Single-arm study of bimiralisib in head and neck squamous cell carcinoma (HNSCC) patients (pts) harboring NOTCH1 loss of function (LOF) mutations.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. TPS6590-TPS6590
Author(s):  
Faye M. Johnson ◽  
Filip Janku ◽  
J. Jack Lee ◽  
Debora Schmitz ◽  
Henk Streefkerk ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Lines ◽  
Clonal Evolution ◽  
Mutant Cell ◽  
Objective Response ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Null Cell ◽  
Second Stage ◽  
Notch1 Mutations

TPS6590 Background: Effective targeted therapies are needed for HNSCC that is lethal despite recent advances with immunotherapy. A major challenge to personalize treatment is that most genomic alterations are in tumor suppressors, including NOTCH1 that is mutated in ~20% of HNSCC. We recently published that HNSCC cell lines harboring NOTCH1 LOF mutations undergo cell death in vivo and in vitro following PI3K inhibition, in contrast to PIK3CA mutant cell lines that merely undergo cell cycle arrest when exposed to the same drugs. Based on these results we initiated a novel genomic biomarker-driven phase II clinical trial treating NOTCH1 mutant HNSCC pts with the dual PI3K/mTOR inhibitor bimiralisib (PQR309). Methods: The primary objective is to determine the objective response rate (ORR) of recurrent/metastatic HNSCC harboring NOTCH1 LOF mutations to bimiralisib. Pts who have already received standard platinum chemotherapy and immunotherapy will receive bimiralisib orally twice per wk unless progression or intolerable toxicity occurs. Tumors will be evaluated using RECIST q 6 wks. A Simon’s optimal two-stage design is used. To have 80% power to detect an ORR of 30%, (one-sided α = 0.05, β = 0.20) 10 pts will be enrolled in the first stage. If ≤1 pts respond, the trial will be closed for futility. If ≥2 pts have an OR, the study will enroll an additional 19 pts in the second stage. The null hypothesis (ORR ≤ 10%) will be rejected if ≥ 6 in 29 pts have an OR. Seven pts have enrolled. The algorithm for determining NOTCH1 mutation function is based on the patterns of mutations in HNSCC vs. leukemia where mutations are activating. It may be difficult to determine whether NOTCH1 mutations are homo- or heterozygous due to normal cell contamination. Therefore, levels of activated NOTCH1 in pretreatment tumors may be assessed by IHC with an antibody against activated NOTCH1 (NICD). In parallel with the trial, to further confirm NOTCH1 LOF, we can use site-directed mutagenesis to re-create NOTCH1 mutations from trial pts that will then be introduced into NOTCH1-null cell lines to assay for NICD and growth inhibition with culture on NOTCH1 ligand. All pts will have serial collection of blood for pharmacokinetics and for ctDNA to examine clonal evolution associated with acquired resistance. Samples with high NOTCH1 mutation ctDNA VAF will be analyzed by WES and compared with pretreatment tissue. In the second stage, IHC and WES may be performed on pre- and post- treatment (day 15 and progression) tissue to examine pharmacodynamics and mechanisms of resistance. Clinical trial information: NCT03740100 .

Genomic biomarkers of response to lenvatinib/pembrolizumab (Len/Pembro) in patients with advanced renal cell carcinoma.

2020 ◽  
Vol 38 (6_suppl) ◽  
pp. 733-733 ◽  
Author(s):  
Chung-Han Lee ◽  
Renzo G. DiNatale ◽  
Diego Chowell ◽  
Chriag Krishna ◽  
Vladimir Makarov ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Renal Cell Carcinoma ◽  
Antigen Presentation ◽  
Cell Carcinoma ◽  
Tumor Antigen ◽  
Renal Cell ◽  
Objective Response ◽  
Evolutionary Divergence ◽  
Loss Of Function ◽  
Genomic Biomarkers

