EMBR-01. CLASS I HDAC INHIBITORS AND PLK1 INHIBITORS SYNERGIZE IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract Background Medulloblastoma (MB) is one of the most common malignant pediatric CNS tumors. Patients with Group 3 MBs harboring MYC amplification exhibit low survival rates. Surviving patients suffer from therapy-induced sequelae, which calls for new targeted therapy strategies. We and others have previously shown the sensitivity of MYC-amplified MB to class I histone deacetylase (HDAC) inhibition. After demonstrating that the MYC target gene PLK1 is significantly downregulated upon class I HDACi treatment, we hypothesized that inhibition of both HDACs and PLK1 could have synergistic effects. Methods Cell metabolic activity changes upon HDAC and PLK1 inhibitor treatment were measured in MYC-amplified and non-amplified MB cell lines, as well as in an additional MYC-inducible cell line. The interaction effect of both inhibitors was determined by computation of the combination index (CI) using the Chou-Talalay method. Results were validated assessing cell viability, cell cycle, and apoptosis induction. Transcription profile changes after combination treatment were evaluated. Results MYC-amplified MB cell lines were more sensitive than non-amplified cell lines to PLK1i treatment, showing IC50 in clinically achievable concentration ranges. Inhibition of class I HDACs and PLK1 synergistically reduced cell metabolic activity in lower concentrations in MYC-amplified compared to non-amplified MB cell lines. We also observed a significant loss of viability and cells in G1 phase, as well as induction of apoptosis after combination treatment in MYC-amplified cells. MYC target gene sets were significantly downregulated in the MYC-amplified cell line HD-MB03 after treatment with combination. We demonstrated reduction of MYC protein levels upon PLK1i treatment. In vivo evaluation of combination treatment using orthotopic Group 3 MYC-amplified MB PDX models is ongoing. Conclusion Our data suggest that MYC-amplification is a predictive marker for PLK1i treatment in MB. The combination of HDACi and PLKi could be a candidate therapy for future clinical trials for MYC-amplified group 3 MB.

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Molecular definition of a polymorphic antigen (LA45) of free HLA-A and -B heavy chains found on the surfaces of activated B and T cells.

Journal of Experimental Medicine ◽

10.1084/jem.174.5.1085 ◽

1991 ◽

Vol 174 (5) ◽

pp. 1085-1095 ◽

Cited By ~ 67

Author(s):

J A Madrigal ◽

M P Belich ◽

R J Benjamin ◽

A M Little ◽

W H Hildebrand ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Cell Types ◽

Class I ◽

Activated T Cells ◽

Heavy Chains ◽

Barr Virus ◽

Beta 2 Microglobulin

A monomoprhic monoclonal antibody (LA45 antibody) reactive with "a new activation-induced surface structure on human T lymphocytes" (LA45 antigen) that resembled free class I heavy chains has recently been described (Schnabl, E., H. Stockinger, O. Majdic, H. Gaugitsch, I.J.D. Lindley, D. Maurer, A. Hajek-Rosenmayr, and W. Knapp. 1990. J. Exp. Med. 171:1431). This antibody was used to clone a class I-like heavy chain (LA45 gene) from the HUT 102 tumor cell, which paradoxically did not give rise to the LA45 antigen on transfection into monkey COS cells. We show here that the LA45 gene is HLA-Aw66.2, a previously uncharacterized allele of the HLA-A locus. The previously determined LA45 sequence differs from that of HLA-Aw66.2, from HUT 102, and the CR-B B cell line derived from the same individual as HUT 102 by substitution of tryptophan for serine at position 4 in the alpha 1 domain. Transfection of HLA-Aw66.2, and of a mutant of this gene with serine 4 substituted for tryptophan, into a human B cell line (C1R) both resulted in expression of the LA45 epitope. Furthermore, we find expression of the LA45 epitope on Epstein Barr virus-transformed B cell lines as well as lectin-activated T cells, but not on long-term T cell lines or unstimulated peripheral blood T cells. The specificity of the LA45 antibody is polymorphic and the presence of the LA45 epitope is precisely correlated with the sequence arginine, asparagine (RN) at residues 62 and 63 of the helix of the alpha 1 domain. The LA45 epitope is broadly distributed, being associated with half the alleles of both HLA-A and -B loci but none of the HLA-C locus. All the results are consistent with the presence of pools of free HLA-A and -B heavy chains at the surfaces of certain cell types but not others. Such molecules are probably responsible for the HLA-associated class I alloantigens of lectin-activated T cells. We hypothesize the free heavy chains result from dissociation of beta 2-microglobulin from subpopulations of empty HLA-A,B molecules, or molecules with weakly bound peptides, that vary in size depending on cellular activation and peptide supply.

