cells, including CD3+, CD20+, and CD14+subsets, from patients with rFSGS, healthy donors, transplant patients and patients with nephrotic syndrome due to diabetes mellitus, and in KHM2 cells.Results: CASK was produced mostly by monocytes in patients with rFSGS but not by T or B lymphocytes. It was not detectein cells from control patients. CASK was also produced and secreted by M2 polarized macrophages and KMH2 cells, but not by M1 polarized macrophages. CASK secretion was not not inhibited by brefeldin A, suggesting an absence of classical secretion pathway involvement. Within cells, CASK was partly colocalized with ALIX, a molecule involved in exosome development, and these two molecules were coprecipitated from M2 macrophages. Moreover, exosomes derived from M2 macrophages induced podocyte cytoskeleton alterations and increased podocyte motility.Conclusion: These results suggest that the soluble permeability factor CASK is secreted by monocytes and M2 macrophages, via exosomes, to alter the glomerular filtration barrier in rFSGS.

Lysosomal P-gp-MDR1 Confers Drug Resistance of Brentuximab Vedotin and Its Cytotoxic Payload Monomethyl Auristatin E in Tumor Cells.

Antibody-drug conjugates (ADCs) are composed of an antibody linked to cytotoxic anticancer payloads. ADCs recognize tumor-specific cell surface antigens and are internalized into lysosomes where proteolytic enzymes release the cytotoxic payloads. Efflux transporters on plasma membrane that play a significant role on multi-drug resistance in chemotherapy can be internalized on lysosomal membrane and sequester the cytotoxic payloads. In the present study, ATP binding cassette (ABC) efflux transporters including breast cancer resistance protein (BCRP), P-glycoprotein (P-gp-MDR1), multidrug resistance protein (MRP) 2, MRP3 and MRP4 in lysosomal, and plasma membrane of tumor cells were quantified by targeted quantitative proteomics. The cytotoxicity of brentuximab vedotin and its cytotoxic payload monomethyl auristatin E (MMAE) to the tumor cell lines in the presence and absence of elacridar (P-gp-MDR1 inhibitor) or chloroquine (lysosomotropic agent) were evaluated. MMAE is a substrate for P-gp-MDR1, as the apparent efflux ratio in MDR1 transfected MDCK cell monolayers was 44.5, and elacridar abolished the MMAE efflux. Cell lines that highly express P-gp-MDR1 show higher EC50s toward the cell killing effects of MMAE. Co-incubation with chloroquine or elacridar resulted in left shift of MMAE EC50 by 2.9–16-fold and 4.2–22-fold, respectively. Similarly co-incubation with chloroquine or elacridar or in combination of chloroquine and elacridar increased cytotoxic effects of brentuximab vedotin by 2.8- to 21.4-fold on KM-H2 cells that express a specific tumor antigen CD30 and P-gp-MDR1. These findings demonstrate important roles of P-gp-MDR1 on cytotoxic effects of brentuximab vedotin and its payload MMAE. Collectively, ABC transporter-mediated drug extrusion and/or sequestration needs to be early assessed for selection of optimal payloads and linkers when developing ADCs.

Targeting Hodgkin and Reed-Sternberg Cells with an Inhibitor of Heat-Shock Protein 90: Molecular Pathways of Response and Potential Mechanisms of Resistance.

Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed–Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol’s toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research.

Abstract 2716: A multi-parameter assay for the detection of rare malignant cells in classical Hodgkin lymphoma

