Browsing by Author "Kirsch, David G"

Soft Tissue Sarcomas are a rare group of mesenchymal tumors with over 50 recognized subtypes. These tumors are a diverse group of malignancies that primarily arise from the connective tissue, fat and muscle. In the United States, there are estimated to be approximately 11,000 new diagnoses a year with an annual mortality rate approaching 40%. Unfortunately, with such a diversity of subtypes of soft tissue sarcoma, and the relative scarcity of patient samples, there is a need for animal models that faithfully recapitulate the biology of these tumors. Such animal models would be useful for dissecting the underlying biology of soft tissue sarcomas and to evaluate novel therapies. One such model is the LSL-KrasG12D; p53Flox/Flox mouse model of soft tissue sarcoma. These tumors are generated in a spatial and temporally restricted fashion and closely mimic the natural history of human soft tissue sarcomas, including a predilection to develop lung metastases. Here I will characterize this model of soft tissue sarcoma by: 1) performing cross species genomic comparisons to show that the LSL-KrasG12D; p53Flox/Flox mouse model of soft tissue sarcoma most closely resembles Undifferentiated Pleomorphic Sarcoma , 2) utilizing this mouse model to identify cathepsin proteases as molecular markers of soft tissue sarcoma. I will then use cathepsin activated imaging probes for intraoperative molecular imaging to identify microscopic residual cancer in real time. Finally, 3) I identify a novel mechanism through which MAPK signaling regulates miRNA biogenesis and the development of distant metastases in the LSL-KrasG12D; p53Flox/Flox mouse model of soft tissue 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.

AbstractFor over 30 years the cancer biology field has scrutinized the mechanisms behind p53 and RB mediated tumor suppression (Fields and Jang 1990; Kern et al. 1991; Raycroft, Wu, and Lozano 1990; Dyson 1998; Classon and Harlow 2002; Knudsen and Knudsen 2008) Together, these genes regulate complex interconnecting pathways responsible for the regulation of cell growth, cell death, and genomic integrity (Sherr and McCormick 2002). Not surprisingly, the pathways regulated by these two tumor suppressors are almost universally disrupted during the development of cancer (Hanahan and Weinberg 2000). By nature, tumor suppressors play a key role in preserving the integrity of the genome. Whether in response to injury or in maintaining stable gene expression patterns, p53 and RB have been well established as two of the most important factors preventing genomic instability, epigenetic deregulation, transformation, and tumorigenesis. However, the precise mechanism by which p53 or RB mediate tumor suppression remains unclear (Tiwari, Jones, and Abrams 2018). Over the last decade, several studies in genetically engineered mouse models have demonstrated that the canonical functions of these two tumor suppressors fail to fully explain their tumor suppressive capabilities (Mello and Attardi 2018; Janic et al. 2018; T. Li et al. 2012). Moreover, expanding literature continues to highlight a strong correlation between transposable element de-repression and several types of cancer, however, no definitive link between transposable elements and tumorigenesis has been established (Tiwari, Jones, and Abrams 2018; Wylie et al. 2016; Tiwari et al. 2020; Rodriguez-Martin et al. 2020). At present, the role that genomic repetitive elements play in tumorigenesis remains an open question. Here, to elucidate a possible mechanism of transformation we sought to investigate the link between the concurrent loss of p53 and RB and the de-repression of transposons. While individually, p53 and RB have been implicated in separate transposable element defense mechanisms, each responsible for preventing the expression of transposons (Ishak et al. 2016; Wylie et al. 2016; Tiwari et al. 2020; Dick et al. 2018; Tiwari, Jones, and Abrams 2018), my thesis work seeks to understand the role these repetitive elements play in transformation. Specifically, I investigate how the combined loss of p53 and RB affect the expression of transposons and how this relates to the development of cancer. Using genetically engineered mouse models we derived several lines of mouse embryonic fibroblasts (MEFs) which, through cre-lox technology, allowed for the deletion of floxed tumor suppressors (H. Kim et al. 2018). We derived MEFs from mice containing floxed p53, RB, both p53 and RB as well as several p53 and RB mutants. Generating primary MEFs allowed us to interrogate the loss of p53 and RB in a non-cancerous context, free of additional mutations (Todaro and Green 1963; Xu 2005). Moreover, by choosing MEFs as a model system, we could harness and observe the transformation process in a minimally manipulated system. Accordingly, we transformed normal Wild Type (WT) MEF cells and induced transformation by recombining loxP sites flanking p53 and RB. In dissecting whether loss of p53 and RB affected transposable element expression we showed that loss of p53 or RB alone each partially derepress long interspersed nuclear element 1 (LINE1) transposable elements, but remarkably co-deletion of p53 and RB together not only transform cells but also simultaneously induce massive expression of LINE1. Additionally, through the use of p53 transactivation domain mutants, we showed that the ability of p53 to repress transposable elements is a non-canonical function linked to tumor suppression. Interrogation of short interspersed nuclear elements (SINEs) produced similar findings. Furthermore, the derepression of both LINEs and SINEs appears to be regulated by the modification of the H3K9me3 histone mark. These results further correlate the expression of transposable elements to transformation and tumorigenesis. Complementary work revealed that loss of both p53 and RB in MEFs not only derepressed transposable elements and transformed cells, but in the process, massively rearranged the three-dimensional (3-D) chromatin landscape. This change in 3-D chromatin architecture resulted in the loss of intrachromosomal loops and the subsequent overexpression of several oncogenes. To assess the scope of the rearrangement of chromatin architecture, we developed a UCSC genome browser based atlas, mapping thousands DNA loops stemming from promoters, enhancers and silencers. We find that while the general structure of topologically associated domains are largely stable (Dixon et al. 2012), following deletion of p53 or RB, local chromatin contacts are vastly reorganized. Moreover, the reorganization of DNA loops was found to affect gene expression in a context dependent manner. These results support a model of p53 and RB as guardians of the genome that cooperate to maintain the chromatin architecture required for normal cellular function and prevent changes in DNA topology that promote uncontrolled growth associated with cellular transformation. More than half of all cancer patients have mutations in p53 or RB and many harbor evidence of deregulated transposable elements amidst a disordered chromatin landscape. Here, we explore the distinct and overlapping genomic regulatory processes that p53 and RB cooperatively maintain to preserve genomic integrity and prevent cancer. Overall, my work implicates transposable element derepression as a mechanism that can promote the development of cancer and moreover, provides a genome wide atlas of how p53 and RB can affect the chromatin landscape to regulate gene expression in a context dependent manner. Moreover, my work provides mechanistic insight into the tissue specific functions of p53 and RB. Taken together, this thesis provides new insights into the mechanisms by which p53 and RB prevent cancer.