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EMBR-11. SYNERGISTIC DRUG COMBINATIONS FOR THE TREATMENT OF MYC AMPLIFIED GROUP 3 MEDULLOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab090.029 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i7-i8

Author(s):

Simon Zeuner ◽

Johanna Vollmer ◽

Heike Peterziel ◽

Romain Sigaud ◽

Sina Oppermann ◽

...

Keyword(s):

Clinical Trials ◽

Cell Lines ◽

Hdac Inhibitors ◽

Drug Combinations ◽

Class I ◽

Drug Screen ◽

Synergistic Activity ◽

Sensitivity Score ◽

Group 3 ◽

Synergistic Drug Combinations

Abstract Background Medulloblastoma (MB) is a highly aggressive brain tumour in children. Patients with Group 3 MB harbouring a MYC-amplification (subtype II) show a particularly poor outcome despite high-intensity multimodal therapy. We and others have previously shown that MYC amplified Group 3 MB cells are highly susceptible towards treatment with class I histone deacetylase (HDAC) inhibitors such as entinostat. However, in clinical trials HDACi as a monotherapy show only modest efficacy in solid tumours. We propose to increase the efficacy of class I HDACi by drug combinations. Methods To identify synergistic drug combinations (entinostat + X) for the treatment of MYC amplified MB we performed a drug screen with a library of n=75 clinically available compounds as single agents and in combination with entinostat in n=3 MYC amplified vs. n=1 MYC-non amplified cell lines. Synergistic behaviour of the six most promising drug combinations was validated by metabolic activity assays, cell count experiments and gene expression profiling. Synergy was assessed by the Loewe additivity model using a combination of ray design and checkerboard matrix. Results The drug screen revealed n=20/75 drugs that were particularly effective (drug sensitivity score ≥10) in combination with entinostat treatment in all three MYC amplified cell lines. Synergy assessment of the top n=6 drugs confirmed strong synergistic activity with entinostat for n=2 drugs (navitoclax, irinotecan). The BCL-2 family inhibitor navitoclax showed the most robust synergy with entinostat in subsequent validation experiments. Conclusion Several drugs either clinically available or currently in clinical trials, including the BCL-2/Xl/w inhibitor navitoclax, show promising effects in a combination therapy with entinostat for the treatment of MYC amplified Group 3 MB.

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The synergistic effect between celecoxib and luteolin and dependence on estrogen receptor in human breast cancer cells.

Journal of Clinical Oncology ◽

10.1200/jco.2015.33.28_suppl.135 ◽

2015 ◽

Vol 33 (28_suppl) ◽

pp. 135-135

Author(s):

Ye-Won Jeon ◽

Youngjin Suh

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Combination Treatment ◽

Breast Cancer Cells ◽

Synergistic Effects ◽

Breast Cancer Cell Lines ◽

Human Breast ◽

Mcf 7

135 Background: The anti-cancer effects of celecoxib and luteolin are well known. Although our previous study demonstrated that the combination of celecoxib and luteolin synergistically inhibits breast tumor growth compared with each of the treatments alone, we did not uncover the molecular mechanisms of these effects. The aims of our present study were to compare the effects of a celecoxib and luteolin combination treatment in four different human breast cell lines and to determine the mechanisms of action in vitro and in vivo. Methods: Using MCF-7, MCF7/HER18, MDA-MB-231 and SkBr3 human breast cancer cells, proliferation assay, apoptosis assay, inhibition assay with MEK and PI3K inhibitor in addition to western blotting and xenograft study after treatment with celecoxib and luteolin. Results: The synergistic effects of a celecoxib and luteolin combination treatment yielded significantly greater cell growth inhibition in all four breast cancer cell lines compared with the single agents alone. In particular, combined celecoxib and luteolin treatment significantly decreased the growth of MDA-MB-231 cancer cells in vivo compared with either agent alone. The celecoxib and luteolin combination treatment induced synergistic effects via Akt inactivation and extracellular signal-regulated kinase (ERK) signaling inhibition in MCF-7 and MCF7/HER18 cells and via Akt inactivation and ERK signaling activation in MDA-MB-231 and SkBr3 cells. Conclusions: These results demonstrate the synergistic anti-tumor effect of the celecoxib and luteolin combination treatment in different four breast cancer cell lines, thus introducing the possibility of this combination as a new treatment modality.

