scholarly journals Random clonal expansion as a limiting factor in transplantable in vivo CRISPR/Cas9 screens

2021 ◽  
Author(s):  
Tet Woo Lee ◽  
Francis W Hunter ◽  
William R Wilson ◽  
Stephen MF Jamieson
Keyword(s):  
Gene Function ◽  
Fold Change ◽  
Effect Sizes ◽  
Whole Genome ◽  
Library Size ◽  
Log2 Fold Change ◽  
Nsg Mice ◽  
Change Effect ◽  
Tumour Initiation

Transplantable in vivo CRISPR/Cas9 knockout screens, in which cells are transduced in vitro and inoculated into mice to form tumours in vivo, offer the opportunity to evaluate gene function in a cancer model that incorporates the multicellular interactions of the tumour microenvironment. In this study, we sought to develop a head and neck squamous cell carcinoma (HNSCC) tumour xenograft model for whole-genome screens that could maintain high gRNA representation during tumour initiation and progression. To achieve this, we sought early-passage HNSCC cell lines with a high frequency of tumour initiation-cells, and identified the pseudodiploid UT-SCC-54C line as a suitable model from 23 HNSCC lines tested based on a low tumourigenic dose for 50% takes (TD50) of 1100 cells in NSG mice. On transduction with the GeCKOv2 whole-genome gRNA library (119,461 unique gRNAs), high (80-95%) gRNA representation was maintained in early (up to 14 d) UT-SCC-54C tumours in NSG mice, but not in UT-SCC-74B tumours (TD50=9200). However, loss of gRNA representation was observed in UT-SCC-54C tumours following growth for 38-43 days, which correlated with a large increase in bias among gRNA read counts due to stochastic expansion of clones in the tumours. Applying binomial thinning simulations revealed that the UT-SCC-54C model would have 40-90% statistical power to detect drug sensitivity genes with log2 fold change effect sizes of 1-2 in early tumours with gRNA libraries of up to 10,000 gRNAs and modest group sizes of 5 tumours. In large tumours, this model would have had 45% power to detect log2 fold change effect sizes of 2-3 with libraries of 2,000 gRNAs and 14 tumours per group. Based on our findings, we conclude that gRNA library size, sample size and tumour size are all parameters that can be individually optimised to ensure transplantable in vivo CRISPR screens can successfully evaluate gene function.

A Whole Genome In Vivo Crispr Screen in Primary ALL Predicts Leukaemic Relapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2619-2619
Author(s):  
Katherine Dormon ◽  
Elda S Latif ◽  
Matthew Bashton ◽  
Deepali Pal ◽  
Matthew Selby ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Whole Genome ◽  
Competitive Situation ◽  
Nsg Mice ◽  
A Genome ◽  
Crispr Screen