Over half of all cancer patients receive radiation therapy, and it contributes to over 40% of cancer cures. Within the last decade, cancer immunotherapy has become a pillar of cancer therapy, along with surgery, chemotherapy, and radiation therapy. The most commonly used type of immunotherapy is immune checkpoint blockade, which can increase immune cell activity by blocking inhibitory signals. Preclinical studies with transplanted tumors demonstrate high cure rates with either immune checkpoint blockade, radiotherapy, or combination treatment. These studies have led to hundreds of clinical trials testing checkpoint blockade and radiotherapy alone or in combination with other therapies, but emerging results are disappointing. My thesis work seeks to develop novel mouse models of cancer that recapitulate human disease, understand mechanisms of tumor resistance to radiation and immunotherapy, and identify novel immunologic targets that can enhance patient responses to radiation therapy.

Using complex genetically engineered mouse models of cancer, we investigated the contributions of the tumor microenvironment to therapeutic resistance. First, we explored the role of neutrophils in mediating radiation response. We showed that elevated neutrophil levels were associated with poor local control and survival in cervical cancer patients treated with definitive chemoradiation. Furthermore, in a genetically engineered mouse model of sarcoma, we demonstrated that genetic and antibody-mediated depletion of neutrophils increases radiosensitivity and decreases a mitogen-activated protein kinase transcriptional program. These results demonstrate that neutrophils promote tumor resistance to radiotherapy.

In complementary work using novel genetically engineered mouse models of sarcoma, we found that cells with high tumor mutational burden transplanted into syngeneic mice were cured by immune checkpoint blockade and radiation therapy, but the identical treatment failed in autochthonous sarcomas. To understand the mechanisms by which primary tumors were resistant to tumor cure by radiation and immunotherapy, we generated a single cell atlas of tumor-infiltrating immune cells from transplant and primary sarcomas treated with radiation and immunotherapy, which revealed marked differences in their immune landscapes. We found that radiation therapy remodeled myeloid cell phenotypes in primary and transplant sarcomas, but only transplant tumors were enriched for the effector CD8+ T cells that mediate response to combination therapy. In contrast, mice with autochthonous sarcomas demonstrated tumor-specific tolerance. These results indicate that radiation and immunotherapy cooperate to promote immunity within the tumor microenvironment, but identify immune tolerance in autochthonous tumors that must be overcome for this promising combination treatment to cure cancers that co-evolve with the immune system.