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The Multikinase Inhibitor Sorafenib Inhibts Proliferation in B- and T-Lymphoblastic Cell Lines Via the PI3K/Akt Pathway.

Blood ◽

10.1182/blood.v112.11.1910.1910 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1910-1910

Author(s):

Catrin Schult ◽

Meike Dahlhaus ◽

Sonja Boldt ◽

Änne Glass ◽

Sabine Ruck ◽

...

Keyword(s):

Gene Expression ◽

Differential Gene Expression ◽

Cell Lines ◽

Metabolic Activity ◽

Synergistic Effects ◽

Cytotoxic Drugs ◽

Akt Pathway ◽

High Grade ◽

Multikinase Inhibitor ◽

Differential Gene

Abstract Background: The phosphatidylinositol 3 Kinase (PI3K)/Akt signalling pathway is involved in the regulation of proliferation, apoptosis and angiogenesis. Dysregulation of PI3K/Akt has been described for several solid and hematological tumors. Its role in acute lymphoblastic leukemogenesis (ALL) is unkown. Here, we investigated whether the inhibition of PI3K/Akt kinases influences apoptosis, necrosis and cell proliferation. Patients and Methods: ALL and high grade NHL cell lines with different cytogenetics and phenotypes were used (SEM, RS4;11, REH, Jurkat, DOGKIT). Phosphorylationstatus of Akt (Ser473, Thr308) and FOXO3A (Thr32) were determined by western blot (WB). Cells were incubated for 96h with LY29004 (5μM, 12.5μM, 25μM; PI3K inhibitor) or sorafenib (0.73μM, 7.3μM). In order to detect synergistic effects with other cytotoxic drugs cells were treated with combinations of sorafenib and doxorubicine, cytosin-arabinoside, and mTOR inhibitor (RAD001), respecitvely. Cell number, apoptosis, necrosis, protein phosphorylation and metabolic activity were determined at 4h, 24h, 48h, 72h, and 96h by microscopy, flow cytometry, WB and WST-1 testing. Whole genome Affymetrix gene expression arrays (U133 Plus 2.0) were performed in order to detect differential gene expression compared to controls treated with DMSO (dimethylsulfoxid) only. Results: In all ALL cell lines phosphorylated Akt (pAKT) was detected. Levels of pAkt and pFOXO3A differed between cell lines significantly, with Jurkat and SEM demonstrating high activation levels. PI3K inhibition by LY29004 led to decreased proliferation in SEM cells with only slight increases in apoptosis and moderate increase in necrosis rates (20%). Sorafenib inhibited the proliferation of SEM, Jurkat and RS4;11 significantly, with most pronounced effects at 96h. Maximal apoptosis and necrosis rates increased and ranged from 11 to 39% and 53 to 84%, respectively. Metabolic activity decreased significantly already after 24h. As early as 0.5h after treatment complete disappearance (SEM, RS4;11) or marked decrease (Jurkat) in levels of pAkt and pFOXO occurred. Combination of sorafenib with conventional cytotoxic drugs failed to demonstrate synergistic effects. Sorafenib treatment induced differential gene expression in several genes e.g. genes involved in apoptosis such as BIMBAM, caspases and CDKs. Conclusions: Inhibition of the PI3K/Akt pathway seems to be a potential therapeutic target in ALL and high grade lymphoma cells. Sorafenib as a multikinase inhibitor approved for clincial application in solid tumors displayes significant antileukemic activity in vitro and might be a potential drug for a targed therapy approach in ALL.

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Combination Treatment With Trabectedin and Irinotecan or Topotecan Has Synergistic Effects Against Ovarian Clear Cell Carcinoma Cells

International Journal of Gynecological Cancer ◽

10.1097/igc.0000000000000143 ◽

2014 ◽

Vol 24 (5) ◽

pp. 829-837 ◽

Cited By ~ 4

Author(s):

Mahiru Kawano ◽

Seiji Mabuchi ◽

Toshiko Kishimoto ◽

Takeshi Hisamatsu ◽

Yuri Matsumoto ◽

...