Abstract Although paediatric acute lymphoblastic leukaemia (ALL) has a favourable prognosis, a number of cases will invariably relapse. One of the major problems associated with relapse is drug resistance, in particular to glucocorticoids, the mainstay of ALL treatment. Examining the underlying mechanisms is complicated by clonal heterogeneity within a patient and the potential impact of the leukaemic niche. To address mechanisms of drug resistance in a patient-relevant setting, we performed a genome-wide in vivo CRISPR screen in primary ALL material. To that end, we took advantage of primografted material from patient L707, who initially presented with a Dexamethasone (DEX) sensitive t(17;19) ALL, but relapsed 5 months after initial diagnosis. We transduced DEX sensitive presentation cells with the full genome GeCKOv2 CRISPR library, before transplantation into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were subsequently treated with DEX by oral gavage (15mg/kg for 5 weeks, 10mg/kg thereafter). DNA from several engrafted sites in the mouse was extracted and PCR amplified before being sequenced on the Illumina HiSeq2500. Changes in pool complexity were analysed using MaGEcK software to determine which sgRNAs were significantly enriched or depleted. By far the most significantly enriched sgRNAs were those targeting NR3C1, the gene encoding the glucocorticoid receptor. In addition, two of the top five significantly depleted sgRNAs targeted the Plexins, PLXNA1 and PLXND1. Whilst PLXNA1 is expressed at low levels, PLXND1 is highly expressed and has been linked to dexamethasone resistance. Notably, the matched relapsed material from L707 was highly DEX resistant both in tissue culture and when transplanted into NSG mice. SNP 6.0 analysis revealed a 5q deletion in the relapse, spanning 5 genes including NR3C1. Whole genome sequencing showed this was comprised of 2 deletions both targeting NR3C1, with different breakpoints for each allele. The differential gene expression between the L707 presentation and relapse established that NR3C1 was the most significant of all the genes lost at relapse, based on gene set enrichment analysis (GSEA). This contrasts with many ALL cases, where one of the downstream effectors of apoptosis is lost as opposed to NR3C1. Growth of the relapse material in vivo and in vitro was slower than the presentation in a competitive situation, but with DEX treatment the relapse phenotype began to emerge with a small percentage of cells showing a heterozygous deletion of NR3C1. These combined data strongly suggest that the NR3C1 deletion is the main driver of DEX resistance in the L707 relapse. Moreover, it proves that our in vivo CRISPR screen predicted the leukaemic relapse. These results confirm NR3C1 deletion as a driver in glucocorticoid resistance and demonstrate the power of in vivo CRISPR screens to predict mechanisms of gain of drug resistance and subsequent relapse. The parallels that can be drawn between the relapse and the CRISPR screen are striking, giving the indication that the progression from presentation to relapse may follow the same path in a patient derived xenograft setting as it did in the patient. Disclosures No relevant conflicts of interest to declare.

Antitumor activity of pazopanib (P) and trametinib (T) in preclinical models of osteosarcoma (OS).

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e22509-e22509
Author(s):  
Giulia Chiabotto ◽  
Maria Laura Centomo ◽  
Alessandra Merlini ◽  
Lorenzo D'Ambrosio ◽  
Dario Sangiolo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antitumor Activity ◽  
Western Blot ◽  
Cell Lines ◽  
Fold Change ◽  
Janus Kinase ◽  
Preclinical Models ◽  
Log2 Fold Change ◽  
In Vivo Antitumor Activity

e22509 Background: Receptor tyrosine kinases (RTKs) and their signal transducers are suitable targets for the treatment of advanced OS. We evaluated the antitumor activity of the RTK inhibitor P and the MEK inhibitor T and deeply investigated molecular mechanisms behind their activity and potential escape. Methods: Flow cytometry and western blot analyses were carried out in 7 OS cell lines to study the expression of RTK P targets and the activation of their pathways, respectively. Cell viability and colony growth were evaluated after 72h and 7-day treatment respectively, with scalar doses of both single agents and their constant combination. Cell cycle distribution and apoptosis were evaluated by flow cytometry after 72h. In vivo antitumor activity was studied in NOD/SCID mice bearing MNNG-HOS xenografts after 3 weeks of treatment. Cell migration was studied by scratch assays. The involvement of MAPK-PI3K pathway key transducers was explored by Vantage 3D RNA Panel and Nanostring technology, validated by western blot and confirmed by silencing experiments. Results: P targets are expressed on OS cell lines and their pathways are activated. P+T have synergistic antitumor activity (combination index < 1) in OS cell lines by inducing apoptosis (6/7) and inhibiting both ERK1/2(7/7) and AKT (7/7). Furthermore, in vivo antitumor activity was shown in OS bearing mice (tumor volume: P+T/untreated = 0.036, p = 0.002). P+T significantly down-modulated RTK EphaA2 (mean log2 fold change RNA P+T/untreated = -2.02±0.50) and induced Janus kinase MEK6 (mean log2 fold change RNA P+T/untreated = 2.9±0.51). EphA2 silencing reduced cellular proliferation and migration of OS cells. Impeding MEK6-up-regulation in P+T treated cells significantly increased the antitumor effect (51.5±14.3%) of the studied drugs. Conclusions: P+T exert antitumor activity in OS preclinical models through ERK and AKT inhibition and EphA2 downmodulation. MEK6-upregulation after P+T is likely implied in escape mechanism.

