Multimodal single-cell profiling reveals the enhanced antitumor activity of FGFR4/CD276 BiCisCAR T cells in rhabdomyosarcoma
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA948361 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23956025.fastq.gz | Fastqsanger.gz | |||
| SRR23956026.fastq.gz | Fastqsanger.gz | |||
| SRR23956027.fastq.gz | Fastqsanger.gz | |||
| SRR23956028.fastq.gz | Fastqsanger.gz | |||
| SRR23956029.fastq.gz | Fastqsanger.gz |
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Project description:We have generated a clinical grade CAR T targeting FGFR4 with a CD8 hinge and transmembrane domain (HTM) and a 4-1BB co-stimulatory domain (CSD) that is currently being developed for a clinical trial at the NCI. This standard FGFR4-CAR demonstrated potency in low burden RMS disease but has a more modest effect for bulky disease in RMS orthotopic model. Thus, we developed optimized FGFR4-CARs by modification of HTM or CSD to improve its efficacy. However, these modified FGFR4-CARs, even FGFR4.28HTM.28z CAR were unable to eradicate solid tumors in an more aggressive RMS559 model. CAR T cells targeting another cell surface protein CD276, a direct target of the RMS core-regulatory transcription factor MYOD1 is potent and effective, also could not eradicate the tumor. Therefore, we engineered a bicistronic CAR (BiCisCAR) to target both FGFR4 and CD276, with CD28 and 4-1BB CSDs, respectively, which showed remarkable and rapid tumor eradication compared with either single CAR alone. Here, we utilized a multimodal approach including simultaneous protein and transcriptome measurement at single cell level to characterize each FGFR4 or CD276 CAR T-cell infiltrating in RMS orthotopic xenografts. This assay further determined the molecular basis of superior antitumor effect of FGFR4.28HTM.28z-CD276.8HTM.BBz BiCisCAR T cells compared with other CARs.
2024-05-23 | GSE228127 | GEO
Project description:Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by a fusion protein (e.g. PAX3-FOXO1) or by mutations in key signalling molecules (e.g. RAS or FGFR4). Despite the latter giving potential for precision medicine approaches in RMS, there are no such treatments implemented in the clinic yet. In order to identify and test novel precision therapy strategies, appropriate cellular and mouse models are crucial. We have here thoroughly characterized a RMS patient-derived cell line model, RMS559, which harbours a FGFR4 V550L activating mutation with high allelic frequency (0.8). Importantly, we show that RMS559 cells are oncogenically dependent on FGFR4 signalling by treatment with the pan-FGFR inhibitor LY2874455. Phosphoproteomic analysis identified RAS/MAPK and PI3K/AKT as the major druggable signalling pathways downstream of FGFR V550L. Inhibitors against these pathways inhibited cell proliferation. Furthermore, we found that FGFR4 V550L is dependent on HSP90 and inhibitors targeting HSP90 efficiently restrain proliferation. Recently, FGFR4 specific inhibitors have been developed. While two of these, BLU-9931 and H3B-6527, did not efficiently inhibit FGFR4 V550L, probably because of the gatekeeper mutation (V550L), one of them, FGF401 inhibited FGFR4 V550L and cell proliferation at low nanomolar concentrations. Finally, we developed a mouse model using RMS559 cells and tested the in vivo efficacy of LY2874455 and FGF401. While LY2874455 inefficiently inhibited growth, FGF401 completely abrogated tumour growth in vivo.