Keyword(s):

Cell Carcinoma ◽

Cell Lines ◽

Combination Treatment ◽

Clear Cell ◽

Clear Cell Carcinoma ◽

Synergistic Effects ◽

Chemotherapeutic Agents ◽

Ovarian Clear Cell Carcinoma ◽

Combination Treatments

ObjectivesThe objective of this study was to investigate the chemotherapeutic agents that produce the strongest synergistic effects when combined with trabectedin against ovarian clear cell carcinoma (CCC), which is regarded as an aggressive chemoresistant histological subtype.MethodsUsing 4 human CCC cell lines (RMG1, RMG2, KOC7C, and HAC2), the cytotoxicities of trabectedin, SN-38, topotecan, doxorubicin, cisplatin, and paclitaxel as single agents were first assessed using the MTS assay. Then, the cytotoxicities of combination treatments involving trabectedin and 1 of the other 4 agents were evaluated by isobologram analysis to examine whether these combinations displayed synergistic, additive, or antagonistic effects. The antitumor activities of the combination treatments were also examined using cisplatin-resistant and paclitaxel-resistant CCC sublines, which were derived from the parental CCC cells by continuously exposing them to cisplatin or paclitaxel. Finally, we determined the effect of everolimus on the antitumor efficacy of trabectedin-based combination chemotherapy.ResultsConcurrent exposure to trabectedin and SN-38 or topotecan resulted in synergistic interactions in all 4 CCC cell lines. Among the tested combinations, trabectedin plus SN-38 was the most effective cytotoxic regimen. The combination of trabectedin plus SN-38 also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Treatment with everolimus significantly enhanced the antitumor activity of trabectedin plus SN-38 or topotecan.ConclusionsCombination treatment with trabectedin and SN-38 displays the greatest cytotoxic effect against ovarian CCC. Our in vitro study provides the rationale for future clinical trials of trabectedin plus irinotecan with or without everolimus in patients with ovarian CCC in both the front-line chemotherapy setting and as a second-line treatment of recurrent CCC that had previously been treated with cisplatin or paclitaxel.

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HDAC Screening Identifies the HDAC Class I Inhibitor Romidepsin as a Promising Epigenetic Drug for Biliary Tract Cancer

Cancers ◽

10.3390/cancers13153862 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3862

Author(s):

Christian Mayr ◽

Tobias Kiesslich ◽

Sara Erber ◽

Dino Bekric ◽

Heidemarie Dobias ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Biliary Tract ◽

Biliary Tract Cancer ◽

Histone Deacetylases ◽

Class I ◽

In Vivo Evaluation ◽

Tract Cancer

Inhibition of histone deacetylases (HDACs) is a promising anti-cancer approach. For biliary tract cancer (BTC), only limited therapeutic options are currently available. Therefore, we performed a comprehensive investigation of HDAC expression and pharmacological HDAC inhibition into a panel of eight established BTC cell lines. The screening results indicate a heterogeneous expression of HDACs across the studied cell lines. We next tested the effect of six established HDAC inhibitors (HDACi) covering pan- and class-specific HDACis on cell viability of BTC cells and found that the effect (i) is dose- and cell-line-dependent, (ii) does not correlate with HDAC isoform expression, and (iii) is most pronounced for romidepsin (a class I HDACi), showing the highest reduction in cell viability with IC50 values in the low-nM range. Further analyses demonstrated that romidepsin induces apoptosis in BTC cells, reduces HDAC activity, and increases acetylation of histone 3 lysine 9 (H3K9Ac). Similar to BTC cell lines, HDAC 1/2 proteins were heterogeneously expressed in a cohort of resected BTC specimens (n = 78), and their expression increased with tumor grading. The survival of BTC patients with high HDAC-2-expressing tumors was significantly shorter. In conclusion, HDAC class I inhibition in BTC cells by romidepsin is highly effective in vitro and encourages further in vivo evaluation in BTC. In situ assessment of HDAC 2 expression in BTC specimens indicates its importance for oncogenesis and/or progression of BTC as well as for the prognosis of BTC patients.

