Potent Efficacy of BCL2 Inhibition with ABT-199 in High-Risk Aggressive B-Lymphoma Models When Combined with Knockdown of MCL1

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 506-506
Author(s):  
Lingxiao Li ◽  
Praechompoo Pongtornpipat ◽  
Timothy Tiutan ◽  
Samantha L. Kendrick ◽  
Soyoung Park ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Double Hit Lymphoma ◽  
Apoptotic Protein ◽  
Resistant Lines ◽  
Double Hit ◽  
Factor C

Abstract Avoiding apoptosis is a hallmark of cancer. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and carries a poor prognosis in cases at high-risk of failing up-front R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Frequent expression of the anti-apoptotic protein BCL2 is well-described in DLBCL in numerous studies and is clear a negative prognostic marker when co-expressed with the oncogenic transcription factor c-MYC. Expression of the anti-apoptotic protein MCL1 also is found in about half of cases. BCL2 and MCL1 have redundant function in protecting cells from apoptosis. Direct inhibitors of MCL1 are not clinically available, but its short half-life permits knock-down through inhibition of cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation. Older multi-CDK inhibitors have anti-tumor activity from loss of MCL1 but are not approved clinically due to off-target toxicities. Dinaciclib is a more potent and specific multi-CDK inhibitor with activity against CDK9. We tested dinaciclib against a panel of >20 DLBCL cell lines and found high potency, with IC50 < 20 nM in most lines. Both in vitro and in vivo, dinaciclib results in rapid loss of MCL1 protein and corresponding induction of apoptosis. Interestingly, both sensitive and resistant lines show loss of MCL1 in response to the compound. Thus, we hypothesized BCL2 activity compensates for loss of MCL1 in resistant lines. Correspondingly, over-expression of BCL2 in sensitive cells renders them completely insensitive to dinaciclib without effecting MCL1 knockdown. In 59 DLBCL cases with known BCL2 status, we assessed MCL1 protein by immunohistochemistry and found no significant difference in MCL1 expression between BCL2 positive (66%, 10/15) and negative (57%, 25/44) cases (p=0.5576). Expression of MCL1, BCL2, or both in DLBCL and the proteins’ redundant function led to the hypothesis that knockdown of MCL1 combined with direct BCL2 inhibition would synergize in the killing of high-risk DLBCL tumors. ABT-199 is a third-generation BH3 mimetic direct inhibitor of BCL2, which has shown remarkable clinical activity in chronic lymphocytic leukemia but less activity in DLBCL and other more aggressive lymphomas. We found ABT-199 combines potently and synergistically with dinaciclib in DLBCL cell lines with none of 23 lines resistant to the combination. We confirmed this in vivo using the line U2932, which is resistant in vitro to both drugs as single agents. U2932 xenografts showed dramatic reduction of tumor burden in response to the combination, a response far superior to either drug alone. We next evaluated a genetically defined immunocompetent mouse model of MYC-BCL2 double-hit lymphoma, based on MYC expression in the VavP-Bcl2 transgenic model, replicating the genetics, pathology, and aggressive clinical behavior of the human disease. Tumors from this model in vitro, interestingly, show little response to single-agent ABT-199, but the combination with dinaciclib is again synergistic. Treatment of tumor-bearing mice in vivo showed animals treated with either drug alone had no significant survival difference from vehicle-treated controls, while those treated with the combination had dramatically improved survival by Kaplan-Meier analysis (p<0.0001). Finally, we assessed the effect of combining ABT-199 with standard lymphoma chemotherapy drugs that are thought to affect MCL1 protein levels due to global effects on transcription. Doxorubicin, etoposide, and cytarabine all result in loss of MCL1 at peak in vivo attainable concentrations and synergize with ABT-199 to kill DLBCL cells otherwise resistant to the single agents. In sum, we propose therapeutic strategies combining direct inhibition of BCL2 with knockdown of MCL1 expression will be effective and tolerable for poor-prognosis lymphomas such as high-risk DLBCL and double-hit lymphoma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Impact of extracellular matrix properties on neuroblastoma genomic heterogeneity

2020 ◽  
Author(s):  
Amparo López-Carrasco ◽  
Susana Martín-Vañó ◽  
Rebeca Burgos-Panadero ◽  
Ezequiel Monferrer ◽  
Ana P Berbegall ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
High Risk ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Chromosome 9 ◽  
Future Research ◽  
Knock Out ◽  
Specific Distribution