scholarly journals CSIG-19. CYTOMEGALOVIRUS INFECTION LEADS TO NF-kB DEPENDENT UPREGULATION OF c-MET AND MGMT IN GLIOBLASTOMA AND RESISTANCE TO TEMOZOLOMIDE IN VIVO

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi48-vi48
Author(s):  
Mykola Zdioruk ◽  
Harald Krenzlin ◽  
M Oskar Nowicki ◽  
Charles H Cook ◽  
Ennio Chiocca ◽  
...  
Keyword(s):  
Median Survival ◽  
Fold Change ◽  
Tyrosine Kinase Receptor ◽  
P Value ◽  
Human Populations ◽  
Cmv Infection ◽  
Log2 Fold Change ◽  
Human Gbm

Abstract INTRODUCTION Cytomegalovirus (CMV), a member of the betaherpesvirinae subfamily widespread in human populations has been reported to be detectable in glioblastoma (GBM). We have recently shown that CMV latent infection promotes GBM growth in a mouse model. The mechanisms involved are not well-defined and treatment responses have not been evaluated. METHODS To investigate the effects of CMV on GBM cells we performed an RNAseq study. Differentially expressed genes were identified and validated in vitro and in vivo. RESULTS RNAseq analysis showed multiple alterations induced by CMV infection of GBM cells including significant upregulation of c-MET (8.2 log2 fold change) and MGMT (3.9 log2 fold change) transcripts after CMV infection. c-MET is a tyrosine kinase receptor known to promote GBM growth, and MGMT expression leads to resistance to the alkylating agent temozolomide (TMZ). These findings were validated in vitro in both mouse and human GBM cells, and shown to be dependent on CMV-induced upregulation of NF-kB by siRNA knockdown of the essential RelA NF-kB subunit. The c-MET co-receptor CD44 was also upregulated suggesting a role for the c-MET signalling axis in CMV induced tumor progression. CMV-infected tumor cells were resistant to TMZ treatment as expected due to MGMT upregulation. Treatment of our murine GBM model with TMZ led to a significant increase in survival in control mock-infected mice (median survival 38 days control, 48 days TMZ), whereas the CMV infected mice did not respond (median survival 35 days control, 35 days TMZ; p value between TMZ treated groups = 10–4). CONCLUSIONS Our results define novel and relevant mechanisms by which CMV may influence both GBM growth and resistance to treatment. Further therapeutic and mechanistic studies are underway to investigate these pathways in the context of CMV in GBM. These data support further study of CMV targeting in GBM.

scholarly journals Whole-Genome CRISPR Screen Reveals the Mechanism of Relapse in Patient-Derived Cells Representing High-Risk Paediatric ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3952-3952
Author(s):  
Katarzyna Szoltysek ◽  
Helen Blair ◽  
Sirintra Nakjang ◽  
Ricky Tirtakusuma ◽  
Mankaran Singh ◽  
...  
Keyword(s):  
High Risk ◽  
Research Funding ◽  
Ex Vivo ◽  
Mtor Inhibitors ◽  
Whole Genome ◽  
Nsg Mice ◽  
Genome Wide ◽  
Crispr Screen ◽  
Dexamethasone Resistance