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844 Immunomodulatory activity of epigenetic drugs combinations in mesothelioma: laying the ground for new immunotherapeutic strategies

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0844 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A896-A896

Author(s):

Sara Cannito ◽

Health Biology ◽

Ornella Cutaia ◽

Carolina Fazio ◽

Maria Fortunata Lofiego ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Hla Class I ◽

Class I ◽

Immunomodulatory Activity ◽

Epigenetic Drugs ◽

Hla Class I Antigens ◽

Hla Class I Molecules ◽

Mesothelioma Cell Line ◽

Mesothelioma Cell

BackgroundGrowing evidence are demonstrating the therapeutic efficacy of immune checkpoint inhibitors (ICI) in mesothelioma; however, a limited percentage of patients benefits from this therapeutic approach. Epigenetic modifications play a relevant role in negatively regulating the cross-talk between neoplastic and immune cells, and in contributing to the highly immunosuppressive mesothelioma microenvironment. A better understanding of mesothelioma epigenetic landscape could open the path to novel and potentially more effective approaches combining ICI and epigenetic drugs. We investigated the immunomodulatory potential of epigenetic agents by comparing the activity of DNA hypomethylating agents (DHA) with histone deacetylases inhibitors (HDACi) and EZH2 inhibitors (EZH2i), alone or combined with DHA, in mesothelioma cells.MethodsFour mesothelioma cell lines were treated with the DHA guadecitabine 1μM, or with the HDACi, Valproic Acid (VPA) 1mM, or the EZH2i, EPZ-6438 1μM, alone or combined with guadecitabine. We investigated the expression of HLA class I molecules by flow-cytometry and of PD-L1, cancer testis antigens (CTA: NY-ESO, MAGE-A1), Natural Killer Group 2 member D Ligands (NKG2DLs: MIC-A, MIC-B, ULBP2) and EMT-regulating cadherins (CDH1, CDH2) by quantitative Real-Time PCR. Fold change (FC) expression for each treatment vs untreated cells was reported as mean values (FCm) among investigated cell lines. A positive modulation of the expression was considered if FCm>1.5.ResultsGuadecitabine upregulated the expression of HLA class I antigens (FCm=1.75), PD-L1 (FCm=2.38), NKG2DLs (MIC-A FCm=1.96, MIC-B FCm=2.57, and ULBP2 FCm=3.56), and upregulated/induced CTA expression. Similarly, VPA upregulated HLA class I antigens (FCm=1.67), PD-L1 (FCm=3.17), NKG2DLs (MIC-A FCm=1.78, MIC-B FCm=3.04, and ULBP2 FCm=3.75) expression; however, CTA expression was modulated only in 1 mesothelioma cell line. Conversely, EPZ-6438 up-regulated only NY-ESO-1 and MIC-B expression in 1 mesothelioma cell line.The addition of both VPA and EPZ-6483 to guadecitabine strengthened its immunomodulatory activity. Specifically, guadecitabine plus VPA or EPZ-6438 upregulated the expression of HLA class I antigens FCm=2.55 or 2.69, PD-L1 FCm=8.04 or 2.65, MIC-A FCm=3.81 or 2.26, MIC-B FCm=8.00 or 3.03, ULBP2 FCm=6.24 or 4.53, respectively. Higher levels of CTA upregulation/induction were observed with combination treatments vs guadecitabine alone.Cadherins modulation was mesothelioma histotype-related: CDH1 expression was induced in the 2 constitutively-negative sarcomatoid mesothelioma cells by guadecitabine alone or combined with VPA or EPZ-6438; CDH2 expression was upregulated by VPA alone (FCm=1.53) or plus guadecitabine (FCm=2.54).ConclusionsCombination of DHA-based immunotherapies with other classes of epigenetic drugs could be an effective strategy to be pursued in the mesothelioma clinic.

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WT1 and DNMT3A Play an Essential Function and Represent Therapeutic Vulnerabilities in Certain AML Samples, As Shown By CRISPR/Cas9 Mediated Knockout in PDX Models In Vivo

Blood ◽

10.1182/blood-2021-150305 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 377-377

Author(s):