733 Background: A phase 1b/2 clinical trial indicated that len/pembro shows promise in the treatment of renal cell carcinoma (RCC) in both PD-1/PD-L1 immune checkpoint blockade (ICB)-naïve and pretreated patients (NCT02501096). The combination is being further investigated in a phase 3 clinical trial in RCC (NCT02811861). Tumor antigen presentation depends on multiple factors, including HLA diversity, which can be measured by HLA evolutionary divergence (HED). HED quantitates the capacity of a patient’s HLA genotype to present different peptide antigens. This study is investigating the genomic components of tumor antigen presentation in ICB-naïve patients who have RCC and are treated with len/pembro. Methods: Whole exome sequencing (WES) was performed on pretreatment tumor-derived DNA. Somatic mutations, tumor mutation burden (TMB), neoantigen (NA) load, germline HLA zygosity and somatic loss of heterozygosity (LOH), and HED were correlated with objective response rate (ORR) and progression-free survival (PFS). An updated clinical cutoff was March 29, 2019. Results: Twenty four (80%) of 30 ICB-naïve patients underwent WES. A top-quartile cutoff was used. Increased mean HED was associated with improved PFS, while HLA homozygosity or LOH trended toward worse PFS. Loss-of-function mutations in PBRM1 (PBRM1 LOF) trended toward improved PFS. However, TMB and NA load were not correlated to PFS. No genomic biomarkers were correlated to ORR. Conclusions: Increased HLA diversity was associated with improved PFS, while decreased HLA diversity may be associated with worse PFS. PBRM1 mutation may be associated with improved PFS; however, TMB and NA load were not correlated to outcomes. These findings warrant further examination in larger datasets to rule out possible artifacts from multiple testing in a small cohort. Clinical trial information: NCT02811861. [Table: see text]

A synonymous UPF3B variant causing a speech disorder implicates NMD as a regulator of neurodevelopmental disorder gene networks

2020 ◽  
Vol 29 (15) ◽  
pp. 2568-2578
Author(s):  
Deepti Domingo ◽  
Urwah Nawaz ◽  
Mark Corbett ◽  
Josh L Espinoza ◽  
Katrina Tatton-Brown ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rna Splicing ◽  
Gene Networks ◽  
Neurodevelopmental Disorder ◽  
Mutant Cell ◽  
Premature Termination Codon ◽  
Loss Of Function ◽  
Altered Expression ◽  
Functional Studies ◽  
Language Disabilities

Abstract Loss-of-function mutations of the X-chromosome gene UPF3B cause male neurodevelopmental disorders (NDDs) via largely unknown mechanisms. We investigated initially by interrogating a novel synonymous UPF3B variant in a male with absent speech. In silico and functional studies using cell lines derived from this individual show altered UPF3B RNA splicing. The resulting mRNA species encodes a frame-shifted protein with a premature termination codon (PTC) predicted to elicit degradation via nonsense-mediated mRNA decay (NMD). UPF3B mRNA was reduced in the cell line, and no UPF3B protein was produced, confirming a loss-of-function allele. UPF3B is itself involved in the NMD mechanism which degrades both PTC-bearing mutant transcripts and also many physiological transcripts. RNAseq analysis showed that ~1.6% of mRNAs exhibited altered expression. These mRNA changes overlapped and correlated with those we identified in additional cell lines obtained from individuals harbouring other UPF3B mutations, permitting us to interrogate pathogenic mechanisms of UPF3B-associated NDDs. We identified 102 genes consistently deregulated across all UPF3B mutant cell lines. Of the 51 upregulated genes, 75% contained an NMD-targeting feature, thus identifying high-confidence direct NMD targets. Intriguingly, 22 of the dysregulated genes encoded known NDD genes, suggesting UPF3B-dependent NMD regulates gene networks critical for cognition and behaviour. Indeed, we show that 78.5% of all NDD genes encode a transcript predicted to be targeted by NMD. These data describe the first synonymous UPF3B mutation in a patient with prominent speech and language disabilities and identify plausible mechanisms of pathology downstream of UPF3B mutations involving the deregulation of NDD-gene networks.