Abstract Background Increased tissue stiffness is a common feature of malignant solid tumors, often associated with metastasis and poor patient outcomes. Vitronectin, as an extracellular matrix anchorage glycoprotein related to a stiff matrix, is present in a particularly increased quantity and specific distribution in high-risk neuroblastoma. Furthermore, as cells can sense and transform the proprieties of the extracellular matrix into chemical signals through mechanotransduction, genotypic changes related to stiffness are possible. Methods We have applied high density SNPa and NGS techniques to in vivo and in vitro models (orthotropic xenograft vitronectin knock-out mice and 3D bioprinted hydrogels with different stiffness) using two representative neuroblastoma cell lines (the MYCN amplified SK-N-BE(2) and the ALK mutated SH-SY5Y), to discern how tumor genomics patterns and clonal heterogeneity of both cell lines are affected. Results We describe a remarkable subclonal selection of some genomic aberrations in SK-N-BE(2) cells grown in knock-out vitronectin xenograft mice that also emerged when cultured for long times in stiff hydrogels. Specially, we detected an enlarged subclonal cell population with chromosome 9 aberrations in both models. Similar abnormalities were found in human high-risk neuroblastoma with MYCN amplification. Genomics of the SH-SY5Y cell line remained stable when cultured in both models. Conclusions Focus on heterogeneous intratumor segmental chromosome aberrations and mutations, as a mirror image of tumor microenvironment, is a vital area of future research.

scholarly journals Paics Inhibition Is a Potential Therapeutic Strategy for MYC-Positive DLBCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 396-396
Author(s):  
Kohta Miyawaki ◽  
Takuji Yamauchi ◽  
Takeshi Sugio ◽  
Kensuke Sasaki ◽  
Hiroaki Miyoshi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Poor Prognosis ◽  
De Novo ◽  
Lymphoma Cells ◽  
Purine Synthesis

Diffuse large B-cell lymphoma (DLBCL) is among the most common hematological malignancies with varying prognosis. As many as forty percent of patients eventually experience relapsed/refractory disease after combinatorial chemo-immunotherapies, R-CHOP, and prognosis after relapse is dismal. MYC is among the most established prognostic factors and associated with clinically-distinct subsets of DLBCL with poor prognosis: double-expressor lymphoma (DEL) and double-hit lymphoma (DHL). MYC is co-expressed with BCL2 in DEL, which consists of 60% of activated B-cell type DLBCL (ABC-DLBCL) cases, while DHL, defined by coexistence of MYC and BCL2/BCL6 rearrangements, were reportedly observed in 15% of germinal center B-cell like DLBCL (GCB-DLBCL). Considering that MYC-positive DLBCLs exhibit dismal outcomes, pharmacological inhibition of MYC activity is highly demanded; however, direct targeting of MYC has been proven challenging. Here we show that PAICS (phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase), which catalyzes a critical step in de novo purine synthesis, functions downstream of MYC in DLBCL cells. We further show MRT252040, a newly-developed PAICS inhibitor, effectively suppresses proliferation of MYC-driven DLBCL cells in vitro and in vivo. Through the nCounter-based transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) tissues from 170 untreated DLBCL patients, we found that MYC and PAICS were co-expressed and their mRNA levels were among the most predictive for poor prognosis after standard R-CHOP therapy. Their expression levels were particularly high in a subset of ABC-DLBCL and extranodal DLBCL, namely in DEL and DHL cases. Importantly, these findings were validated using three independent cohorts (Schmitz et al. NEJM, 2018). MYC and PAICS expression levels were high in most DLBCL lines and low in normal B cells in the lymph nodes, while they were variable in primary DLBCL tissues, revealed by nCounter and immunofluorescence. This trend was more evident in PAICS due presumably to active de novo purine biosynthesis in highly-proliferative cell lines and a subset of DLBCLs, including MYC-positive DLBCLs. These findings were also validated using the DepMap, a publicly-available genome-wide CRISPR/Cas9 dropout screen datasets. PAICS was among the top-ranked essential genes for the survival of DLBCL cell lines. Since co-expression of MYC and PAICS in a subset of DLBCL were indicative of a functional relationship between the two factors, we explored publicly-available ChIP-seq datasets to see if MYC directly regulates PAICS expression. As expected, MYC ChIP-seq signals were highly enriched near the PAICS promoter in a series of cancer cell lines. Furthermore, shRNA-mediated MYC knockdown led to reduced levels of PAICS mRNA in MYC-positive DLBCL cells and significantly slowed their growth. Collectively, these data suggest that PAICS is a direct transcriptional target of MYC, playing a key role in proliferation of MYC-positive DLBCL cells. To assess the feasibility of PAICS-inhibition as a therapeutic option for MYC-positive DLBCLs, we tested MRT252040 for its anti-lymphoma activity in vitro and in vivo. To do so, we first assessed cell cycle status and Annexin positivity upon MRT252040 treatment using a series of DLBCL cell lines. As expected, MRT252040-mediated PAICS inhibition induced cell cycle arrest and apoptosis. Furthermore, MRT252040 treatment significantly delayed proliferation of DLBCL cell lines, namely those harboring MYC rearrangements. Finally, to assess anti-lymphoma activity of MRT252040 in vivo, we tested MRT252040 efficacy using patient-derived xenograft DLBCL. After xenotransplantation, proportions of lymphoma cells per total mononuclear cells in peripheral blood were examined over time by FACS, and MRT252040 (or vehicle) treatment was initiated once lymphoma cells constituted &gt;0.1%. MRT252040-treated mice survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of MRT252040 monotherapy against DLBCL in vivo. Our data suggest that MYC regulates the de novo purine synthesis pathway via directly transactivating PAICS expression. We propose that MRT252040, a newly-developed PAICS inhibitor, warrants attention as a novel therapeutic approach for MYC-positive DLBCLs, which otherwise exhibit poor clinical outcomes. Disclosures Ohshima: SRL, Inc.: Consultancy; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding. Akashi:Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

