Preclinical evaluation of KZR-261, a novel small molecule inhibitor of Sec61.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3582-3582
Author(s):  
Eric Lowe ◽  
R. Andrea Fan ◽  
Jing Jiang ◽  
Henry W. B. Johnson ◽  
Christopher J. Kirk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Expression Profiling ◽  
Single Agent ◽  
Tumor Types ◽  
Anti Tumor Activity

3582 Background: Secreted and transmembrane proteins play key roles in malignant transformation and growth, including in autocrine growth factor expression, receptor oncogene signaling, and immune system evasion. Biogenesis of these proteins involves translocation of the nascent polypeptides into the endoplasmic reticulum (ER) through the Sec61 channel, providing an untapped therapeutic target for a broad spectrum of malignancies. Here we describe preclinical activity of KZR-261 and related inhibitors of Sec61-dependent protein secretion. Methods: Sec61 inhibition with KZR-261 and related analog KZR-834 were evaluated using cell lines overexpressing proteins of interest tagged with luciferase. In vitro anti-tumor activity was assessed against a panel of 346 cell lines across 25 tumor types. Quantitative proteomic profiling by mass spec and gene expression profiling by RNAseq were conducted following treatment in multiple solid and heme tumor cell lines. Anti-tumor efficacy was evaluated in athymic nude mice implanted with the cancer cell lines H82 (SCLC), HT29 (CRC), BxPC3 (Pancreatic), 22RV1 (Prostate), H929 (Myeloma) and RL (NHL). Activity was also evaluated in a MC38 syngeneic colon tumor model. Results: KZR-261 and KZR-834 exhibited nanomolar potency against many therapeutic targets, including immune checkpoints, VEGF-A, VEGFR and EGFR. Broad in vitro anti-cancer activity was observed with KZR-834, which potently decreased cell viability across both solid and heme tumor types including CRC, Pancreatic, HNSCC, HCC, Lymphoma and Myeloma. Global proteomic analysis observed more than 1.5 fold downregulation of < 10% of detected Sec61 client proteins following treatment, while gene expression profiling revealed upregulation of ER stress response genes in sensitive versus resistant cell lines. Analysis of the TCGA database also found these genes upregulated in a number of different tumor types. In vivo, weekly IV administration was well tolerated and induced a dose dependent anti-tumor response at doses below the MTD in solid and heme xenograft models. In the syngeneic MC38 model, administration of KZR-834 in combination with anti-PD1 antibody resulted in greater anti-tumor activity than either single agent. Conclusions: Novel Sec61 inhibitors potently block expression of secreted and membrane proteins, translating into anti-tumor activity against many tumor types in vitro and in vivo, suggesting broad therapeutic potential. Clinical trials are being planned with KZR-261 to understand safety and early efficacy of this novel compound and therapeutic target.

Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13569-e13569
Author(s):  
Enrica Marchi ◽  
Matko Kalac ◽  
Danielle Bongero ◽  
Christine McIntosh ◽  
Laura K Fogli ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Cell Lines ◽  
Molecular Level ◽  
Cell Lymphoma ◽  
Single Agent ◽  
T Cell Lymphoma ◽  
Cytotoxicity Assays

e13569 Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.

Gene expression profiling of in vitro-derived and in vivo-derived bovine blastocysts using microarray analysis

2011 ◽  
Vol 22 ◽  
pp. S53-S54
Author(s):  
Digdem Aktoprakligil Aksu ◽  
Cansu Agca ◽  
Soner Aksu ◽  
Haydar Bagis ◽  
Tolga Akkoc ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Microarray Analysis ◽  
Expression Profiling

scholarly journals Gene expression profiling of human oocytes following in vivo or in vitro maturation

2008 ◽  
Vol 23 (5) ◽  
pp. 1138-1144 ◽  
Author(s):  
Gayle M. Jones ◽  
David S. Cram ◽  
Bi Song ◽  
M. Cristina Magli ◽  
Luca Gianaroli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
In Vitro Maturation ◽  
Human Oocytes

Gene expression profiling of liver cells after copper overload in vivo and in vitro reveals new copper-regulated genes

2007 ◽  
Vol 12 (4) ◽  
pp. 495-507 ◽  
Author(s):  
Patricia Muller ◽  
Harm van Bakel ◽  
Bart van de Sluis ◽  
Frank Holstege ◽  
Cisca Wijmenga ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Liver Cells ◽  
Copper Overload

scholarly journals Hyperbaric Oxygen Increases Sensitivity to Chemotherapy in Acute Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1399-1399
Author(s):  
Tara L. Lin ◽  
Amanda Wise ◽  
Abigale Berry ◽  
Joseph Fontes ◽  
Partha Kasturi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Cell Lines ◽  
Expression Profiling ◽  
Chemotherapy Resistance ◽  
Leukemia Cells ◽  
P Value ◽  
Significant Challenge ◽  
Increased Sensitivity