Phase II trial of olaparib in combination with ceralasertib in patients with recurrent osteosarcoma.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. TPS11575-TPS11575
Author(s):  
Suzanne J. Forrest ◽  
Michael D. Kinnaman ◽  
J Andrew Livingston ◽  
Kieuhoa Tran Vo ◽  
Priscilla Merriam ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Overall Survival ◽  
Cell Lines ◽  
Phase Ii ◽  
Objective Response ◽  
Parp Inhibitors ◽  
Clinical Activity ◽  
Osteosarcoma Cell ◽  
In Vitro Susceptibility ◽  
Correlative Studies

TPS11575 Background: Osteosarcoma is the most common primary bone tumor, occurring in children, adolescents, and young adults. In contrast to advances in treatment for most childhood cancers, there have been no significant improvements in osteosarcoma outcomes in the past 40 years. Forty percent of osteosarcoma patients will, at some point, have advanced disease which has a very poor outcome with a 5-year overall survival of approximately 20%. Genomic alterations and signatures associated with sensitivity to treatment with DNA damage response (DDR) inhibitors are observed frequently in osteosarcoma. Many osteosarcomas have a unique mutational signature (signature 3) similar to that seen in BRCA1 deficient cancer and response to PARP inhibitors has been seen in osteosarcoma cell lines. Additionally, ATRX, a protein involved in the alternative lengthening of telomeres (ALT), is often inactivated in osteosarcoma. Defects in the ALT pathway may sensitize tumor cells to ATR inhibitors and osteosarcoma cell lines have been shown to be sensitive to ATR inhibition. In vitro susceptibility of osteosarcoma cell lines to ATR and PARP inhibitors, the presence of mutations in genes involved in DDR and the presence of signature 3 in a subset of osteosarcomas serves as the basis for the development of this trial. Methods: This is an ongoing open label, multicenter, phase II clinical trial to evaluate the clinical activity of PARP inhibitor, olaparib, in combination with ATR inhibitor, ceralasertib, in 2 cohorts of patients aged 12-40 with recurrent osteosarcoma (NCT04417062). Patients with unresectable disease are enrolled into Cohort 1. Patients with resectable disease limited to the lung are enrolled into Cohort 2. Patients in both cohorts receive olaparib 300mg orally twice a day on days 1-28 and ceralasertib 160 mg orally once a day on days 1-7 of a 28-day cycle (adult maximum tolerated dose for the combination). For patients in Cohort 2, study treatment also includes surgical resection of lung metastases at protocol-specified timepoints. Patients can remain on treatment for up to 2 years if they have not progressed. For Cohort 1, the primary objective is to determine whether the combination treatment improves the 4-month event-free rate as compared to a historical benchmark from Children’s Oncology Group (COG) trials using a Simon’s two-stage design. In the first stage, ≥3 of 19 patients must be event-free at 4 months to proceed to stage 2. Enrollment into Cohort 2 continues as long as Cohort 1 enrollment is ongoing. For Cohort 2, the primary endpoint is the submission of paired pre- and post-treatment tumor samples for correlative studies. Secondary endpoints include objective response rate, event-free survival, and overall survival. Integrated correlative studies will assess tumor tissue for biomarkers of treatment response and measure circulating tumor DNA longitudinally. Enrollment began November 24, 2020 and is ongoing. Clinical trial information: NCT04417062.

scholarly journals Haploinsufficiency and Deletions of G3BP1 on Chromosome 5q Result in Induction of TP53

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 784-784
Author(s):  
Naoko Hosono ◽  
Mahfouz Reda ◽  
Bartlomiej P Przychodzen ◽  
Chantana Polprasert ◽  
Latifa Zekri ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Board Of Directors ◽  
Cell Lines ◽  
Lower Risk ◽  
Chromosomal Instability ◽  
Somatic Mutations ◽  
Clonal Evolution ◽  
Loss Of Function ◽  
Advisory Committees ◽  
Low Expression