Acute lymphoblastic leukemia (ALL) is the most common type of childhood leukaemia. Recently improved risk stratification resulted in therapy optimization and extended survival for the majority of cases. Unfortunately, there is still a significant number of patients either relapsing or not responding to treatment with response to glucocorticoids being one of the most important prognostic indicators of treatment outcome. In order to investigate the mechanism of dexamethasone resistance, we performed genome-wide CRISPR screens in patient derived xenotransplant (PDX) material from t(17;19)-positive ALL. Primary material was obtained from the patient at the presentation and at relapse stage of disease and corresponding PDX samples were generated in immunocompromised NSG mice. PDX cells were lentivirally transduced with the CRISPR knockout pooled 'Brunello' library and then subjected to dexamethasone pressure both ex vivo and in vivo. For the in vivo screen, CRISPR-modified cells were intrafemorally injected into immunodeficient NSG mice followed by either 7.5mg/kg dexamethasone or vehicle treatment. In parallel, PDXs were co-cultured with mesenchymal and endothelial-like human stromal cells generated from human bone marrow-derived iPSCs. Data analysis performed with the MAGeCKFlute software identified the glucocorticoid receptor gene NR3C1 as a main driver of chemoresistance-mediated relapse in this high-risk ALL. Notably, a homozygous deletion of NR3C1 was present in the relapse PDX sample. Furthermore, we identified that loss of the NR3C1 gene in those cells was associated with an inferior engraftment potential in the absence of dexamethasone. Interestingly, the whole-genome CRISPR screen in the relapse sample identified BCL2 and several genes associated with the mTOR pathway as crucial for leukaemic propagation. Knockout of NR3C1 in the diagnostic PDX also established dexamethasone resistance and further enhanced the already significant sensitivity towards mTOR inhibitors. To explore a potential synergism between BCL2 and mTOR inhibition, we assessed the effect of the BCL2 inhibitor ABT-199 and several mTOR inhibitors in both presentation and relapse PDX samples. PDX samples were co-cultured with MSCs and treated with drug combinations in a matrix format for 96 hrs followed by high-throughput fluorescence microscopy-based analysis. These experiments revealed substantial synergism of ABT-199 and mTOR inhibitors associated with increased cell death and prolonged growth inhibition in both presentation and relapse samples. In conclusion, our studies (i) demonstrate that genome-wide CRISPR screens are feasible in PDX material both ex vivo and in vivo, (ii) provide an explanation for the relative rarity of NR3C1 mutations in relapsed material and (iii) identify drug combinations effective in both diagnostic and relapse PDX for further preclinical evaluation. Disclosures Vormoor: Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.

Does Expression Of CD200 Provide a Target For LIC In Childhood ALL?

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2894-2894
Author(s):  
Charlotte Victoria Cox ◽  
Paraskevi Diamanti ◽  
Allison Blair
Keyword(s):  
Flow Cytometry ◽  
Functional Ability ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Treatment Strategies ◽  
Whole Genome ◽  
Childhood All ◽  
Nsg Mice ◽  
Flow Cytometric