Abstract Background: Classical Hodgkin Lymphoma (CHL) ranks among the most frequently occurring lymphomas and is marked by Hodgkin and Reed-Sternberg (HRS) cells, frequently multi-nucleated neoplastic cells that are derived from germinal center B cells. The genetic alterations involved in the pathogenesis of this lymphoma have been difficult to study because HRS cells are rare and are surrounded by robust, non-neoplastic infiltrate, therefore they frequently represent less than 0.1% of cells within involved lymph nodes. Consequently, methods that isolate these rare cells are required to understand pathobiology of this lymphoma. We used the AccuCyte® - CyteFinder® system (RareCyte) to develop a method for detection and isolation of HRS cells. Methods: A 6-parameter immunofluorescence assay was designed to identify HRS cells that included HRS markers (CD40, CD30, CD15), CD3, combined CD20/CD14 and a cell membrane permeability viability dye. The assay was tested using the KMH2 Hodgkin lymphoma cell line spiked into PBMC and transferred to well slides for imaging by CyteFinder. This instrument utilizes 6-color scanning microscopy coupled with predefined algorithms to identify rare cell populations. The assay was then applied to a frozen fine needle aspirate (FNA) sample taken from the lymph node of a patient with histologically confirmed CHL. The FNA sample was thawed, prepared into a suspension, stained and analyzed as above. Cells identified with HRS phenotype were individually retrieved using the integrated CytePicker module. PCR of framework regions of the immunoglobulin heavy chain gene (IGH) was performed on single HRS cells (InvivoScribe) to assess clonality. Results: Of ~200 KMH2 cells spiked into blood, the assay identified 180 cells (90%) with the correct HRS phenotype. HRS phenotype cells were identified in the patient lymph node FNA sample. Two individual cells analyzed by IGH framework region PCR demonstrated identical size PCR products, indicating clonality of the B cell receptor rearrangement. Conclusion: A multi-parameter assay was successfully developed for identifying rare HRS cells with the CyteFinder system. Cells with HRS phenotype in a lymph node FNA sample were demonstrated to have clonal IGH rearrangements, demonstrating that actual HRS cells were identified. This method may be a useful tool for the diagnosis of CHL by identification of rare HRS cells from FNA specimens, as well as to investigate the molecular genomics of CHL. Citation Format: Lance U'Ren, David Wu, Jonathan R. Fromm, Jackie Stilwell, Eric Kaldjian. A multi-parameter assay for the detection of rare malignant cells in classical Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2716. doi:10.1158/1538-7445.AM2017-2716

Characterization of Alstrom Syndrome 1 (ALMS1) Transcript Variants in Hodgkin Lymphoma Cells.

The Alstrom syndrome gene (ALMS1) is one of the largest disease associated genes identified today in the human genome and is implicated in cell cycle control, ciliogenesis, endosome recycling and intracellular transport mechanisms. ALMS1 mutations cause Alstrom syndrome, a rare genetic disorder. However, its function is not completely understood. DNA microarray analysis suggested that ALMS1 might be differentially expressed between Hodgkin lymphoma (HL) cells and normal tissues. By using reverse transcription-polymerase chain reaction (RT-PCR) we detected low but variable expression of ALMS1 in HL cell lines with highest expression in KM-H2 cells. Immunofluorescence indicated centrosomal accumulation of ALMS1 protein in HL cells. Knock-down of ALMS1 in KM-H2 cells had no impact on viability or cytotoxic drug sensitivity of these cells. Sequencing of RT-PCR products from HL cell lines identified three variable regions in ALMS1 transcripts that affect exons 2, 13, and 23. One of these variants was characterized by splicing out of exon 13. The other variants are characterized by two alternative 5 prime ends or alternative 3 prime ends. Structure prediction of the corresponding RNAs and proteins suggest that the different transcript variants might affect posttranscriptional regulation and ligand binding.

Treatment of Hodgkin Lymphoma Xenografts with the Novel PI3K δ/γ Inhibitor RP6530 Suppresses M2 Macrophage Polarization and Results in Potent Antitumor and Antiangiogenic Effects