Abstract Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. The extent of individual defects vary, which may account for observed clinical diversity. Del(5q) pathogenesis has been related to haploinsufficiency of genes contained in the common deleted regions (CDR), including RPS14, miR-145/146a and SPARC. Driver mutations or pathogenic microdeletions were not identified for these genes, suggesting that multiple genes must function in combination to promote clonal evolution and phenotypic heterogeneity. Hence, we performed a comprehensive analysis of somatic mutations in genes located on chromosome 5 (chr5), both in patients with diploid 5q and in those with del(5q), to clarify the role of germline and somatic mutations in disease pathogenesis. In parallel, expression analysis was performed to correlate haploinsufficiency with the frequency of mutational events, in particular for diploid 5q cases. Applying SNP-array karyotyping to samples from 146 patients with del(5q), the lesion was identified in 5q31.1q33.1. Two retained regions (CRRs) were also observed in q11.1q14.2 (CRR1) and q34qter (CRR2). Lower-risk MDS is frequently affected by CDR, while in higher-risk MDS and secondary AML CRR1/2 are commonly co-involved. Using whole exome sequencing, we identified 11 hemizygous mutations located within the deleted area in del(5q) (N=59), while in cases diploid for 5q (N=330), 243 heterozygous mutations were found. One of the mutations discovered on chr5q afflicted a gene G3BP1 (5q33.1), located within the CDR and present in 2 patients. Both were missense mutations (one heterozygous and the other homo/hemizygous). A mutant case showed good responses to lenalidomide even though diploid 5. In addition, other somatic mutations of driver genes including TET2, CUX1 and EZH2 were concomitantly observed. Whole transcriptome sequencing demonstrated hemizygous loss of G3BP1 resulting in haploinsufficiency. G3BP1 was haploinsufficient in 48% of RAEB as well as low-risk MDS cases with del(5q). Overall, defective G3BP1 is associated with shorter overall survival (P<.001) in AML, consistent with the reports that del(5q) is a worse prognostic factor in myeloid neoplasms with aggressive phenotype. G3BP1 is a nuclear RNA-binding protein and is ubiquitously expressed in bone marrow, CD34+ progenitors and leukemic cell lines. Furthermore, G3BP1 binds to TP53 and its expression leads to the redistribution of TP53 from the nucleus to the cytoplasm. Similar to RPS14, haploinsufficient of G3BP1 resulted in TP53 up-modulation. Moreover, low expression of G3BP1 in diploid 5q cases was indeed associated with higher TP53 expression. Next, we generated haploinsufficient G3BP1 cell lines using shRNA transduction. Decreased expression of G3BP1 led to growth inhibition and impaired colony formation by transduced cells lines and hematopoietic progenitor cells, respectively. Knockdown of G3BP1 in K562 cell line increased TP53 in the nucleus, and when treated with CPT11, DNA-damaged induced G1-arrest was more prominent in knockdown cells. Furthermore, after knockdown of G3BP1 in TP53-null HL60 cells, we observed increased aneuploidy, suggesting that the loss of function of G3BP1 and TP53 may result in chromosomal instability. Most significantly, G3bp1-/+ mice showed lower blood counts and defective, dysplastic hematopoiesis, similar to lower-risk MDS. As previously described, TP53 defects are associated with advanced disease but recently it became apparent that TP53 may be one of the most common somatic lesions found in the context of del(5q). We stipulate that loss of TP53 function might overcome TP53 tumor suppressor effects and induce leukemic evolution in the defective G3BP1 status. In our cohort, TP53 mutations were more frequently present in high-risk phenotype with G3BP1 haploinsufficient expression. In conclusion, novel somatic mutations of G3BP1 suggest that it could be a candidate gene associated with the clonal evolution of del(5q). Loss of function or low expression of G3BP1 has been shown to up-modulate TP53 and result in dysplasia and growth inhibition, hallmarks of early stages of MDS. Additional events constitute loss of function of TP53, resulting in chromosomal instability, which is associated with leukemogenesis. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.