Abstract Although the survival of children with acute lymphoblastic leukaemia (ALL) has improved considerably, there are still around 20% of cases that go on to relapse. These patients do not respond well to current therapies and there is a need to develop treatment strategies to directly target the cells that initiate the leukaemia to allow eradication of the disease. Leukaemia initiating cells (LIC) have been shown to be present in several immunophenotypic subpopulations evaluated in NSG assays. Identifying a marker that is expressed by all LIC populations may allow their discrimination from normal haemopoietic stem cells and more specific targeting of these cells. Whole genome expression studies have recently highlighted genes, including CD58, CD97 and CD99 that were over expressed in B-ALL cases when compared to normal CD19+/CD10+ cells. The addition of these markers to the minimal residual disease (MRD) flow cytometric panel increased the sensitivity of detection by 10-fold to one leukaemia cell in 105 bone marrow (BM) cells. We have previously investigated the functional capacity of ALL cells sorted for CD58, CD97 and CD99 in combination with CD34 in vivo. However, none of these antigens were specific for LIC. CD200 has been described as another marker that improved MRD sensitivity by flow cytometry and consequently we investigated its expression in LIC populations and the functional ability of these cells in vivo. Expression of CD34, CD19 and CD200 was assessed in 12 B-ALL cases (6 pre B-ALL, 6 c-ALL) and 6 cord blood samples (CB) by flow cytometry. Whole genome microarray analysis was also performed on unsorted cells and sorted CD34+/CD19+, CD34+/CD19-, CD34-/CD19+ and CD34-/CD19- subpopulations to compare expression of genes between these LIC and the bulk leukaemia. Cells from 5 of these patients were also sorted on the basis of expression of CD34 and CD200 and the functional ability of the sorted subpopulations was assessed in NSG mice. The microarray analyses demonstrated that CD200 was over expressed in all the subpopulations and was especially high in the CD34+/CD19+ and the CD34-/CD19+ subpopulations. CD200 was significantly over expressed in the CD34+/CD19+ subpopulation when compared to its expression in the CD34+/CD19- and CD34-/CD19- subpopulations (p=0.009, F=6.3, Fcritical=4.5). Flow cytometric analyses confirmed that CD200 was over expressed in unsorted B-ALL cells compared to unsorted CB (54.6%±8.1% vs 0.07±0.09% respectively, p=0.0002) and expression was higher in CD34+/CD19+ (68.4%±10.8% p=0.0006), CD34+/CD19- (39%±9.2% p=0.01), CD34-/CD19+ (48.4%±8.6% p=0.002) and CD34-/CD19- (5%±1.8% p=0.3) subpopulations compared to CD34+/CD38- CB cells (2%±1.1%). Over expression of CD200 on these ALL subpopulations indicates that it may be useful in characterisation of LIC. When cells from 5 of these cases were sorted, the CD34+/CD200+ subpopulation accounted for the greatest proportion of cells (60.5%±16.1%). Expression of CD19 was also high in this subpopulation (65±11%). CD34+/CD200- cells represented the smallest subpopulation (0.5%±0.1%) and these cells had low expression of CD19 (4±7%). The CD34-/CD200+ and CD34-/CD200- subpopulations represented 18%±11.4% and 21%±120.6% of the bulk cells, respectively. CD19 expression was higher in the CD34-/CD200+ subpopulation (30±14%) than in CD34-/CD200- cells (7±5%). Results from 4 cases to date indicate that engraftment was achieved with the CD34+/CD200+ subpopulation in every case (4-92% human leukaemia). In 3 cases, comparable engraftment levels were also achieved using CD34-/CD200+ cells (10.4-86.1%). No engraftment was detected using the CD34+/CD200- or CD34-/CD200- subpopulations, with the exception of 1 case where engraftment levels of 18-48% were achieved. These levels were lower than those achieved with CD200+ cells from the same patient. Interestingly when BM from mice engrafted with CD200- cells from this patient were analysed they now expressed CD200 (12-35%), indicating cell differentiation had occurred in vivo. Assessment of self-renewal capability of these LIC populations is ongoing. These data suggest that CD200 may be a useful marker for discriminating LIC cells from normal haemopoietic cells, irrespective of expression of CD34 and CD19 in these cases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA Methylation Profiling for the Diagnosis and Prognosis of Patients with Nontuberculous Mycobacterium Lung Disease

2021 ◽  
Vol 43 (2) ◽  
pp. 501-512
Author(s):  
Jee Youn Oh ◽  
Young Kyung Ko ◽  
Jeong-An Gim
Keyword(s):  
Dna Methylation ◽  
Lung Disease ◽  
Poor Prognosis ◽  
Wnt Signaling Pathway ◽  
Fold Change ◽  
Nontuberculous Mycobacterium ◽  
Healthy Controls ◽  
Log2 Fold Change ◽  
Diagnosis And Prognosis ◽  
Ntm Lung Disease