scholarly journals UPF1 Promotes Chemoresistance to Oxaliplatin Through Maintenance of Stemness by Increasing Phosphorylated TOP2A in Colorectal Cancer.

2020 ◽  
Author(s):  
Congcong Zhu ◽  
Long Zhang ◽  
Senlin Zhao ◽  
Weixing Dai ◽  
Yun Xu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Overall Survival ◽  
Cell Lines ◽  
Clinical Relevance ◽  
Poor Prognosis ◽  
Therapy Strategy ◽  
Underlying Mechanisms

Abstract Background: UPF1 is proved to dysregulate in multiple tumors and influence carcinogenesis. However, the role of UPF1 on oxaliplatin resistance in colorectal cancer (CRC) remains unknown.Methods: Firstly, we investigated the clinical relevance of UPF1 in CRC patients. Then, we explored the influence of UPF1 on chemoresistance to oxaliplatin in vitro and in vivo. Finally, we disclosed the underlying mechanisms of oxaliplatin resistance induced by UPF1.Results: UPF1 is upregulated in CRC and overexpression of UPF1 more likely results in recurrence in CRC patients and predicts a poorer overall survival (OS). UPF1 maintains stemness in CRC cell lines and promotes chemoresistance to oxaliplatin in CRC. UPF1-induced oxaliplatin resistance can be associated with interaction with TOP2A and increasing phosphorylated TOP2A.Conclusions: UPF1 was overexpressed and predicted a poor prognosis in CRC. UPF1 enhanced the stemness and chemoresistance to oxaliplatin by interaction with TOP2A and increase of phosphorylated TOP2A in CRC, which may provide a new therapy strategy for chemoresistance to oxaliplatin in CRC patients.

scholarly journals NOX1 Inhibition Attenuates the Development of a Pro-Tumorigenic Environment in Experimental Hepatocellular Carcinoma

2020 ◽  
Author(s):  
Astrid Vandierendonck ◽  
Helena Degroote ◽  
Bart Vanderborght ◽  
Lindsey Devisscher ◽  
Hans Van Vlierberghe
Keyword(s):  
Cell Lines ◽  
Poor Prognosis ◽  
Macrophage Polarization ◽  
Tumor Formation ◽  
Advanced Hcc ◽  
Treatment Regimens ◽  
Metavir Score ◽  
Systemic Treatments