Abstract Introduction: Chemotherapy resistance remains a significant challenge in the treatment of adults with Acute Myeloid Leukemia (AML). Although new targets and novel agents are under clinical investigation, the majority of AML patients are treated with conventional induction chemotherapy consisting of an anthracycline and cytarabine, with little modification in over 40 years. Insights into the biology of resistance to cytarabine and anthracyclines may suggest novel strategies in the treatment of AML. Hyperbaric oxygen (HBO) treatment is commonly used in the treatment of decompression sickness from scuba diving, wound healing and infections. 100% oxygen is administered at increased pressure. Limited published data suggest a role for HBO to cause in vitro apoptosis of leukemia cell lines, but no mechanism has been described. We examined the role of HBO in AML sensitivity to chemotherapy, reactive oxygen species (ROS) generation, glycolytic pathway activation and gene expression. Methods: Human AML cell lines HL-60 and MV-411 were treated with escalating doses of cytarabine or daunorubicin as single agents followed by HBO treatment for 2 hours or normobaric conditions. HBO treatment consisted of exposure to 100% oxygen at 2.5 atmosphere absolutes for a total of 2 hours in a specifically designed in vitro HBO chamber. At 24 hours after chemotherapy dosing, leukemia cells were analyzed for proliferation using Cell Titer 96 Aqueous MTS assay (Promega). Apoptosis was measured using Annexin V-FITC and analyzed by flow cytometry. In order to understand potential mechanisms of HBO activity on leukemia cells, studies of HBO-treated cells without use of chemotherapy were also performed. CellROX Oxidative Stress assay (Life Technologies) was performed at 24 hours. CellROX Green and Deep Red reagents measured ROS level in the DNA and cytoplasm. RNA was isolated and real-time PCR was performed using CFX384 Real-time PCR system using primer sets specific for human hexokinase2 and phosphofructokinase to examine the effects of HBO on glycolysis. Gene expression profiling was performed using the Human Transcriptome Array 2.0 (Affymetrix) and examined differences in gene expression following HBO treatment of HL-60 cells and bone marrow from an AML patient. Results: HBO-treated leukemia cells had increased sensitivity to cytarabine (Table 1, Figure 1) (MV411 response to cytarabine: IC50 2649nM without HBO treatment, 1921nM with HBO, p=0.02; HL60 response to cytarabine: linear response without HBO, IC50 1625nM with HBO, p<0.035). Decreased proliferation was also seen with HBO-treated cells in response to daunorubicin but was not statistically significant (Table 1). Similarly, apoptosis from cytarabine in HL-60 cells was increased with HBO treatment (p<0.02), but the results with daunorubicin were not significant. Significant increases in ROS levels were observed at 24 hours post-HBO for both cell lines when compared to non-treated cells (HL60 DNA p<0.0001, HL60 cytoplasm p<0.01, MV411 DNA p<0.01, MV411 cytoplasm p<0.01). Glycolysis pathway components human hexokinase 2 and phosphofructokinase were upregulated in HL60 and MV411 cells treated with HBO compared to controls. Gene expression profiling showed that approximately 200 genes in coding regions were upregulated in HBO-treated HL-60 cells; specific piRNA clusters were downregulated in both AML cell line HL60 and a bone marrow sample from a patient with relapsed AML when treated with HBO. Conclusions: HBO treatment significantly increases the sensitivity to cytarabine AML cell lines in vitro. Increased ROS, upregulation of glycolysis-related enzymes and changes in gene expression profiling provide insight into the potential mechanisms of HBO enhanced chemotherapy sensitivity. Given the safety and commonplace use of HBO for other indications, and the significant challenge of chemotherapy resistance, it is important to pursue further studies to understand the biology of HBO treatment in AML. Table 1. IC50 (nM) of chemotherapy agents in AML cell lines with and without co-treatment with HBO. Cytarabine Daunorubicin no HBO HBO p-value no HBO HBO p-value MV411 2649 1921 0.0214 709.1 641.1 ns HL60 * 1625 0.0346 639 406.5 ns * linear response, no IC50 available Figure 1. Increased sensitivity to cytarabine in AML cell lines following treatment with HBO. Figure 1. Increased sensitivity to cytarabine in AML cell lines following treatment with HBO. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sirt1 Is a Novel Therapeutic Target in T-ALL

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2221-2221
Author(s):  
Olga Lancho ◽  
Amartya Singh ◽  
Victoria da Silva-Diz ◽  
Patricia Renck Nunes ◽  
Luca Tottone ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Gene Expression Profiling ◽  
Therapeutic Target ◽  
Expression Profiling ◽  
Conditional Knockout ◽  
Dependent Manner ◽  
Notch1 Signaling ◽  
Novel Therapeutic

