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Rakheja, D.

Publications and source records attributed to Rakheja, D..

6 recordsLinked to original sources

A Visually Interpretable Histopathology-Based Immune Model Predicts T-effector Biology and Response to Immune checkpoint inhibition in Clear Cell Renal Cell Carcinoma Clinical Trial and Contemporary Real-World Datasets

Immune checkpoint inhibitors (ICI) are central to the treatment of metastatic clear cell renal cell carcinoma (ccRCC), yet only a subset of patients derive durable benefit, and clinically deployable predictive biomarkers remain an unmet need. RNA-based T-effector signatures capture cytotoxic immune biology and have been associated with ICI response in clinical trial cohorts; however, their clinical implementation is limited by the marked spatial heterogeneity of ccRCC, as well as cost, long turnaround time, sample quality requirements, and limited accessibility. Here, we developed a visually interpretable deep learning (DL) model that predicts a T-cell-enriched immune score directly from hematoxylin and eosin (H&E)-stained whole-slide images. To overcome the inability of H&E morphology alone to distinguish lymphocyte subsets, we trained the model using multimodal spatial supervision from CD8, PAX8, and ERG IHC, which respectively identified cytotoxic T-cell-rich regions, tumor cells, and endothelial cells, thereby constraining immune predictions to relevant tumor microenvironmental niches. The resulting H&E DL Immune score was validated by pathologist review, comparison with held-out CD8 IHC annotations, and independent datasets. The H&E DL Immune score correlated with T-effector RNA scores across independent institutional and IMmotion150 clinical trial cohorts (spearman correlations of 0.726; p=5.90x10-15 and 0.706; p=4.04x10-19). As a proof of principle, the score was used to characterize associations with key biological features across large cohorts, including sarcomatoid differentiation, BAP1 and PBRM1 mutation status, and additional transcriptomic signatures. In IMmotion150 clinical trial cohort, a median-dichotomized H&E DL Immune score, similar to RNA-based T-effector score, was significantly associated with clinical benefit from atezulumab therapy. In contemporary institutional cohorts of patients treated with frontline ipilimumab plus nivolumab or in initial 3 lines of nivolumab monotherapy, patients in the top quartile of H&E DL Immune score had significantly longer progression-free survival. Collectively, these findings support a scalable and interpretable H&E-based biomarker that captures T-effector biology and can help identify patients with ccRCC more likely to benefit from ICIs.

bioinformatics↗

Melanoma antigens in pediatric medulloblastoma contribute to tumor heterogeneity and species-specificity of group 3 tumors

BackgroundMedulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. MethodsTo determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Childrens Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. ResultsOur RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. ConclusionsThese results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key PointsO_LISeveral Type I MAGE CTAs are expressed in >60% of group 3 MBs. C_LIO_LIType I MAGEs affect MB cell proliferation and apoptosis. C_LIO_LIMAGEs are potential biomarkers and therapeutic targets for group 3 MBs. C_LI Importance of the StudyThis study is the first comprehensive analysis of all Type I MAGE CTAs (MAGEA, -B, and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/594201v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1ac274aorg.highwire.dtl.DTLVardef@1479944org.highwire.dtl.DTLVardef@19f5aaforg.highwire.dtl.DTLVardef@fad069_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

Target-directed microRNA degradation regulates developmental microRNA expression and embryonic growth in mammals

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. Target-directed miRNA degradation (TDMD), a pathway in which miRNAs that bind to specialized targets with extensive complementarity are rapidly decayed, has emerged as a potent mechanism of controlling miRNA levels. Nevertheless, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8, which encodes an essential TDMD factor. Loss of Zswim8 resulted in developmental defects in heart and lung, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in co-transcribed clusters and examples in which TDMD underlies arm switching, a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two miRNAs, miR-322 and miR-503, rescued growth of Zswim8 null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. These data illuminate the broad landscape and developmental role of TDMD in mammals.