Abstract Background The poor prognosis of advanced HCC and limited efficacy of current systemic treatments emphasize the need for new or combined targeted therapies. The development of HCC is a multistage process in which liver injury appears in a complex microenvironment associated with oxidative stress. NOX enzymes are the main source of ROS during hepatocarcinogenesis and NOX1 in particular has shown correlation with poor prognosis of HCC patients. This study evaluates the effect of pharmacological NOX1 inhibition on the development and progression of HCC and its effect on the tumor microenvironment.Methods The in vitro cytotoxic effects of the NOX1 inhibitor GKT771 (Genkyotex) on human Huh7 and Hep3B and murine Hepa1-6 HCC cell lines, and murine macrophages were evaluated via MTT, LDH activity and CaspGlo® assays. In order to induce in vivo HCC, male SV129 wild-type mice received weekly IP injections of diethylnitrosamine (DEN) (35 mg/kg) for 20-25 weeks. Mice were treated with vehicle or GKT771 (30 mg/kg) via oral gavage. Treatment duration and frequency (daily or twice daily) varied in the preventive and therapeutic studies. mRNA transcript levels in the tumor and liver tissue were determined by RT-qPCR. Fibrosis was visualized by Sirius Red staining and quantified by the Metavir​ score. Results A concentration-dependent reduction in cellular activity of the human HCC cell lines without cytotoxicity was observed. GKT771 treatment reduced LPS-induced pro-inflammatory bone-marrow derived macrophage polarization. DEN injections resulted in 100% tumor formation and the induction of HCC markers which could be reduced by twice daily dosing of GKT771 at early onset of advanced HCC. DEN-induced HCC resulted in an upregulation of pro-inflammatory, angiogenic and fibrotic markers which was less pronounced in GKT771 treated mice in all treatment regimens. In line, liver fibrosis was induced in HCC mice and this to a lesser extend upon GKT771 treatment.Conclusion NOX1 inhibition showed to be safe and well tolerated and was able to attenuate the induction of a pro-inflammatory, angiogenic and pro-fibrotic microenvironment suggesting that this might be a promising adjuvant therapeutic strategy in the treatment of advanced HCC.

Development of a Novel Phthalimide Derivative, Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5718-5718
Author(s):  
Yutaka Hattori ◽  
Maiko Matsushita ◽  
Noriko Tabata ◽  
Hirokazu Shiheido ◽  
Hiroshi Yanagawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Cell Lines ◽  
Pharmacokinetic Study ◽  
Bone Disease ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Phthalimide Derivative