Abstract T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy that affects both children and adults. Still, 20%-50% of patients show primary resistance or relapse after treatment, and ultimately die from their disease. Aberrant NOTCH1 signaling has a major role in the pathogenesis of T-ALL, as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. In this context, small-molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH1 activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH1 signaling, are being tested in clinical trials for the treatment of relapsed and refractory T-ALL. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by limited and delayed therapeutic response to these drugs, underscoring the need to identify novel therapeutic targets and to develop more effective drugs for the treatment of this disease. We previously demonstratedthe importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs). Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation. Indeed, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in T-ALL. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Moreover, through the integration of GSI-washout experiments, epigenetic profiling and CRISPR/Cas9-induced experiments, we have identified a distal enhancer of Sirt1 that is bound and controlled by NOTCH1, which might help explain the broad upregulation of Sirt1 observed in T-ALL patients. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with EX-527 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemiasleads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo. Mechanistically, acetyl-proteomics analyses revealed that acute deletion of Sirt1 consistently leads to hyperacetylation of Kat7 and Brd1, which are both part of a histone acetyltransferase complex. Indeed, Sirt1 loss results in global epigenetic changes including decreased levels of H4K12ac, which is a Kat7-target mark. Moreover, gene expression profiling analyses upon Sirt1 loss in leukemia in vivo revealed broad transcriptional changes. Gene-set enrichment analyses revealed that the transcriptional signature upon Sirt1 loss significantly correlates with the one obtained upon Kat7 loss, overall suggesting that Sirt1 loss leads to hyperacetylation of Kat7, which might be less active. Finally, our gene expression analyses also revealed a marked block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, glutaminolysis and TCA, with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal an oncogenic role for Sirt1 in T-ALL generation and progression, demonstrate that Sirt1 contributes to mediate resistance to anti-NOTCH1 therapies, identify a novel Notch1-Sirt1-Kat7 link and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification And Validation of Pivotal Genes Related To Meniscus Senescence Based On Gene Expression Profiling Analysis And In Vivo And In Vitro Models Detection

2021 ◽  
Author(s):  
Ming Chen ◽  
Siqi Zhou ◽  
Huasong Shi ◽  
Hanwen Gu ◽  
Yinxian Wen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Hub Genes ◽  
Age Related ◽  
Meniscus Cells ◽  
Toxic Drugs ◽  
Profiling Analysis

Abstract Background: The compositional change in the meniscus with aging would increase the tissue vulnerability of the meniscus, which would induce meniscus tearing. Here, we investigated the molecular mechanism of age-related meniscus degeneration with gene expression profiling analysis, and validate pivotal genes in vivo and in vitro models.Methods: The GSE45233 dataset, including 6 elderly meniscus samples and 6 younger meniscus samples, was downloaded from the Gene Expression Omnibus (GEO) database. To screen the differential expression of mRNAs, identify the miRNAs targeting hub genes, and forecast the potentially toxic drugs, we completed a series of bioinformatics analyses, including functional and pathway enrichment, protein-protein interaction network, hub genes screening, construction of a lncRNA–miRNA–mRNA network, and molecular docking of potential drugs. Furthermore, crucial genes were examined in human senescent menisci, mouse senescent meniscus tissues and mouse meniscus cells stimulated by IL-1β.Results: In total, the most significant 4 hub genes (RRM2, AURKB, CDK1, and TIMP1), 5 miRNAs (hsa-miR-6810-5p, hsa-miR-4676-5p, hsa-miR-6877-5p, hsa-miR-8085, and hsa-miR-6133) that regulated such 4 hub genes, and potential toxic drugs (Cladribine, Danusertib, Barasertib, Riviciclib, and Dinaciclib) that had a targeting effect on these genes, were finally identified. Moreover, these hub genes were decreased in meniscus cells in vitro and meniscus tissues in vivo, which indicated that hub genes were related to meniscus senescence and could serve as potential biomarkers for age-related meniscus tearing.Conclusions: In short, the integrated analysis of gene expression profile, co-expression network, and models detection identified pivotal genes, which elucidated the possible molecular basis underlying the senescence meniscus and also provided prognosis clues for early-onset age-related meniscus tearing.

scholarly journals Selinexor (KPT-330) Is Not Cross-Resistant with Chemotherapy and Demonstrates Strong Synergy with Targeted Therapy in DLBCL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4503-4503
Author(s):  
Jingda Xu ◽  
R Eric Davis ◽  
Zhiqiang Wang ◽  
Jason R. Westin
Keyword(s):  
Gene Expression ◽  
Targeted Therapy ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Cell Lines ◽  
Expression Profiling ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Dose Titration ◽  
Ic50 Values