molecular biology↗

VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis

Clinical sequencing efforts are uncovering numerous fusion genes in childhood solid tumors, yet few methods exist to delineate fusion-oncogenes from structural changes of unknown significance. One such novel fusion gene is VGLL2-NCOA2, which was described by us and others in patients with congenital sarcomas but has lacked functional validation. To determine if this fusion is an oncogene, and how it is driving disease, we developed a vertebrate zebrafish model and mouse allograft model of human VGLL2-NCOA2 driven sarcomagenesis. We found that VGLL2-NCOA2 is indeed an oncogene and is sufficient to generate mesenchymal tumors that recapitulate the human disease at the histological and transcriptional level. Zebrafish VGLL2-NCOA2 tumors display features of arrested skeletal muscle development, and a subset transcriptionally cluster with somitogenesis in developing embryos. By comparing tumor and embryonic gene expression signatures, we identified developmentally regulated targets that VGLL2-NCOA2 potentially leverages for tumorigenesis. These targets highlight the core biology of the disease and could represent therapeutic opportunities. Specifically, a RAS family GTPase, arf6/ARF6, involved in actin remodeling and rapid cycling of endocytic vesicles at the plasma membrane, is highly expressed during zebrafish somitogenesis and in VGLL2-NCOA2 tumors. In zebrafish tumors, arf6 protein is highly expressed and is absent from mature skeletal muscle. In VGLL2-NCOA2 mouse allograft models and patient tumors, ARF6 mRNA is overexpressed as compared to skeletal muscle or normal controls. More broadly, ARF6 is overexpressed in adult and pediatric sarcoma subtypes as compared to mature skeletal muscle. Overall, our cross-species comparative oncology approach provides evidence that VGLL2-NCOA2 is an oncogene which leverages developmental programs for tumorigenesis, and that one of these programs, the reactivation or persistence of arf6/ARF6, could represent a therapeutic opportunity.

cancer biology↗

CIC-DUX4 expression drives the development of small round cell sarcoma in transgenic zebrafish: a new model revealing a role for ETV4 in CIC-mediated sarcomagenesis

CIC-DUX4 sarcoma is a rare subtype of sarcoma characterized by a devastating prognosis and resistance to conventional therapeutic strategies. So far, only few models of the disease have been reported, and its biological mechanisms remain to be elucidated. We established mosaic transgenic zebrafish expressing the human CIC-DUX4 fusion under the control of the {beta}-actin promoter. CIC-DUX4 transgenic fish rapidly developed aggressive soft tissue tumors with a high penetrance. RNAseq profiling revealed that fish tumors shared major common targets with human tumors and cell lines, including the overexpression of the Pea3 transcription factors, etv4 and etv5. Tumor development was strongly impaired in etv4-deficient zebrafish, implicating Etv4 as a critical effector of CIC-DUX4-mediated oncogenesis. Altogether, we report here the first in vivo model of CIC-DUX4 sarcoma in zebrafish, which will represent a major tool for future preclinical research.

cancer biology↗

miR-16 suppresses growth of rhabdoid tumor cells

BackgroundRhabdoid tumor is a highly aggressive pediatric cancer characterized by biallelic loss and/or mutation of SMARCB1. Outcomes remain poor, and there are no established ways to target the tumorigenic pathways driven by SMARCB1 inactivation. SMARCB1 loss leads to an increase in cyclin D transcription.\n\nProcedureWe characterized the cell line WT-CLS1, which has been described previously as Wilms tumor, by whole-exome sequencing, RNA-seq, and xenograft histology. We measured the effect of microRNA overexpression on WT-CLS1, BT-12, and CHLA-06-ATRT.\n\nResultsWe found that WT-CLS1 demonstrates the histological, mutational, and transcriptional hallmarks of rhabdoid tumor. Because the microRNAs let-7 and miR-16 can target cyclin D genes, we next overexpressed each of these microRNAs in WT-CLS1. We found that miR-16 reduced cell accumulation. This was accompanied by a decrease in proliferation markers and an increase in apoptosis markers. These results were replicated in the BT-12 and CHLA-06-ATRT cell lines.\n\nConclusionsThe loss-of-function SMARCB1 mutation found in WT-CLS1, in conjunction with immunohistochemical and gene expression analysis, warrants reclassification of this cell line as rhabdoid tumor. Proliferation of WT-CLS1 and other rhabdoid tumor cell lines is significantly abrogated by miR-16 overexpression. Further studies are necessary to gain insight into the potential for miR-16 to be used as a novel therapeutic in rhabdoid tumor.\n\nAbbreviations

cancer biology↗