Maryam Ghalandary ◽

Yuqiao Gao ◽

Martin Becker ◽

Diana Amend ◽

Klaus H. Metzeler ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Line ◽

Cell Lines ◽

Target Gene ◽

Target Genes ◽

Nsg Mice ◽

Essential Function ◽

Pdx Models

Abstract Background: The prognosis of patients with acute myeloid leukemia (AML) remains poor and novel therapeutic options are intensively needed. Targeted therapies specifically address molecules with essential function for AML and deciphering novel essential target genes is of utmost importance. Functional genomics via CRISPR\Cas9 technology paves the way for the systematic discovery of novel essential genes, but was so far mostly restricted to studying cell lines in vitro, lacking features of, e.g., primary tumor cells and the in vivo tumor microenvironment. To move closer to the clinical situation in patients, we used the CRISPR\Cas9 technology in patient-derived xenograft (PDX) models of AML in vivo. Methods: Primary tumor cells from seven patients with AML were transplanted into immunocompromised NSG mice and serially transplantable PDX models derived thereof. PDX models were selected which carry the AML specific mutations of interest at variant allele frequencies close to 0.5. PDX cells were lentivirally transduced to express the Cas9 protein and a sgRNA; successfully transduced PDX cells were enriched by flow cytometry gating on a recombinant fluorochrome or by puromycin. The customized sgRNA library was designed using the CLUE (www.crispr-clue.de) platform and cloned into a lentiviral vector with five different sgRNAs per target gene, plus positive and negative controls (Becker et al., Nucleic Acids Res. 2020). PDX cells were lentivirally transduced with the CRISPR/Cas9 sgRNA library, transplanted into NSG mice, grown in vivo and cells re-isolated at advanced AML disease. sgRNA distribution was measured by next generation sequencing and compared to input control using the MAGeCK pipeline. Interesting dropout hits from PDX in vivo screens were validated by fluorochrome-guided competitive in vivo experiments in the PDX models, comparing growth of PDX AML cells with knockout of the gene of interest versus control knockout in the same mouse. PDX cells were transduced with lentiviral vectors expressing a single sgRNA, using in parallel three different sgRNAs per target gene. Targeting and control sgRNAs were marked by different fluorochromes; PDX cells expressing targeting or control sgRNA were mixed at a 1:1 ratio, injected into NSG mice and PDX models competitively grown until advanced disease stage, when cell distributions was determined by flow cytometry. Human AML cell lines were studied in vitro for comparison. Results: In search for genes with essential function in AML, we cloned a small customized sgRNA library targeting 34 genes recurrently mutated in AML and tested the library in two PDX AML models in vivo. From the dropouts, we validated most interesting target genes using fluorochrome-guided competitive in vivo assays. Knockout of NPM1 abrogated in vivo growth in all PDX AML models tested, reproducing the known common essential function of NPM1. KRAS proved an essential function in PDX AML models both with and without an oncogenic mutation in KRAS, although with a stronger effect upon KRAS mutation, suggesting that patients with tumors both with and without KRAS mutation might benefit from treatment inhibiting KRAS. Surprising results were obtained for WT1 and DNMT3A. Both genes are frequently mutated in AML, but most AML cell lines tested in vitro do not show an essential function of any of the two genes, in published knockdown or knockout data, including from the Cancer Dependency Map database. On the contrary, knockout of either WT1 or DNMT3A was shown to enhance growth of AML cell lines and increase leukemogenesis in certain models. In PDX models in vivo, we found a clearly essential function for DNMT3A in all AML samples and WT1 in most samples tested and PDX in vivo results were discordant to cell line in vitro data, suggesting that cell line inherent features and/or the in vivo environment influence the function of WT1 and DNMT3A. Conclusion: We conclude that functional genomics in PDX models in vivo allows discovering essentialities hidden for cell line in vitro approaches. WT1 and DNMT3A harbor the potential to represent attractive therapeutic targets in AML under in vivo conditions, warranting further evaluation. Disclosures No relevant conflicts of interest to declare.

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RNA Cytosine Methyltransferases NSUN1 and NSUN2 Mediate the Lineage-Associated Resistance to Venetoclax in Leukemia

Blood ◽

10.1182/blood-2020-141684 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 13-14

Author(s):

Shaun Wood ◽

Amber Willbanks ◽

Jason Xiaojun Cheng

Keyword(s):