Abstract Introduction: XPO1 (CRM1, Exportin 1) is the sole transporter of many tumor suppressor proteins (including MYC, BCL2, BCL6, BTK, IkB) and is elevated in non-Hodgkin Lymphoma. Selinexor (Sel, KPT-330) is an oral covalent inhibitor of XPO1, the first clinical molecule of the Selective Inhibitors of Nuclear Export drug class. The phase I clinical trial of Sel in hematologic malignancies showed promising early single-agent efficacy with modest toxicity in relapsed Diffuse Large B-cell Lymphoma (DLBCL, Gutierrez et al, ASCO 2014). DLBCL, the most common lymphoid malignancy, is currently cured in only 10% of relapsed patients, and consists of 2 subtypes based on putative cell of origin (COO): activated B-cell (ABC) and germinal center B-cell (GCB). We performed preclinical studies of Sel, modeling its single-agent efficacy in frontline and relapsed DLBCL and its potential synergy with other clinically relevant therapeutics. Methods: To model drug resistant DLBCL, resistant subpopulations of 12 patient-derived DLBCL cell lines were created by in vitro intermittent exposure to active congeners of cyclophosphamide, doxorubicin, and vincristine (ivCHOP), approximating clinical practice. To determine if CHOP-resistant DLBCL is also resistant to other agents, we determined single-agent dose response curves and IC50 values for both parental and ivCHOP resistant (CHOP-res) subclones of 4 of these lines at submission (HBL1 & TMD8 of ABC subtype, OCI-Ly7 & HT of GCB subtype, with 8 lines in progress) with Sel, chemotherapy (CT, ivCHOP, DHAP, and ICE), and targeted therapy (TT, ibrutinib, ABT-199, idelalisib, everolimus, MLN0128, alisertib, lenalidomide, bortezomib, I-BET151, and ONC201). Viability was assessed with CellTiter-Glo (Promega) after a 3 day cell culture. IC50 values were determined using GraphPad Prism. Based on these results, we evaluated the ability of Sel to synergize with other agents or restore sensitivity in CHOP-res with a combination “checkerboard” (orthogonal dose titration for each drug). The Combination Index (CI) for pairs at all concentrations was calculated with ComboSyn, with CI values <1 indicating synergy. Gene expression profiling with Illumina HT12v4 arrays will compare parental and CHOP-res of 12 DLBCL lines. Results: All CHOP-res lines of both COO types had higher IC50 for both ivCHOP (mean, 3.7x) and DHAP (4.5x) as compared to parental cells (Table 1). In contrast, the IC50 of Sel is unchanged between parental and CHOP-res, for both COO types. Other targeted agents displayed variable activity between parental and CHOP-res and between COO types, with the IC50 of ibrutinib being nearly 2 log lower in ABC lines. CI values showed that Sel was generally a strong synergizer (Table 1), especially with TT and in ABC lines. Sel had lower CI values with CT, but restored sensitivity to ivCHOP in HBL1 (Figure 1). Bortezomib and Sel were moderately antagonistic, although further tests are ongoing. Gene expression profiling, comparing parental vs. CHOP-res and Sel synergizing vs. non-synergizing lines, is ongoing. Conclusions: Our data suggest that Sel: 1) is equally active, and thus not cross-resistant, in cell lines made resistant to standard chemotherapeutics, 2) is a potent, broadly active synergizer with targeted therapy against lines modeling relapsed DLBCL, and 3) has greater synergy in ABC DLBCL, in which it may be able to reverse acquired resistance to frontline therapy. This behavior fits with the broad effects of XPO1 inhibition. The cross-resistance of CHOP-res lines to DHAP models clinical outcomes, and re-sensitization of CHOP-res lines with Sel suggests the potential for relapsed and frontline clinical trials. Further work with our model may discover more synergies of Sel, suggesting future clinical combinations and biomarkers associated with response. Table 1HBL1TMD8OCI-Ly7HTABCGCBIC50SR ΔSR ΔSR ΔSR ΔivCHOP2E-62.31E-75.51E-63.78E-63.2DHAP6E-73.65E-85.52E-73.71E-75.2Selinexor5E-80.66E-81.57E-80.94E-71.3Ibrutinib8E-81.02E-70.43E-60.92E-61.0ABT-1992E-60.53E-61.23E-60.38E-940.3Bortezomib4E-100.61E-101.03E-102.84E-100.9MLN01282E-71.22E-89.44E-85.42E-73.4CI with selinexorivCHOP 0.27 0.27 1.26 3.24DHAP 0.65 0.65 2.23 0.49Ibrutinib 0.06 0.06 0.02 0.95ABT-199 0.47 0.47 0.26 0.89Bortezomib 3.23 3.23 3.53 10Dexamethasone 0.19 0.19 0.39 2.09MLN0128 0.11 0.35 0.47 0.09 ivCHOP sensitive=S, Resistant=R, Δ fold change from S to R Disclosures No relevant conflicts of interest to declare.

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