INTRODUCTION: Primary refractory and early-relapsed patients with Hodgkin lymphoma (HL) experience poor responses to salvage chemotherapy and dismal long-term disease control. Therefore, they represent an unmet medical need urgently requiring not only new diagnostic tools but also novel therapeutic agents to overcome the chemo-refractory phenotype.Tumor-associated macrophages (TAMs) have been implicated in the pathogenesis of HL and have been suggested to negatively impact clinical outcome. The phosphatidylinositol 3-kinase (PI3K) is a major effector pathway mediating tumor and endothelial cell survival and functions, as well as macrophage activation. As PI3Kδ and PI3Kγ are constitutively activated in several hematologic malignancies resulting in cancer development and progression, we hypothesize that the novel dual PI3K δ/γ inhibitor,RP6530, might represent a unique therapeutic opportunity for HL patients. This study aimed at investigating the activity and mechanism(s) of action of RP6530 in preclinical HL models.METHODS: The effects of RP6530 on HL cell lines (L-540, KM-H2, L-428) were investigated by assaying cell proliferation, cell death, and gene expression profiling. Upon treatment with RP6530, human macrophages co-cultured with HL cell lines were assessed by flow cytometry for cell death and expression of M1/M2 macrophage markers. The antitumor efficacy and mechanism(s) of action of RP6530 were also analyzed in NOD/SCID mice bearing HL cell line xenografts. Tumor vasculature was analyzed by in vivo biotinylation of vascular endothelial proteins.RESULTS: Exposure of HL cell lines to RP6530 (1.25 - 10 µM) induced a marked, early, and dose-dependent dephosphorylation of PI3K/Akt and MAPK pathways that was associated with inhibition of cell proliferation (L-540: 40 ± 4%, KM-H2: 33 ± 5% and L-428 30 ± 5%, mean ± SEM) and S phase cell cycle arrest (3-fold reduction, compared to vehicle-controls). Significant levels of caspase-dependent cell death were observed in L-540 (20µM: 61 ± 5 vs. 15 ± 2%), KM-H2 (20µM: 41 ± 4 vs. 12 ± 1%) and L-428 (20µM: 31± 4% vs. 12 ± 1%) cells that were associated with mitochondrial depolarization (up to 40%). Interestingly, microarray gene expression analysis of KM-H2 cell line showed that a 24-hour treatment with RP6530 decreased the amplitude of several gene signatures, including “PI3K signaling in B lymphocytes”, “Tumor cell proliferation”, “Tumor angiogenesis”, and “Macrophages activation”, while genes involved in positive regulation of cell death were upregulated. In KM-H2 co-culture, besides observing cell death of human macrophages (up to 40% following a 24-hour exposure), we found that RP6530 inhibited M2 macrophage differentiation, as suggested by decreased CD209 and CD163 expression, and increased M1 macrophage differentiation by upregulating CD40 and CD80 expression. Compared to vehicle-controls, analysis of RP6530-treated tumor xenografts showed a marked reduction of TAM (-44% and -50%, P ≤.0001 in L-540 and KM-H2, respectively), as well as tumor vessel density (average decrease: 80%), consistent with the role of PI3K signaling in the macrophage-dependent promotion of tumor growth and tumor-induced angiogenesis. This finding was paralleled by the histological detection of tumor vasculature disruption, and inhibition of phospho-Akt and -Erk on both endothelial and tumor cells. Finally, RP6530 treatment (150 mg/kg/BID/3wks) of NOD/SCID mice significantly reduced the growth of KM-H2 nodules (-60%, P ≤.0001) compared to vehicle-controls. Reduction of tumor growth was associated with a strong decrease in Ki-67 expression by tumor cells, increase in tumor apoptosis (1.2- and 12-fold in L-540 and KM-H2, respectively) and tumor necrosis (average increase: 7-fold, P≤.0001).CONCLUSIONS: These results demonstrate that the therapeutic potential of RP6530 in HL is likely due to a reduction of M2 macrophages polarization and inhibition of tumor angiogenesis. Our findings warrant further investigation in future clinical trials. DisclosuresViswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals: Employment, Equity Ownership. Santoro:Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; ArQule: Membership on an entity's Board of Directors or advisory committees. Carlo-Stella:Boehringer Ingelheim: Consultancy; Rhizen Pharmaceuticals: Research Funding.

Restoration of Brain Acid Soluble Protein 1 Inhibits Proliferation and Migration of Thyroid Cancer Cells.

