Browsing by Subject "Sarcoma"

The adaptive immune system plays a crucial role in combating tumors through immunosurveillance and responding to immunotherapies. However, many studies investigating the anti-tumor immune response focus primarily on tumor-infiltrating lymphocytes (TILs), which often lack specificity for the antigenic epitopes presented on tumor cells, making them less relevant for effective anti-tumor immunity. To gain insights into novel immunotherapeutic targets and biomarkers of response, it is essential to characterize the phenotypic features and dysfunctional mechanisms of tumor-specific T cell populations. This requires the use of tumor models that express known neoantigens in order to study tumor-specific T cell responses in vivo. While transplant models with known neoantigen expression are widely used, there is a limitation in the availability of autochthonous tumor models where the tumor coevolves with the immune system. In this dissertation, various approaches to studying the tumor-specific immune response in the autochthonous setting are presented and discussed. One such approach that has been developed involves combining CRISPR/Cas9 and sleeping beauty transposase technology to create an autochthonous orthotopic murine sarcoma model. This model incorporates key genetic elements such as oncogenic KrasG12D, functionally impaired p53, and the expression of known MHCI and MHCII sarcoma neoantigens. By utilizing MHC tetramer flow cytometry, a tumor-specific immune response in the peripheral blood was identified as early as 10 days after tumor induction, leading to effective tumor clearance. Interestingly, when CD8 and CD4 T cells were co-depleted, tumors developed at a high penetrance. However, depleting either CD8 or CD4 T cells alone was insufficient to permit tumor growth. These findings indicate that both CD8 and CD4 T cells can independently contribute to immunosurveillance and participate in the clearance of sarcomas expressing MHCI and MHCII neoantigens. Understanding the tumor-specific immune response in autochthonous models is crucial for uncovering new targets for immunotherapy and identifying biomarkers of response. The development of the autochthonous orthotopic murine sarcoma model described in this dissertation provides a valuable tool for investigating the mechanisms and characteristics of tumor-specific T cell responses in an in vivo setting.

Myxoid liposarcoma (MLPS) is a malignant soft tissue sarcoma characterized by a pathognomonic t(12;16)(q13;p11) translocation that produces a fusion oncoprotein, FUS-CHOP. This cancer is remarkably sensitive to radiotherapy and exhibits a unique pattern of extrapulmonary metastasis. However, the mechanism for its radiosensitivity is unknown. In order to further understand the biological mechanisms underlying MLPS response to radiotherapy we studied the fusion oncoprotein FUS-CHOP and linked its role in sarcomagenesis to the radiosensitivity phenotype.

Here we investigate a molecular mechanism of radiosensitization that couples ionizing radiation to inhibition of translocation-driven sarcomagenesis in myxoid liposarcoma. We performed co-immunoprecipitation (co-IP) to identify proteins interacting with FUS-CHOP. Incucyte assays measured cell proliferation after knockdown of interacting proteins fusion-negative and fusion-positive primary murine sarcoma cell lines from a novel FUS-CHOP genetically engineered mouse model (GEMM). ChIP-seq/CUT&RUN mapped genome-wide binding sites of FUS-CHOP and identified DNA-binding motifs for the fusion oncoprotein. Co-IP of irradiated human MLPS cell lines were performed to evaluate post-translational modification of FUS-CHOP after irradiation, and to investigate regulation of protein-protein interactions by these modifications.

We detected functionally important interactions between FUS-CHOP and multiple chromatin remodeling complexes via co-IP including a new interaction with SNF2H, the ATPase subunit of the imitation switch (ISWI) complex. Using knockdown systems, we demonstrated that these interacting chromatin remodelers are functionally important for proliferation specifically in FUS-CHOP-driven, but not Kras-driven murine sarcoma cells. ChIP-seq and CUT&RUN profiling of human MLPS cell lines identified DNA-binding motifs and genomic loci targeted by FUS-CHOP, which co-localized with SNF2H and H3K27ac marks of active chromatin. We further hypothesized that post-translational modification of the FUS-CHOP PrLD may regulate the protein-protein interactions between FUS-CHOP and chromatin remodelers. Using irradiated human MLPS cell lines, we show that FUS-CHOP is a target of phosphorylation by the DNA damage response kinases DNA-PK and ATM after irradiation. Finally, we show that phosphorylation of the PrLD of FUS-CHOP diminishes protein-protein interactions with chromatin remodeling complexes and the ability for FUS-CHOP to transform NIH-3T3 cells.

We also report the generation and characterization of a spatially and temporally restricted mouse model of sarcoma driven by FUS-CHOP. Using different Cre-drivers in the adipocyte lineage, we initiated in vivo tumorigenesis by expressing FUS-CHOP in Prrx1+ mesenchymal progenitor cells. In contrast, expression of FUS-CHOP in more differentiated cells does not form tumors in vivo, and early expression of the oncoprotein during embryogenesis is lethal. We also employ in vivo electroporation and CRISPR technology to rapidly generate spatially and temporally restricted mouse models and cell lines of high grade FUS-CHOP-driven sarcomas for preclinical studies.

Soft tissue sarcomas (STSs) are a heterogeneous group of mesenchymal tumors comprised of >70 subtypes. An important question is how the cell of origin and the pathways to tumor development shape the broad array of STS subtypes. By forcing identical tumor-promoting mutations to different cell types in Genetically Engineered Mouse Models (GEMMs) of STS, I have a unique model system to investigate this question. In the process of performing these experiments I observed that genetic mutations are necessary, but not sufficient for rapid sarcoma formation. However, tissue injury dramatically accelerates sarcoma formation in our GEMM of STS. For my thesis, I have worked to understand how cell of origin affects sarcoma subtype and how the microenvironment in our models promotes transformation. I have observed that cell of origin plays an important, but not the only, role in defining STS subtype. Additionally, I have concluded that the microenvironment, and specifically the HGF/c-MET signaling pathway play a crucial role in promoting sarcoma development after acute tissue injury.