2022-10-19 | PXD029719 | Pride
Project description:BackgroundRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood, whose prognosis is still poor especially for metastatic, high-grade, and relapsed RMS. New treatments are urgently needed, especially systemic therapies. Chimeric Antigen Receptor T cells (CAR Ts) are very effective against hematological malignancies, but their efficacy against solid tumors needs to be improved. CD276 (B7-H3) is a target upregulated in RMS and detected at low levels in normal tissues. FGFR4 is a very specific target for RMS. Here, we optimized CAR Ts for these two targets, alone or in combination, and tested their anti-tumor activity in vitro and in vivo.MethodsFour different single-domain antibodies were used to select the most specific FGFR4-CAR construct. RMS cell killing and cytokine production by CD276- and FGFR4-CAR Ts expressing CD8α or CD28 HD/TM domains in combination with 4-1BB and/or CD28 co-stimulatory domains were tested in vitro. The most effective CD276- and FGFR4-CAR Ts were used to generate Dual-CAR Ts. Tumor killing was evaluated in vivo in three orthotopic RMS mouse models.ResultsCD276.V-CAR Ts (276.MG.CD28HD/TM.CD28CSD.3ζ) showed the strongest killing of RMS cells, and the highest release of IFN-γ and Granzyme B in vitro. FGFR4.V-CAR Ts (F8-FR4.CD28HD/TM.CD28CSD.3ζ) showed the most specific killing. CD276-CAR Ts successfully eradicated RD- and Rh4-derived RMS tumors in vivo, achieving complete remission in 3/5 and 5/5 mice, respectively. In CD276low JR-tumors, however, they achieved complete remission in only 1/5 mice. FGFR4 CAR Ts instead delayed Rh4 tumor growth. Dual-CAR Ts promoted Rh4-tumors clearance in 5/5 mice.ConclusionsCD276- and CD276/FGFR4-directed CAR Ts showed effective RMS cell killing in vitro and eradication of CD276high RMS tumors in vivo. CD276low tumors escaped the therapy highlighting a correlation between antigen density and effectiveness. FGFR4-CAR Ts showed specific killing in vitro but could only delay RMS growth in vivo. Our results demonstrate that combined expression of CD276-CAR with other CAR does not reduce its benefit. Introducing immunotherapy with CD276-CAR Ts in RMS seems to be feasible and promising, although CAR constructs design and target combinations have to be further improved to eradicate tumors with low target expression.
| S-EPMC10625270 | biostudies-literature
Project description:Fibroblast growth factor receptor 4 (FGFR4) aberrant expression and activity have been linked to the pathogenesis of a variety of cancers including rhabdomyosarcomas (RMS). We found that treatment of alveolar rhabdomyosarcoma (aRMS) cells with Guadecitabine (SGI-110), a next-generation DNA methyltransferase inhibitor (DNMTi), resulted in a significant reduction of FGFR4 protein levels, 5 days post treatment. Chromatin immunoprecipitation-sequencing (ChIP-seq) in aRMS cells revealed attenuation of the H3K4 mono-methylation across the FGFR4 super enhancer without changes in tri-methylation of either H3K4 or H3K27. These changes were associated with a significant reduction in FGFR4 transcript levels in treated cells. These decreases in H3K4me1 in the FGFR4 super enhancer were also associated with a 240-fold increase in KDM5B (JARID1B) mRNA levels. Immunoblot and immunofluorescent studies also revealed a significant increase in the KDM5B protein levels after treatment in these cells. KDM5B is the only member of KDM5 (JARID1) family of histone lysine demethylases that catalyzes demethylation of H3K4me1. These data together suggest a pleiotropic effect of DNMTi therapy in aRMS cells, converging to significantly lower FGFR4 protein levels in these cells.
2021-05-14 | GSE150602 | GEO
Project description:Neuroblastoma (NB) is the most common extra-cranial solid tumor in children and remains deadly in patients with high-risk disease. Single chimeric antigen receptor T-cell therapies (CAR-T) for solid tumor have shown poor efficacy in clinical trials due, in part, to T cell exhaustion and uneven expression of target antigens in tumors. Here, we attempted to target Glypican-2(GPC2) and CD276(B7-H3), both highly but heterogeneously expressed on NB cell surface, to overcome these challenges in single CAR T- therapies. First, to identify optimal binders for CAR T- cell targeting each antigen, we made individual CAR constructs using multiple binders against these antigens and utilized a multimodal approach including simultaneous protein and transcriptome measurement at single cell level to characterize each individual CAR T- cell in a pooled strategy. Combined with cell expansion and cytotoxicity assays, we identified the most effective CAR construct for each target.