Abstract BACKGROUND: Despite recent advances in the use of newly developed drugs including immune-modulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide and proteasome inhibitors such as bortezomib, carfilzomib, and MLN9708, MM is still an incurable disease. In particular, MM patients harboring 17p deletion, t(14;16), t(14;20), or t(4;14) are classified as a high-risk group and have shown significantly shorter survival. With the goal of helping prolong the survival of these high-risk MM patients, we screened 29 synthetic phthalimide derivatives and found a novel compound, 2-(2,6-diisopropylphenyl)-5-amino-1H-isoindole-1,3-dione (TC11), which induced the apoptosis of KMS34 cells with t(4;14) and del17p13. PURPOSE:The purpose of this project is to clarify preclinical effects of the synthetic phthalimide derivative, TC11, on high-risk MM cell lines and osteoclasts. Namely, anti-myeloma and anti-osteoclastogenic activities and pharmacokinetic study in mice were shown. We also try to isolate directly binding molecules. Safety issues including hematological toxicities and teratogenicity were also discussed. METHODS AND RESULTS: TC11 significantly inhibited growth of MM cell lines (IC50 4-8μM) including KMS34 and KMS11 cells which have high-risk chromosomal abnormalities. TC11 also suppressed the proliferation of all of the bone marrow cells obtained from the MM patients, in a dose-dependent manner. TC11 increased annexin V-positive fraction and induced apoptosis. TC11 was injected intraperitonealy into myeloma (KMS34 and KMS11 cells)-bearing lcr/SCID mice, and anti-myeloma activity was evaluated in vivo. Twenty mg/kg of TC11 significantly inhibited growth of KMS34 or KMS11-derived plasmacytomas. Apoptosis of MM cells was observed by histopathological examination. In order to evaluate hematological toxicity of TC11, growth of colony-forming cells was examined. In the presence of 5μM of TC11, formation of CFCs was not significantly suppressed, suggesting low hematopoietic toxicity. In the pharmacokinetic analyses using lcr mice, the plasma concentrations of TC11 was examined; Cmaxwas 18.1μM at 1.5hr (Tmax), and T1/2 was 2.5hr, when 100mg/kg of TC11 was injected. If 20mg/kg was injected, Cmaxwas 2.1μM at 1.0hr (Tmax), and T1/2 was 1.2hr. Oral administration of TC11 to Icr mice was safely carried out, and results of pharmacokinetic study will be shown. Aiming at the therapeutic use of TC11 to bone disease, anti-osteoclastogenic activity was examined. Mouse bone marrow mononuclear cells were incubated in the presence of M-CSF and RANK-ligand. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts was reduced in number in the presence of 1μM of TC11. It was also found that 1μM of TC11 inhibited bone resorption by pit assay. We have identified nucleophosmin 1 (NPM1) and α-tubulin as TC11-binding molecules using our unique in vitro selection system using mRNA display, in vitro virus (IVV) method. However, cereblon (CRBN) was not detected as a TC11-binding protein by this method. The immunofluorescent analysis showed that TC11-treated cells exhibited elevated levels of α-tubulin fragmentation. Together with our previous observation of induction of centrosomal disruption of HeLa cells by NPM1-knock down, TC11 may cause anti-myeloma effects via mitotic catastrophe. CONCLUSION: We have demonstrated that TC11, a novel phthalimide derivative, has anti-tumor activity against MM cells with high-risk genetic abnormality including del 17p and t(4;14), in vitro and in vivo. This novel compound also down-regulates the differentiation and function of osteoclasts. Our data provide a strong preclinical rationale for TC11 as a safe and effective drug for the treatment of high-risk MM patients with bone disease. The actions of this drug relating to α-tubulin and NPM1 remain to be further investigated. TC11 exerts its anti-myeloma effect via molecular interactions which do not involve CRBN. In addition, TC11 does not form racemate and is expected to lack teratogenicity. The results of our present study suggest that new phthalimide derivatives other than thalidomide, lenalidomide and pomalidomide could be developed by drug designing for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals NOX1 inhibition attenuates the development of a pro‐tumorigenic environment in experimental hepatocellular carcinoma

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Astrid Vandierendonck ◽  
Helena Degroote ◽  
Bart Vanderborght ◽  
Xavier Verhelst ◽  
Anja Geerts ◽  
...  
Keyword(s):  
Cell Lines ◽  
Poor Prognosis ◽  
Macrophage Polarization ◽  
Tumor Formation ◽  
Advanced Hcc ◽  
Treatment Regimens ◽  
Systemic Treatments ◽  
Mouse Macrophages

Abstract Background The poor prognosis of advanced HCC and limited efficacy of current systemic treatments emphasize the need for new or combined targeted therapies. The development of HCC is a multistage process in which liver injury appears in a complex microenvironment associated with oxidative stress. NOX enzymes are the main source of ROS during hepatocarcinogenesis and NOX1 in particular has shown correlation with poor prognosis of HCC patients. This study evaluates the effect of pharmacological NOX1 inhibition on the development and progression of HCC and its effect on the tumor microenvironment. Methods The in vitro cytotoxic effects of the NOX1 inhibitor GKT771 (Genkyotex) on human Huh7 and Hep3B and murine Hepa1-6 HCC cell lines, the human THP1 monocyte cell line and mouse macrophages were evaluated via MTT, LDH activity and CaspGlo® assays. In order to induce in vivo HCC, male SV129 wild-type mice received weekly IP injections of diethylnitrosamine (DEN) (35 mg/kg) for 20–25 weeks. Mice were treated with vehicle or GKT771 (30 mg/kg) via oral gavage, daily or twice daily, in preventive and therapeutic studies. The liver damage was evaluated for inflammation, angiogenesis, fibrosis and HCC development via histology, RT-qPCR, multiplex analyses and ROS levels. Results A concentration-dependent reduction in cellular activity of the human HCC cell lines without cytotoxicity was observed. GKT771 treatment reduced LPS-induced pro-inflammatory bone-marrow derived macrophage polarization. DEN injections resulted in 100 % tumor formation and the induction of HCC markers which could be reduced by twice daily dosing of GKT771 at early onset of advanced HCC. DEN-induced HCC resulted in an upregulation of pro-inflammatory, angiogenic and fibrotic markers which was less pronounced in GKT771 treated mice in all treatment regimens. In line, liver fibrosis was induced in HCC mice and this to a lesser extend upon GKT771 treatment. Conclusions NOX1 inhibition showed to be safe and well tolerated and was able to attenuate the induction of a pro-inflammatory, angiogenic and pro-fibrotic microenvironment suggesting that this might be a promising adjuvant therapeutic strategy in the treatment of advanced HCC.

