Biology subjects

Mukherjee, E.

Publications and source records attributed to Mukherjee, E..

5 recordsLinked to original sources

bioRxiv2026

Loss of stromal VEGFR2 protects against ischemic acute kidney injury by preserving the renal microvasculature

Injury to the renal microvasculature, particularly the peritubular capillaries, is a defining feature of ischemic acute kidney injury (AKI). Vascular endothelial growth factor receptor 2 (VEGFR2) signaling is a central regulator of endothelial cell survival, proliferation, migration, and vascular remodeling. Although VEGFR2 has been extensively studied in endothelial cells, accumulating evidence suggests that it is also expressed in non-endothelial cell populations, where it may regulate tissue-specific responses to injury. Here, using publicly available single-cell or single-nucleus RNA sequencing data from human and mouse kidneys, we identified VEGFR2 expression in renal stromal populations. Immunostaining further detected phosphorylated VEGFR2 in the renal stroma of mouse kidneys. To determine the functional role of stromal VEGFR2, we utilized renal stroma-specific Vegfr2 KO mice. Stromal VegfR2 deletion attenuated kidney injury after renal ischemia/reperfusion injury (IRI), as evidenced by reduced tubular injury, preservation of renal blood flow, decreased endothelial oxidative stress, and maintenance of the renal microvasculature. Stromal Vegfr2-deficient kidneys also exhibited increased Angpt1 and Tek levels, enhanced endothelial ERK activation, and preserved endothelial adherent junctions. Taken together, these findings reveal an unexpected role for VEGFR2 in the renal stromal lineage and highlight stromal-endothelial communication as a potential therapeutic target for preserving microvascular integrity after ischemic AKI.

pathology↗

bioRxiv2025

SARC028 samples reveal an interplay between TGF-beta, interferon signaling and low HLA class I expression as contributors to Ewing sarcoma checkpoint blockade resistance

PurposeEwing sarcoma, in contrast to some adult sarcoma subtypes, generally does not respond to single agent immunotherapy targeting PD1. The features of Ewing sarcoma that preclude the effectiveness of immunotherapy remain largely unknown. To address this question, we utilized biopsies from patients with Ewing sarcoma obtained pre- and post-pembrolizumab (anti-PD1) therapy from the phase 2 clinical trial SARC028 to interrogate the Ewing tumor microenvironment and features associated with resistance to checkpoint inhibition. Experimental DesignWe utilize multiplexed immunofluorescence, spatial proteomics and spatial transcriptomics to analyze paired pre- and 8 weeks post-treatment biopsy specimens from patients with Ewing sarcoma enrolled on SARC028. ResultsPembrolizumab therapy did not alter the quantity of immune cell infiltration in Ewing tumor biopsies. Analysis of tumor-associated protein markers revealed increased immunoregulatory markers after pembrolizumab. Spatial transcriptomics identified ten cellular neighborhoods (CN) across patients consisting of specific cell subsets. CN10 was consistently observed across patients with poor response. This cellular neighborhood was enriched for a tumor subpopulation with high TGF-{beta} response, low interferon (IFN) response, and low HLA class I expression. IFN response, HLA class I expression, and overall immune infiltration were correlated. ConclusionsAnalyses of the tumor microenvironment from Ewing sarcoma biopsies reveals an immunosuppressive triad, the disruption of which should be pursued to improve antitumor immunity. This work highlights the unique insight that can be gained from the analysis of paired patient Ewing sarcoma tumor biopsy samples from clinical trials. Statement of translational relevanceSingle agent checkpoint inhibitors have demonstrated limited clinical benefit in bone sarcomas, including Ewing sarcoma (EwS). While in vitro and limited patient tumor analyses have suggested mechanisms of immunotherapy resistance in EwS, tumor biopsies following checkpoint inhibitor administration have not been examined. SARC028 was a phase II study investigating blockade of programmed cell death-1 (PD-1) in patients with relapsed/refractory sarcomas, including EwS. Tumor biopsies were collected prior to and 8 weeks after anti-PD1 therapy. Here, we leverage a unique opportunity to understand EwS immunotherapy resistance through analysis of paired tumor samples from SARC028. Using a multi-omics approach, we identify features of EwS that contribute to primary resistance to anti-PD1 including TGF-{beta}, reduced interferon signaling and low HLA class I expression. This study provides an improved understanding of tumor characteristics driving poor response to immunotherapy in relapsed EwS and will guide the development of future trials to modulate this immunosuppressive axis.

cancer biology↗

bioRxiv2024

Immunocompetent murine model of Ewing sarcoma reveals role for TGFβ inhibition to enhance immune infiltrates in Ewing tumors during radiation