Background:Brain acid soluble protein 1 (BASP1) is identified as a novel potential tumor suppressor in several cancers. However, its role in thyroid cancer has not been investigated yet. In the present study, the antitumor activities of BASP1 against the growth and migration of thyroid cancer cells were evaluated.Methods:BASP1 expression in thyroid cancer tissues and normal tissues were examined by immunohistochemical staining and the association between its expression and prognosis was analyzed. pcDNA-BASP1 carrying full length of BASP1 cDNA was constructed to restore the expression of BASP1 in thyroid cancer cell lines (BHT-101 and KMH-2). The cell proliferation in vitro and in vivo was evaluated by WST-1 assay and xenograft tumor models, respectively. Cell cycle distribution after transfection was analyzed using flow cytometry. Cell apoptosis after transfection was examined by annexin V/propidium iodide assay. The migration was examined using transwell assay.Results:BASP1 expression was abundant in normal tissues while it is significantly decreased in cancer tissues (P = 0.000). pcDNA-BASP1 restored the expression of BASP1 and significantly inhibited the growth of BHT-101 and KMH-2 cells as well as xenograft tumors in nude mice (P = 0.000). pcDNA-BASP1 induced G1 arrest and apoptosis in BHT-101 and KMH-2 cells. In addition, pcDNA-BASP1 significantly inhibited the cell migration.Conclusions:Downregulation of BASP1 expression may play a role in the tumorigenesis of thyroid cancer. Restoration of BASP1 expression exerted extensive antitumor activities against growth and migration of thyroid cancer cells, which suggested that BASP1 gene might act as a potential therapeutic agent for the treatment of thyroid cancer.

Mutations in CD58 and Other Immune System Related Genes in Hodgkin Lymphoma

Hodgkin Lymphoma (HL) is a B cell derived malignancy characterized by a minority of tumor cells, known as Hodgkin Reed-Sternberg (HRS) cells. The background is composed of a wide variety of inflammatory cells with T cells representing the largest population. Chemokines and cytokines produced by HRS cells and by the infiltrating cells shape the environment and provide proliferative and survival signals to the HRS cells. Despite this critical dependence on the microenvironment, HRS cells also need to apply mechanisms to escape from both antigen-dependent and innate immune responses. HRS cells have evolved multiple mechanisms to evade cytotoxic T cell (CTL) and natural killer (NK) cell mediated anti-tumor responses. These mechanisms include secretion of immune-suppressive factors (IL10, TGFβ and others), recruitment of regulatory and helper T cells, expression of PDL1 and CD95 and loss of HLA expression. Recent publications show that mutations in immune system related genes might represent a mechanism of HRS cells to evade detection by immune cells. The aim of this study was to validate whole exome sequencing results of seven HL cell lines focusing on immune system associated genes. We previously showed that B2M mutations affect the ATG start codon in L428 (heterozygous) and DEV (homozygous) cells. B2M mRNA levels were reduced in both cell lines as compared to L1236, whereas HLA-A, HLA-B and HLA-C mRNA levels were in the same range. Consistent with these findings we observed no membranous B2M and HLA class I expression by flow cytometry in the two cell lines with mutated B2M genes. In primary diagnostic HL tissue we showed lack of membranous B2M in 51% of the cases. We now studied two additional genes in more detail. CD58 gene mutations were observed in KMH2 and DEV cells. By manual inspection of the alignments using the Integrative Genomics Viewer (IGV), we also noticed a lack of reads of exons 1, 2 and 3 in SUPHD1. Heterozygous mutations and homozygous loss of exons 1-3 were confirmed for all three cell lines. CD58 mRNA levels were low or absent in SUPHD1 and KMH2 cells and normal in DEV. CD58 protein expression as determined by flow, western blot and IHC was low or absent in all 3 mutated HL cell lines in comparison to four cell lines with wild type CD58. Tumor cells of 36 primary HL cases with good treatment outcome showed a strong CD58 expression in all cases. As HL cell lines are derived from end stage HL patients, we next studied CD58 expression in relapsed HL patients. No or weak CD58 staining was observed in HRS cells in 6 out of 45 patients who experienced a relapse. Our results indicate that mutations in CD58 and loss of CD58 expression are common in HL derived cell lines and that loss of CD58 expression in tumor cells is restricted to relapsed HL patients. Heterozygous CSF2RB mutations in KMH2, SUPHD1, DEV and L1236 were validated by RNA-seq and Sanger-seq. As CSF2RB encodes the common β chain (CD131) shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, we also measured the expression of these 3 α chain receptors. We observed the same expression pattern between CD131 and CD116 (GM-CSF α receptor chain) in HL cell lines by flow cytometry suggesting that these mutations mainly affect the GM-CSF receptor. In conclusion, we show that mutations of immune system genes are common in HL. Deleterious mutations in B2M explain the lack of HLA class I expression, indicating that this genetic alteration is responsible for defective antigen presentation. Deleterious mutations or deletions of CD58 exons result in loss of CD58 protein expression. This will lead to loss of binding to CD2 expressed on T cells and will result in a defect in T cell adhesion and activation. Overall these results indicate that mutations are likely to contribute to the immune escape mechanisms applied by the HRS cells. DisclosuresNo relevant conflicts of interest to declare.