Rna Polymerase Ii ◽

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Synergistic Effects ◽

Leukemia Cell ◽

Resistant Cell ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

Leukemia Cell Lines

Background: Combinations of venetoclax/ABT-199, a small molecule that selectively inhibits anti-apoptotic protein BCL2, with hypomethylating agents (HMAs), such as 5-azacytidine (5-AZA, azacidtine) and decitabine have demonstrated remarkable synergistic effects and resulted in high response rates and significant overall survivals in patients with refractory MDS/AML (Ram, et al. Annual Hematology 2019; DiNardo et al. Blood 2019). However, resistance to venetoclax-based therapies has emerged as a major therapeutic barrier and been linked to monocytic clones in leukemia (Kuusanmaki et al. Haematologica 2020; Pei et al. Cancer Discovery 2020). Our recent study demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, mediate the lineage-associated resistance to 5-AZA through formation of a drug-resistant elongating RNA-Polymerase-II (eRNAPII) complex at nascent RNA (Cheng et al. Nature Communications 2018). This study aims to address the role of NSUN1 and NSUN2 in mediating venetoclax resistance in leukemia. Experimental Design and Methods: Experiments, including drug-induced cell growth inhibition, western blot, and co-immunoprecipitation, were performed on leukemia cell lines with various lineages to assess lineage-associated venetoclax resistance and identify the key factors/proteins involved in such resistance. Venetoclax-resistant cell lines were established from drug sensitive lines in order to elucidate mechanisms underlying resistance and cell lineage plasticity. Knockdown of NSUN1 and NSUN2 expression was performed to determine their roles in venetoclax-resistant cell lines. Results: Our experimental results have demonstrated monocyte differentiation-associated resistance to venetoclax in leukemia cell lines of different lineages (Figure 1A), which is consistent with previous published studies (Kuusanmaki et al. Haematologica 2020; Pei et al. Cancer Discovery 2020). The degree of lineage-associated venetoclax resistance is closely correlated with the expression of eRNAPII, NSUN2 and NSUN1. Importantly, venetoclax strongly induces expression of eRNAPII and NSUN1 (Figure 1B). We established venetoclax-resistant leukemia cell line (K1VR) from original venetoclax-sensitive granulocytic leukemia cell line Kasumi-1 and confirmed the importance of NSUN1 and NSUN2 in mediating venetoclax resistance in those leukemia cells. siRNA knockdown of NSUN1 or NSUN2 expression inhibits growth of leukemia cells and re-sensitizes the venetoclax-resistant K1VR leukemia cells to a low dose of venetoclax (Figure 1C). Conclusion: Our study has demonstrated that RNA cytosine methyltransferases NSUN1 and NSUN2 mediate monocyte-associated resistance to venetoclax in leukemia cells. We are currently extending our study to clinical specimens. Our study may lead to development of novel RNA epigenetics-driven strategies to predict and overcome the resistance to venetoclax-based therapies. Disclosures No relevant conflicts of interest to declare.

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Epitopes on H-2Dd somatic cell mutants recognized by cytotoxic T cells.

Journal of Experimental Medicine ◽

10.1084/jem.158.4.1061 ◽

1983 ◽

Vol 158 (4) ◽

pp. 1061-1076 ◽

Cited By ~ 12

Author(s):

T A Potter ◽

M A Palladino ◽

D B Wilson ◽

T V Rajan

Keyword(s):

T Cell ◽

Somatic Cell ◽

Cell Line ◽

Cell Lines ◽

Cytotoxic T Cells ◽

Mutant Cell ◽

Class I ◽

Mouse Strains ◽

Parental Cell Line ◽

Mutant Lines

We have generated several cell lines that express an altered H-2Dd molecule. These cell lines, which were selected for by the failure to express the serological specificity reacting with the monoclonal antibody 34-2-12, have also undergone alterations in epitopes recognized by CTL. One of the mutants, 2.12(-4) was not killed by an allogeneic anti-Dd CTL line, CTLL-A2, even though this line was cytotoxic for the parental cell line and two other 34-2-12- mutant lines. Two of the 34-2-12- mutant lines had an identical serological profile using other monoclonal Dd antibodies, however these two mutants differed markedly in their susceptibility to cytotoxicity by CTLL-A2. In addition to the determinants recognized by allogeneic CTL we also examined the effect of the mutation on the determinants involved in restricting the anti-FITC modified-self-cytotoxic response. An anti-FITC-Dd CTL line did not react with two of the mutants and reacted only weakly with the other mutant, demonstrating not only that the Dd epitopes recognized by this cell line and the allogeneic CTL were different, but also that it is possible for a H-2 class I molecule to express epitopes recognized by allogeneic CTL but not epitopes that function as restricting elements to certain antigens. The observation that both T cell- and B cell-defined determinants were altered in these mutant cell lines is in contrast to the findings, with the mutant mouse strains which were selected for by changes in T cell-defined determinants, which show few, if any, alterations to serological specificities. Characterization of T cell-recognized epitopes expressed on serologically selected somatic cell variants may therefore prove to be most useful for the study of structure-function relationships of H-2 class I molecules.

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