Ewing sarcoma (ES) is an aggressive cancer diagnosed in adolescents and young adults. The fusion oncoprotein (EWSR1::FLI1) that drives Ewing sarcoma is known to downregulate TGFBR2 expression (part of the TGF{beta} receptor). Because TGFBR2 is downregulated, it was thought that TGF{beta} likely plays an inconsequential role in Ewing biology. However, the expression of TGF{beta} in the Ewing tumor immune microenvironment (TIME) and functional impact of TGF{beta} in the TIME remains largely unknown given the historical lack of immunocompetent preclinical models. Here, we use single-cell RNAseq analysis of human Ewing tumors to show that immune cells, such as NK cells, are the largest source of TGF{beta} production in human Ewing tumors. We develop a humanized (immunocompetent) mouse model of ES and demonstrate distinct TME signatures and metastatic potential in these models as compared to tumors developed in immunodeficient mice. Using this humanized model, we study the effect of TGF{beta} inhibition on the Ewing TME during radiation therapy, a treatment that both enhances TGF{beta} activation and is used to treat aggressive ES. Utilizing a trivalent ligand TGF{beta} TRAP to inhibit TGF{beta}, we demonstrate that in combination with radiation, TGF{beta} inhibition both increases ES immune cell infiltration and decreases lung metastatic burden in vivo. The culmination of these data demonstrates the value of humanized models to address immunobiologic preclinical questions in Ewing sarcoma and suggests TGF{beta} inhibition as a promising intervention during radiation therapy to promote metastatic tumor control.

cancer biology↗

bioRxiv2023

Multiomic single-cell sequencing defines tissue-specific responses in Stevens-Johnson Syndrome and Toxic epidermal necrolysis.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but life-threatening cutaneous drug reaction mediated by human leukocyte antigen (HLA) class I-restricted CD8+ T-cells. To obtain an unbiased assessment of SJS/TEN cellular immunopathogenesis, we performed single-cell (sc) transcriptome, surface proteome, and TCR sequencing on unaffected skin, affected skin, and blister fluid from 17 SJS/TEN patients. From 119,784 total cells, we identified 16 scRNA-defined subsets, confirmed by subset-defining surface protein expression. Keratinocytes upregulated HLA and IFN-response genes in the affected skin. Cytotoxic CD8+ T-cell subpopulations of expanded and unexpanded TCR{beta} clonotypes were shared in affected skin and blister fluid but absent or unexpanded in SJS/TEN unaffected skin. SJS/TEN blister fluid is a rich reservoir of oligoclonal CD8+ T-cells with an effector phenotype driving SJS/TEN pathogenesis. This multiomic database will act as the basis to define antigen-reactivity, HLA restriction, and signatures of drug-antigen-reactive T-cell clonotypes at a tissue level.

immunology↗

bioRxiv2020

PARP inhibition in Ewing sarcoma: impact of germline DNA damage repair defects and activation of immunoregulatory pathways

Ewing sarcoma, an oncofusion-driven primary bone tumor, can occur in the setting of various germline mutations in DNA damage repair pathway genes. We recently reported our discovery of a germline mutation in the DNA damage repair protein BARD1 (BRCA1-associated RING domain-1) in a patient with Ewing sarcoma. BARD1 is recruited to the site of DNA double stranded breaks via the poly(ADP-ribose) polymerase (PARP) protein and plays a critical role in DNA damage response pathways including homologous recombination. PARP inhibitors (PARPi) are effective against Ewing sarcoma cells in vitro, though have demonstrated limited success in clinical trials to date. In order to assess the impact of BARD1 loss on Ewing sarcoma sensitivity to PARP inhibitor therapy, we generated the novel PSaRC318 patient-derived Ewing tumor cell from our patient with a germline BARD1 mutation and then analyzed the response of these cells to PARPi. We demonstrate that PSaRC318 cells are sensitive to PARP inhibition and by testing the effect of BARD1 depletion in additional Ewing sarcoma cell lines, we confirm that loss of BARD1 enhances PARPi sensitivity. In certain malignancies, DNA damage can activate the IRF1 (interferon response factor 1) immunoregulatory pathway, and the activation of this pathway can drive immunosuppression through upregulation of the immune checkpoint protein PD-L1. In order to determine the ability of PARPi to alter Ewing tumor immunoregulation, we evaluated whether PARPi results in upregulation of the IRF1-PDL1 pathway. Indeed, we now demonstrate that PARPi leads to increased PD-L1 expression in Ewing sarcoma. Together, these data thus far suggest that while Ewing tumors harboring germline mutations in DNA damage repair proteins may in respond to PARPi in vitro, in vivo benefit of PARPi may only be demonstrated when counteracting the immunosuppressive effects of DNA damage by concurrently targeting immune checkpoint proteins.

cancer biology↗