scholarly journals Mechanisms Regulating Myeloma Cell Responsiveness to Antisense Oligonucleotides Targeting IRF4

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Ryosuke Shirasaki ◽  
Esperanza M Algarín ◽  
Ricardo De Matos Simoes ◽  
Sondra L. Downey-Kopyscinski ◽  
Shizuka Yamano ◽  
...  
Keyword(s):  
High Risk ◽  
Cell Lines ◽  
Cell Response ◽  
Antisense Oligonucleotides ◽  
Research Funding ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Genome Scale

Functional genomics studies from our group and others, including CRISPR screens, have documented that interferon regulatory factor 4 (IRF4) is a critical transcription factor (TF) for multiple myeloma (MM) cells in preclinical in vitro and in vivo models; and one of the top most pronounced and recurrent dependencies for MM cells vs. other neoplasias. IRF4 lacks known ligand-binding pocket(s) amenable to selective inhibition by small-molecule pharmacological agents. IRF4 is thus considered, as many TFs, as "undruggable". Recent progress in antisense oligonucleotides (ASOs) and their in vivo properties motivated us to systematically evaluate the response of MM cells to anti-IRF4 ASOs, to obtain direct insights into their potential therapeutic applications in MM and also as functional probes into IRF4 biology. We observed that a panel of anti-IRF4 ASOs (but not control ASO) exhibited dose- and time-dependent activity against genotypically diverse MM cell lines (sub-uM IC50s for most MM lines vs. &gt;10uM against non-MM cells lacking IRF4, e.g. HS5 bone marrow stromal cells [BMSCs]). Major in vitro anti-MM activity could be observed within 3-5 days of treatment; and was preceded by significant decrease in intracellular IRF4 protein levels. Importantly, 24-hour exposure to IRF4 ASO (followed by washout and subsequent culture in ASO-free media for up to 6 days) can induce similar anti-MM activity as continuous in vitro exposure over 6 days. Combinations of IRF4-ASO with several established (e.g. proteasome inhibitor, thalidomide derivative, glucocorticoids) or investigational (e.g. venetoclax) agents for MM result led to enhanced or even synergistic effects in a panel of 6 MM cell lines. For some of these MM cell lines, their in vitro response to IRF4 ASO had statistically significant attenuation in co-cultures with BMSCs: this effect was partially recapitulated in MM cell monocultures supplemented by conditioned media from BMSCs or (to a lesser extent) rhIL6 treatment. To obtain insights into cell-autonomous and nonautonomous mechanisms regulating MM cell responses to IRF4 ASOs, we examined the transcriptional profiles of MM.1S cells exposed to ASOs; and also performed genome-scale CRISPR studies to define genes whose editing (loss-of-function, LOF) or activation (gain-of-function, GOF) alter the MM cell response to the IRF4 ASOs. The transcriptional signature of MM cell treatment with IRF4 ASO (vs. control ASO) comprised distinct clusters of downregulated genes with preferential expression in normal or malignant plasma cells vs. other lineages; CRISPR-validated roles as dependencies for MM cells in vitro (preferentially essential for MM vs. non-MM tumors; or pan-essential); and/or proximity to large areas of chromatin accessibility (defined by H3K27Ac genome-wide ChIP-Seq or ATAC-Seq). Importantly, several genes downregulated by IRF4 ASO treatment are upregulated in MM cells cocultured with BMSCs, possibly explaining at least in part the impact of co-cultures on anti-MM activity of ASO. In our integrated genome-scale CRISPR studies (and validation of many genes with individual sgRNAs), MM cell response to IRF4 ASOs was attenuated by LOF of endonuclease RNase H1 (RNASEH1; which degrades the target RNA when it establishes intracellular hybrids with the ASOs); LOF of diverse genes involved in ASO endocytosis (e.g. IGF2R, PICALM, SH3GL1, RAB5C) or regulation of chondroitin/heparan sulfate (SLC35B2, B3GAT3, B4GALT7, ALG5 and ALG6); GOF of IRF4 itself (likely reflecting a "stoichiometric" effect, e.g. induction of higher IRF4 mRNA levels may require higher concentrations of ASO to maintain anti-MM effect); and GOF of exocytosis-related genes(e.g. CLU, QPCT). Importantly, LOF or GOF of individual genes typically associated with high-risk MM was not associated with decreased MM cell response to the IRF4 ASOs: therefore, retaining intracellular accumulation of IRF4 ASO and productive knockdown of IRF4 mRNA remains a primary driver of anti-MM activity of ASOs even in the context of biologically aggressive MM cells with "high-risk" features. Our results provide comprehensive integrated assessment of the molecular and functional landscapes associated with dysregulation of IRF4; have direct implications for our mechanistic understanding of the role of IRF4 in MM biology; and provide a framework for IRF4 targeting by ASOs or potentially other therapeutic approaches. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Luo:Ionis Pharmaceuticals, Inc.: Current Employment. Kim:Ionis Pharmaceuticals, Inc.: Current Employment. MacLeod:Ionis Pharmaceuticals, Inc.: Current Employment. Mitsiades:Arch Oncology: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; Karyopharm: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; EMD Serono: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