Signalling by the Platelet C-Type Lectin Receptor CLEC-2 Is Mediated by a Novel Mechanism Involving Syk and a Single YxxL Motif.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 381-381
Author(s):  
Andrew C. Pearce ◽  
Gemma L.J. Fuller ◽  
Katsue Suzuki-Inoue ◽  
Michael G. Tomlinson ◽  
Steve P. Watson
Keyword(s):  
Cell Lines ◽  
Transmembrane Protein ◽  
Mutant Cell ◽  
Consensus Sequence ◽  
Adaptor Proteins ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Tyrosine Residue ◽  
Lectin Receptor ◽  
Platelet Receptor

Abstract We have recently identified the C-type lectin-like receptor CLEC-2 as a novel receptor for the snake toxin rhodocytin on platelets. CLEC-2 is a 32 kDa type II transmembrane protein with a single cytoplasmic tyrosine residue in a YxxL consensus sequence. It is the first C-type lectin receptor that has been shown to activate platelets. The aim of the present study was to investigate the mechanism of signalling by CLEC-2 using model cell lines and to establish the role of the YxxL motif in this process. We have expressed CLEC-2 in wild type DT40 B cells and Jurkat T cells and in mutant derivatives of these cell lines that lack key signalling proteins. We have assayed PLCγ activation using an NFAT-luciferase reporter assay. Site directed mutagenesis of the cytoplasmic tyrosine residue of CLEC-2 has been used to assess its role in CLEC-2 signalling. We have compared the signalling pathway of CLEC-2 with that of the ITAM-coupled collagen receptor GPVI by expressing GPVI in the mutant cell lines and monitoring responses to the GPVI specific agonist convulxin as above. Cells transfected with CLEC-2 respond to rhodocytin. Mutation of the CLEC-2 YxxL motif to FxxL abrogates CLEC-2 signalling. CLEC-2 signalling is abrogated in cells deficient in Src or Syk family kinases or PLCγ . CLEC-2 signalling is partially dependent on Tec family kinases and the adaptors LAT and SLP-76/BLNK. Interestingly, GPVI signalling and CLEC-2 signalling show a differential requirement for the SLP-76 family adaptor proteins SLP-76 or Blnk; GPVI signalling is entirely dependent on SLP-76/Blnk, whereas CLEC-2 signalling is only partially dependent on these proteins. These observations are consistent with a model whereby CLEC-2 recruits Syk via its YxxL motif and initiates a signalling cascade similar to the major platelet glycoproteins GPVI and integrin α IIbβ 3. CLEC-2 is the first platelet receptor shown to regulate Syk through a single YxxL motif. Importantly, despite the similarity in the signalling proteins involved in signalling by the ITAM receptor, the integrin receptor and the C-type lectin receptor, the signalling pathways are organised and initiated differently.

scholarly journals DDRE-06. CELLULAR STRESS RESPONSE IN DIPG THERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii62-ii62
Author(s):  
Sreepradha Sridharan ◽  
Arif Harmanci ◽  
Robert Siddaway ◽  
Tara Dobson ◽  
Jyothishmathi Swaminathan ◽  
...  
Keyword(s):  
Stress Response ◽  
Single Cell ◽  
Synthetic Lethality ◽  
Clonal Evolution ◽  
Single Agent ◽  
Objective Response ◽  
Hdac Inhibitors ◽  
Pediatric Brain Tumor ◽  
Dominant Negative ◽  
Cellular Stress Response