2022-03-18 | GSE181437 | GEO
Project description:Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Prognosis for patients with high grade and metastatic disease is still very poor, and survivors are burdened with long-lasting side effects. Therefore, more effective and less toxic therapies are needed. Surface proteins are ideal targets for antibody-based therapies, like bispecific antibodies, antibody drug conjugates, or chimeric antigen receptor (CAR) T cell. Specific surface targets for RMS are scarce. Here, we performed a surfaceome profiling based on differential centrifugation enrichment of surface/membrane proteins and detection by LC-MS on six fusion-positive (FP) RMS cell lines, five fusion-negative (FN) RMS cell lines, and three RMS patient-derived xenografts (PDXs). 699 proteins were detected in the three RMS groups. Ranking based on expression levels and comparison to expression in normal MRC-5 fibroblasts and myoblasts, followed by statistical analysis, highlighted known RMS targets such as FGFR4, NCAM1, and CD276/B7-H3, and revealed AGRL2, JAM3, MEGF10, GPC4, CADM2, as potential targets for immunotherapies of RMS. L1CAM expression was investigated in RMS tissues and strong L1CAM expression was observed in more than 80% of alveolar RMS tumors, making it a practicable target for antibody-based therapies of alveolar RMS.
2023-03-11 | PXD039480 | Pride
Project description:We performed RNA sequencing (RNA-seq) to compare the gene expression pattern between CD276-high/EpCAM+ and CD276-low/EpCAM+ cells isolated from human HNSCC PDX. We identified a cohort of key oncogenes associate with cancer stemness enriched in CD276-high cells. Suggesting CD276 may play a positive role in maintaining cancer stem cells in human head and neck squamous cell carcinoma.
2021-05-28 | GSE132627 | GEO
Project description:We report the genome-wide maps of PAX3-FKHR binding sites. Chromatin immunoprecipitation was performed against PAX3-FKHR positive (Rh4) and PAX3-FKHR negative (RD) rhabdomyosarcoma cells with a monoclonal antibody (pFM2) specific for the fusion region of PAX3-FKHR. We obtained 4 million sequence tags for both input and ChIP DNA that aligned to the human genome. We identified 1,463 binding sites from ChIP-seq of Rh4 cells, none of which appeared from ChIP-seq of fusion negative RD cells. The PAX3-FKHR binding sites were found to associate with 1,072 genes in RMS cells. The data shows that PAX3-FKHR binds to the same sites as PAX3, at the enhancers for MYF5, FGFR4, and the MYOD core enhancer previously shown to be regulated by PAX3. Moreover, our dataset has the precision for rapid identification and validation of novel and specific sequences required for the enhancer activity of MYOD and FGFR4. The genome wide analysis reveals that the PAX3-FKHR sites are: 1) mostly distal to transcription start sites; 2) conserved; 3) enriched for PAX3 motifs; and 4) strongly associated with genes over-expressed in PAX3-FKHR positive RMS cells and tumors. There is little evidence in our dataset for PAX3-FKHR binding at the promoters. In one instance, we show two intronic enhancer elements for MET, rather than at the previously described promoter. The genome-wide analysis further illustrates a strong association between PAX3 and E-box motifs in these binding sites, suggestive of a common co-regulation for many target genes. The map of PAX3-FKHR binding sites provides new links for PAX3 and PAX3-FKHR functions and new targets for RMS therapy. Examination of PAX3-FKHR binding sites in translocation-positive rhabdomyosarcoma cells via ChIP-seq with an antibody specific for the fusion protein.
2010-08-17 | E-GEOD-19063 | biostudies-arrayexpress
Project description:mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion
| PRJNA883227 | ENA
Project description:70-100% of colorectal, prostate, ovarian, pancreatic and breast cancer specimens show expression of CD276 (B7-H3), a potential immune checkpoint family member. Several studies have shown that high CD276 expression in cancer cells correlates with a poor clinical prognosis. This has been associated with the presence of lower tumor infiltrating leukocytes. Among those, tumor-associated macrophages can comprise up to 50% of the tumor mass and are thought to support tumor growth through various mechanisms. However, a lack of information on CD276 function and interaction partner(s) impedes rigorous evaluation of CD276 as a therapeutic target in oncology. Therefore, we aimed to understand the relevance of CD276 in tumor-macrophage interaction by employing a 3D spheroid coculture system with human cells. Our data show a role for tumor-expressed CD276 on the macrophage recruitment into the tumor spheroid, and also in regulation of the extracellular matrix modulator PAI-1. Furthermore, our experiments focusing on macrophage-expressed CD276 suggest that the antibody-dependent CD276 engagement triggers predominantly inhibitory signaling networks in human macrophages.
2021-07-28 | GSE165610 | GEO