scholarly journals Downregulation of PRAME Suppresses Proliferation and Promotes Apoptosis in Hepatocellular Carcinoma Through the Activation of P53 Mediated Pathway

2018 ◽  
Vol 45 (3) ◽  
pp. 1121-1135 ◽  
Author(s):  
Hanzhang Zhu ◽  
Jingrui Wang ◽  
Junjie Yin ◽  
Bei Lu ◽  
Qijun Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Cell Apoptosis ◽  
Poor Prognosis ◽  
P53 Pathway ◽  
Tissue Samples ◽  
Potential Biomarker

Background/Aims: The expression of PRAME and its role in hepatocellular carcinoma (HCC) remain unknown. The aim of this study was to examine the functional role of PRAME in HCC development and exploring the molecular mechanism. Methods: We first detected PRAME expression in 96 human HCC tissue samples and correlated with clinicopathological characteristics and prognosis of the patients. We then established stable HCC cell lines with PRAME overexpression and knockdown followed by functional analysis in vitro. Further, we examined the relationship between PRAME and p53 pathway in vitro by using Western blotting. Finally, PRAME expression was detected to evaluate its correlation with p-p53 and p53 pathway related apoptotic proteins in xenograft tumor mouse model using immunohistochemistry. Results: PRAME expression was significantly higher in HCC tissues than in adjacent non-tumor tissues and their expression was positively correlated with alpha fetoprotein levels and tumor size. In addition, PRAME expression was associated with AJCC stage and is a potential biomarker of poor prognosis regarding 5-year overall survival in HCC. In vitro studies, we found that PRAME expression was higher in HCC cell lines than in normal hepatic cell line. Inhibited cell proliferation and increased cell apoptosis was observed in PRAME knockdown HCC cells. Futher, increased cell apoptosis was correlated with the proportion of cells in G0/G1 stage, activated p53 mediated apoptosis, and increased cyclin p21 expression. Xenograft analysis in nude mice also found that PRAME knockdown inhibited tumorigenesis while PRAME overexpression had opposite effect. Conclusions: In HCC, PRAME serves as a potential biomarker for poor prognosis and novel therapeutic target in treating this cancer. PRAME is a potential biomarker of poor prognosis in HCC. PRAME surpresses HCC cell death in vitro and in vivo by regulating p53 apoptotic signaling and may serve as a potential therapeutic target in HCC.

scholarly journals NOX1 inhibition attenuates the development of a pro-tumorigenic environment in experimental hepatocellular carcinoma

2021 ◽  
Author(s):  
Astrid Vandierendonck ◽  
Helena Degroote ◽  
Bart Vanderborght ◽  
Xavier Verhelst ◽  
Anja Geerts ◽  
...  
Keyword(s):  
Cell Lines ◽  
Poor Prognosis ◽  
Macrophage Polarization ◽  
Tumor Formation ◽  
Advanced Hcc ◽  
Treatment Regimens ◽  
Systemic Treatments ◽  
Mouse Macrophages