The incidence of nontuberculous Mycobacterium (NTM) lung disease is rapidly increasing; however, its diagnosis and prognosis remain unclear while selecting patients who will respond to appropriate treatment. Differences in DNA methylation patterns between NTM patients with good or poor prognosis could provide important therapeutic targets. We used the Illumina MethylationEPIC (850k) DNA methylation microarray to determine the pattern between differentially methylated regions (DMRs) in NTM patients with good or poor prognosis (n = 4/group). Moreover, we merged and compared 20 healthy controls from previous Illumina Methylation450k DNA methylation microarray data. We selected and visualized the DMRs in the form of heatmaps, and enriched terms associated with these DMRs were identified by functional annotation with the “pathfinder” package. In total, 461 and 293 DMRs (|Log2 fold change| > 0.1 and p < 0.03) were more methylated in patients with four poor and four good prognoses, respectively. Furthermore, 337 and 771 DMRs (|Log2 fold change| > 0.08 and p < 0.001) were more methylated in eight NTM patients and 20 healthy controls, respectively. TGFBr1 was significantly less methylated, whereas HLA-DR1 and HLA-DR5 were more methylated in patients with poor prognosis (compared to those with good prognosis). LRP5, E2F1, and ADCY3 were the top three less-methylated genes in NTM patients (compared with the controls). The mTOR and Wnt signaling pathway-related genes were less methylated in patients with NTM. Collectively, genes related to Th1-cell differentiation, such as TGFBr1 and HLA-DR, may be used as biomarkers for predicting the treatment response in patients with NTM lung disease.

scholarly journals Spatio-temporal biodistribution of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells by PET imaging in a preclinical tumor model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Naomi S. Sta Maria ◽  
Leslie A. Khawli ◽  
Vyshnavi Pachipulusu ◽  
Sharon W. Lin ◽  
Long Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
Real Time ◽  
Pet Imaging ◽  
Dose Group ◽  
Low Dose ◽  
High Dose ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

AbstractQuantitative in vivo monitoring of cell biodistribution offers assessment of treatment efficacy in real-time and can provide guidance for further optimization of chimeric antigen receptor (CAR) modified cell therapy. We evaluated the utility of a non-invasive, serial 89Zr-oxine PET imaging to assess optimal dosing for huLym-1-A-BB3z-CAR T-cell directed to Lym-1-positive Raji lymphoma xenograft in NOD Scid-IL2Rgammanull (NSG) mice. In vitro experiments showed no detrimental effects in cell health and function following 89Zr-oxine labeling. In vivo experiments employed simultaneous PET/MRI of Raji-bearing NSG mice on day 0 (3 h), 1, 2, and 5 after intravenous administration of low (1.87 ± 0.04 × 106 cells), middle (7.14 ± 0.45 × 106 cells), or high (16.83 ± 0.41 × 106 cells) cell dose. Biodistribution (%ID/g) in regions of interests defined over T1-weighted MRI, such as blood, bone, brain, liver, lungs, spleen, and tumor, were analyzed from PET images. Escalating doses of CAR T-cells resulted in dose-dependent %ID/g biodistributions in all regions. Middle and High dose groups showed significantly higher tumor %ID/g compared to Low dose group on day 2. Tumor-to-blood ratios showed the enhanced extravascular tumor uptake by day 2 in the Low dose group, while the Middle dose showed significant tumor accumulation starting on day 1 up to day 5. From these data obtained over time, it is apparent that intravenously administered CAR T-cells become trapped in the lung for 3–5 h and then migrate to the liver and spleen for up to 2–3 days. This surprising biodistribution data may be responsible for the inactivation of these cells before targeting solid tumors. Ex vivo biodistributions confirmed in vivo PET-derived biodistributions. According to these studies, we conclude that in vivo serial PET imaging with 89Zr-oxine labeled CAR T-cells provides real-time monitoring of biodistributions crucial for interpreting efficacy and guiding treatment in patient care.

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