The First-in-Class Alkylating Histone-Deacetylase Inhibitor (HDACi) Fusion Molecule Edo-S101 Exerts Potent Preclinical Activity Against Tumor Cells of Hodgkin Lymphoma (HL) Including Bendamustine-Resistant Clones

Background: The highly unfavorable outcome of patients with recurrent HL, who progress after stem cell transplantation or are ineligible for such procedure make the development of new active agents an impellent medical need in this clinical setting. EDO-S101 is fusion molecule combining the DNA damaging effects of bendamustine (BDM) with the pan-histone deacetylase (HDAC) inhibitor, vorinostat. Given that BDM and HDAC inhibitors are active agents in recurrent HL we investigated the preclinical activity of EDO-S101 in this malignancy.Methods: We assessed the patterns of EDO-S101 cytotoxicity (0.39 to 50 µmol/L) in a panel of HL-derived cell lines (L1236, L428, KMH2, HDLM2, L540) and its regulatory effects on genes involved in DNA-damage/repair response, apoptosis and cell cycle checkpoints. As a further model we exploited an L1236 cell clone (R100) selected for resistance (R) to BDM through continuous exposure to increasing concentrations of the agent. R100 cells display a growth pattern indistinguishable from parental L1236 cells when cultured in the presence of BDM (100 µmol/L). Clonal identity of R100 cells with parental L1236 was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions.Results: EDO-S101 induced a significant time- and dose-dependent inhibition of growth and survival in all HL cell lines. L1236 cells displayed the highest sensitivity to the agent with an IC50, at 48 hrs, of 1.88 µmol/L, as opposed to KMH2, L428, L540 and HDLM2 cells with IC50 of 2.06, 2.53, 2.26 and 16.2 µmol/L, respectively. These values were about 10-fold lower than the IC50 of BDM in the same cell lines. While exposure of L1236 cells to EDO-S101 caused cell accumulation in S-phase, qRT-PCR disclosed that cell death was mainly dependent on triggering of apoptosis, as shown by the early (24 hrs) and sustained (48 hrs) upregulation of NOXA, p21 and p27 genes. Data were confirmed by the significant increase (>150%) of Annexin V-expressing L1236 cells. In contrast, expression levels of PLK1, AKA and cyclin B1 genes remained unchanged or were increased. This excluded induction of the mitotic catastrophe (MC) as a major determinant of cytotoxic activity for EDO-S101 in L1236 cells. Exposure to EDO-S101 induced a strong DNA stress/repair response as shown by the activation of pATR/pATM and increase of the downstream DNA damage checkpoint proteins pCHK1-/-2 and CCNB1, along with the upregulation of the EXO1 gene. Most intriguingly, BDM-resistant L1236 cells (R100) were highly sensitive to EDO-S101, with an IC50 of only 4.56 µmol/L, but less responsive to vorinostat (IC50: 6.17 µmol/L) than parental L1236 cells (IC50: 0.58 µmol/L). Differently from native cells, EDO-S101 induced a late downregulation of transcripts for PLK1 and AKA genes and of cyclin B1 gene and protein in R100 cells, along with the early induction of NOXA and p21, but not p27 genes. In both L1236 and R100 cells, expression of MC-genes was unaffected by exposure to vorinostat. This suggests a more complex mechanism for EDO-S101 in BDM-resistant HL cells involving activation of the both apoptotic and MC pathways. Notably, we documented that EDO-S101 corrected the constitutive ATM/ATR unbalance of R100 cells by triggering the early (24 hrs) upregulation of ATR and a late (48 hrs) downregulation of ATM transcripts and proteins, along with increased levels of EXO1 and MGMT at 24 hrs. Vorinostat induced a similar effect. Finally, while baseline expression levels of HDAC isoforms were comparable among HL cell lines, EDO-S101 caused a significant (>40%) late downregulation of transcripts for all HDAC isoforms (HDAC-1 to -8) in R100 cells but only of HDAC-6 in native L1236. This pattern diverged from results obtained in both L1236, i.e. increase of all HDAC isoform transcripts except HDAC-6, and R100 cells, i.e. upregulation of all isoforms and reduction of HDAC-6, with vorinostat and BDM as single agents.Conclusions: We have described for the first time that EDO-S101 is effective in preclinical models of HL including cells resistant to BDM. The combined functions of in one molecule of a bifunctional alkylator and panHDAC inhibitor confer this agent unique antitumor property different from both of its single drug components. Following a strong DNA damage response, triggering of apoptosis and/or MC may take place in HL cells according to their sensitivity status to BDM. A phase 1/2 study in recurrent HL, including patients pretreated with BDM, is next to be launched. DisclosuresMehrling:4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Pinto:Takeda, Celgene, Roche, TEVA: Honoraria; Takeda: Research Funding.