Abstract Background The poor prognosis of advanced HCC and limited efficacy of current systemic treatments emphasize the need for new or combined targeted therapies. The development of HCC is a multistage process in which liver injury appears in a complex microenvironment associated with oxidative stress. NOX enzymes are the main source of ROS during hepatocarcinogenesis and NOX1 in particular has shown correlation with poor prognosis of HCC patients. This study evaluates the effect of pharmacological NOX1 inhibition on the development and progression of HCC and its effect on the tumor microenvironment.Methods The in vitro cytotoxic effects of the NOX1 inhibitor GKT771 (Genkyotex) on human Huh7 and Hep3B and murine Hepa1-6 HCC cell lines, the human THP1 monocyte cell line and mouse macrophages were evaluated via MTT, LDH activity and CaspGlo® assays. In order to induce in vivo HCC, male SV129 wild-type mice received weekly IP injections of diethylnitrosamine (DEN) (35 mg/kg) for 20-25 weeks. Mice were treated with vehicle or GKT771 (30 mg/kg) via oral gavage, daily or twice daily, in preventive and therapeutic studies. The liver damage was evaluated for inflammation, angiogenesis, fibrosis and HCC development via histology, RT-qPCR, multiplex analyses and ROS levels. Results A concentration-dependent reduction in cellular activity of the human HCC cell lines without cytotoxicity was observed. GKT771 treatment reduced LPS-induced pro-inflammatory bone-marrow derived macrophage polarization. DEN injections resulted in 100% tumor formation and the induction of HCC markers which could be reduced by twice daily dosing of GKT771 at early onset of advanced HCC. DEN-induced HCC resulted in an upregulation of pro-inflammatory, angiogenic and fibrotic markers which was less pronounced in GKT771 treated mice in all treatment regimens. In line, liver fibrosis was induced in HCC mice and this to a lesser extend upon GKT771 treatment.Conclusion NOX1 inhibition showed to be safe and well tolerated and was able to attenuate the induction of a pro-inflammatory, angiogenic and pro-fibrotic microenvironment suggesting that this might be a promising adjuvant therapeutic strategy in the treatment of advanced HCC.

scholarly journals Circ-0005105 activates COL11A1 by targeting miR-20a-3p to promote pancreatic ductal adenocarcinoma progression

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Gang Ma ◽  
Guichen Li ◽  
Wufeng Fan ◽  
Yuanhong Xu ◽  
Shaowei Song ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Cellular Proliferation ◽  
Ductal Adenocarcinoma ◽  
Loss Of Function ◽  
Gain And Loss ◽  
Proliferation And Invasion

AbstractGrowing evidence indicates that circular RNAs (circRNAs) are closely involved in tumorigenesis, but the association between circRNAs and pancreatic ductal adenocarcinoma (PDAC) is far from clear. Here, we focused on the functional investigation of circ-0005105, a newly identified circRNA, in PDAC progression. In the present study, we assessed circ-0005105 expression in PDAC tissues and cell lines with quantitative reverse transcription–polymerase chain reaction (qRT-PCR). The biological functions of circ-0005105 in cellular proliferation and invasion were identified through gain- and loss-of-function experiments in vitro and in vivo. The interaction between circ-0005105 and the microRNA (miR)-20a-3p–COL11A1 (collagen type XI alpha 1) axis was examined using luciferase reporter and RNA immunoprecipitation assays. We found that circ-0005105 expression was upregulated in both PDAC tissues and cell lines. Higher circ-0005105 expression correlated positively with the malignant clinical phenotype and poor prognosis of patients with PDAC. Gain- and loss-of-function analysis showed that circ-0005105 facilitated both in vitro and in vivo cellular proliferation and invasion. Mechanistically, circ-000510 served as a competing endogenous RNA (ceRNA) of miR-20a-3p and indirectly modulated COL11A1 expression, leading to activation of epithelial–mesenchymal transition (EMT). Rescue experiments suggested that the oncogenic activity of circ-0005105 was dependent on the modulation of the miR-20a-3p–COL11A1 axis. More importantly, COL11A1 overexpression was significantly associated with poor prognosis in PDAC, and silencing COL11A1 reduced PDAC cell tumorigenicity and metastasis. Taken together, our findings confirm for the first time that circ-0005105 has critical functions by regulating the miR-20a-3p–COL11A1 axis. In the clinic, circ-0005105 can act as a potential prognostic marker and therapeutic target in PDAC.

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