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor of the pons and brainstem. Therefore, there is a desperate need for new therapeutics. Genomic profiling of tumors identified a highly prevalent dominant negative somatic mutation at lysine (K)-27 in histone genes HIST1H3B and H3F3A. Clonal evolution modeling suggests these mutations are truncal, and studies have demonstrated their contribution to tumorigenesis. ONC201, a first-in-class DRD2 antagonist and ClpP agonist is an anticancer drug developed by Oncoceutics, which targets the unfolded protein response (UPR) and integrated stress response (ISR) signaling and is actively being investigated in patients with recurrent H3 K27M-mutant gliomas. In adults with recurrent glioma, single agent studies showed benign-safety, no dose-limiting toxicities and a durable objective response when administered orally. In addition, intra-tumoral drug levels exceeded therapeutic thresholds, and induced tumor cell apoptosis. Based on this and response seen in a pediatric patient with DIPG for whom compassionate use of ONC201 was approved, a multi-arm, non-randomized multi-institutional Phase I clinical trial (NCT03416530) is actively accruing patients. However, the strength of UPR and ISR in DIPGs and their effect on DIPG response to ONC201 is not known. Our group employed bulk/single cell transcriptomic and single cell proteomic approaches to demonstrate substantial heterogeneity in UPR and ISR signaling in human DIPG samples. Consistent with this, DIPG cell lines exhibited considerable variability in sensitivity to ONC201. Single cell profiling identified tumor sub-populations with significant proliferative capacity even after ONC201 exposure. Incomplete response promotes recurrence. To target these cells, we performed a synthetic lethality screen with a library of 360 FDA-approved CNS penetrant compounds, which identified HDAC inhibitors and DNA damage-inducing chemotherapy as having synergy with ONC201. Thus, we suggest that tumor heterogeneity impacts sensitivity to ONC201 and that this can be reduced by combination treatments.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals Long noncoding RNA LINC00941 promotes pancreatic cancer progression by competitively binding miR-335-5p to regulate ROCK1-mediated LIMK1/Cofilin-1 signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Wang ◽  
Zhiwei He ◽  
Jian Xu ◽  
Peng Chen ◽  
Jianxin Jiang
Keyword(s):  
Pancreatic Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Progression ◽  
Noncoding Rna ◽  
Epithelial Mesenchymal Transition ◽  
Loss Of Function ◽  
Mesenchymal Transition

AbstractAn accumulation of evidence indicates that long noncoding RNAs are involved in the tumorigenesis and progression of pancreatic cancer (PC). In this study, we investigated the functions and molecular mechanism of action of LINC00941 in PC. Quantitative PCR was used to examine the expression of LINC00941 and miR-335-5p in PC tissues and cell lines, and to investigate the correlation between LINC00941 expression and clinicopathological features. Plasmid vectors or lentiviruses were used to manipulate the expression of LINC00941, miR-335-5p, and ROCK1 in PC cell lines. Gain or loss-of-function assays and mechanistic assays were employed to verify the roles of LINC00941, miR-335-5p, and ROCK1 in PC cell growth and metastasis, both in vivo and in vitro. LINC00941 and ROCK1 were found to be highly expressed in PC, while miR-335-5p exhibited low expression. High LINC00941 expression was strongly associated with larger tumor size, lymph node metastasis, and poor prognosis. Functional experiments revealed that LINC00941 silencing significantly suppressed PC cell growth, metastasis and epithelial–mesenchymal transition. LINC00941 functioned as a molecular sponge for miR-335-5p, and a competitive endogenous RNA (ceRNA) for ROCK1, promoting ROCK1 upregulation, and LIMK1/Cofilin-1 pathway activation. Our observations lead us to conclude that LINC00941 functions as an oncogene in PC progression, behaving as a ceRNA for miR-335-5p binding. LINC00941 may therefore have potential utility as a diagnostic and treatment target in this disease.

scholarly journals DDRE-03. IDH1-MUTANT GBM CELLS ARE HIGHLY SENSITIVE TO COMBINATION OF KDM6A/B AND HDAC INHIBITORS

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i6-i7
Author(s):  
Alişan Kayabölen ◽  
Gizem Nur Sahin ◽  
Fidan Seker ◽  
Ahmet Cingöz ◽  
Bekir Isik ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Mutant Cell ◽  
Glioma Cells ◽  
Epigenetic Therapy ◽  
Low Grade ◽  
Orthotopic Model ◽  
Cycle Arrest ◽  
Mutant Cells

Abstract Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

scholarly journals Establishment of Cell Lines from Both Myeloma Bone Marrow and Plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a Single Patient

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Youngil Koh ◽  
Woo-June Jung ◽  
Kwang-Sung Ahn ◽  
Sung-Soo Yoon
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Clonal Evolution ◽  
Myeloma Cell ◽  
Mouse Bone Marrow ◽  
Single Patient ◽  
U266 Cell

Purpose.We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM).Materials and Methods.Mononuclear cells obtained from MM patient’s bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM.Results.After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC.Conclusion.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

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