Hodgkin lymphoma cell lines bind to platelets. Incubation with platelets induces CD15 and P-selectin dependent adhesion of the cell lines to Human Umbilical Vein Endothelial cells (HUVEC)

Hodgkin's lymphoma is believed to spread in an orderly fashion within the lymphatic compartment. In a minority of cases, after reaching the spleen, the neoplasm disseminates, reminiscent of metastasis. In the spleen, the Hodgkin-Reed-Sternberg tumor cells come across platelets in the blood vessels and mainly in the splenic red pulp. Based on this knowledge, we investigated the possibility of platelets inducing cell adhesion in Hodgkin's lymphoma cell lines. We showed that L428 and KMH-2 cells strongly adhere to thrombin-activated platelets. Cell adhesion to platelets is partially dependent on CD15 antigens (LewisX), mainly sialyl-CD15, and P-selectin. KMH-2, as compared to L428 cells, showed increased binding due to its differential high expression of the sialyl-CD15. As a consequence of incubation with platelets, KMH-2 cells also produced increased amounts of tumor necrosis factors α (TNFα) followed by enhanced binding to human vascular endothelial cells (HUVEC). Incubation of both cell lines with activated platelets also induced activation of AP-1 transcription complex. Our findings are consistent with the concept that platelets play a critical role in the dissemination of HRS cells in HL, predominantly in the spleen, by increasing cell adhesion and thus promoting their proliferative and migratory properties beyond the lymphatic system.

Early growth response gene (EGR)-1 regulates leukotriene D4-induced cytokine transcription in Hodgkin lymphoma cells

Classical Hodgkin lymphoma (cHL) has a unique pathological feature characterized by a minority of malignant Hodgkin Reed–Sternberg (H–RS) cells surrounded by numerous inflammatory cells. Cysteinyl-leukotrienes (CysLTs) are produced by eosinophils, macrophages and mast cells in the HL tumor microenvironment. In the present study we have explored the signal transduction pathways leading to leukotriene (LT) D4 induced expression of cytokines in the Hodgkin lymphoma cell line L1236 and KM-H2. Stimulation of L1236 and KM-H2 cells with LTD4 led to a concentration- and time-dependent increase at the transcriptional level of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, chemokine (C-C motif) ligand 3 (CCL3) and CCL4. The expression of several transcription factors was induced upon stimulation of Hodgkin cell lines with LTD4. Among these, EGR-1 was required for cytokine production. Inhibition of EGR-1 expression using shEGR-1 transduced by lentivirus led to suppression of the expression of TNF-α and IL-6. The effect of LTD4 on the expression of transcription factors and cytokines were also blocked by the specific CysLT1 receptor antagonist zafirlukast. These results demonstrate that EGR-1 plays a critical role in LTD4-induced cytokine transcription in Hodgkin cell lines.