Approximately 50% of all patients with cancer receive radiation therapy at some point during the course of their illness. Despite advances in radiation delivery and treatment planning, normal tissue toxicity often limits the ability of radiation to eradicate tumors. The tumor microenvironment consists of tumor cells and stromal cells such as endothelial cells that contribute to tumor initiation, progression and response to therapy. Although endothelial cells can contribute to normal tissue injury following radiation, the contribution of stromal cells to tumor response to radiation therapy remains controversial. To investigate the contribution of endothelial cells to the radiation response of primary tumors, we have developed the technology to contemporaneously mutate different genes in the tumor cells and stromal cells of a genetically engineered mouse model of soft tissue sarcoma. Using this dual recombinase technology, we deleted the DNA damage response gene Atm in sarcoma and heart endothelial cells. Although deletion of Atm increased cell death of proliferating tumor endothelial cells, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. In addition, the ATM inhibitor NVP-BEZ235 selectively radiosensitized primary sarcomas, demonstrating a therapeutic window for inhibiting ATM during radiation therapy. Sensitizing tumor endothelial cells to radiation by deleting Atm prolonged tumor growth delay following a non-curative dose of radiation, but failed to increase local control. In contrast, deletion of Atm in tumor parenchymal cells increased the probability of tumor eradication. These results demonstrate that tumor parenchymal cells rather than endothelial cells are the critical targets that regulate tumor eradicaiton by radiation therapy.

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Lung squamous cell carcinoma (LSCC) comprises ~30% of non-small cell lung cancers, and currently lacks effective targeted therapies. Previous immunohistochemical and microarray studies reported overexpression of Hedgehog (HH)-GLI signaling components in LSCC. However, they addressed neither the tumor heterogeneity nor the requirement for HH-GLI signaling. Here, we investigated the role of HH-GLI signaling in LSCC, and studied the therapeutic potential of HH-GLI pathway suppression.

Gene expression datasets of two independent LSCC patient cohorts were analyzed to study the activation of HH-GLI signaling. Four human LSCC cell lines were examined for HH-GLI signaling components. Cell proliferation and apoptosis were assayed in these cells after blocking the HH-GLI pathway by lentiviral-shRNA knockdown or small molecule inhibitors. Xenografts in immunodeficient mice were used to determine the in vivo efficacy of GLI inhibitor GANT61.

In both patient cohorts, we found that activation of HH-GLI signaling was significantly associated with the classical subtype of LSCC. GLI2 expression level was significantly higher than GLI1, and displayed strong positive correlations with the prominent markers for the classical subtype (SOX2, TP63 and PIK3CA) on chromosome 3q. In cell lines, genetic knockdown of SMO produced minor effects on cell survival, while GLI2 knockdown significantly reduced proliferation and induced extensive apoptosis. Consistently, the SMO inhibitor GDC-0449 resulted in limited cytotoxicity in LSCC cells, whereas the GLI inhibitor GANT61 was very effective. Importantly, GANT61 demonstrated specific in vivo anti-tumor activity in xenograft models of GLI-positive cell lines.

Taken together, we report SMO-independent regulation of GLI in LSCC, and demonstrate an important role for GLI2 in LSCC. Different from standard-of-care chemotherapy or small molecule inhibition of kinase signaling cascades, we present a novel and potent strategy to treat a subset of LSCC patients by targeting the GLI transcriptional network.

Background: TP53 is the most commonly mutated gene in cancer. Canonical TP53 DNA damage response pathways are well characterized and classically thought to underlie the tumor suppressive effect of TP53. Challenging this dogma, mouse models have revealed that p53 driven apoptosis and cell cycle arrest are dispensable for tumor suppression. Here, we investigate two mouse models that seek to further elucidate the necessity of p53 pathways in tumor suppression. These mutants represent a paradoxical signaling of canonical targets and incidence in human cancer. One p53 mutation is deficient in signaling, but not found in human cancer and the other represents the inverse context of a p53 mutation predicted to drive the expression of canonical targets, but is detected in human cancer.

Methods: We established novel mouse models with single amino acid substitutions (GAG>GAC, mouse p53E221D and GCC>GGG, mouse p53G259A) in the DNA-binding domain. The first represents the mouse variant of a p53 mutant that has wild-type function in screening assays, but is paradoxically found in human cancer in Li Fraumeni syndrome. The second mutation has decreased transactivation in screening assays but is not widely found in human cancer. Using mouse models and the analogous human mutants, we evaluated expression, transcriptional activation, and tumor suppression in vitro and in vivo.

Results: Expression of human p53E224D from cDNA translated to a fully functional p53 protein. However, mouse p53E221D/E221D RNA transcribed from the endogenous locus is mis-spliced resulting in nonsense-mediated decay. Moreover, fibroblasts derived from p53E221D/E221D mice do not express a detectable protein product. Mice homozygous for p53E221D/E221D exhibited increased tumor penetrance and decreased life expectancy compared to p53WT/WT animals. Expression of the human p53G262A from cDNA translated into a functionally deficient p53 protein. The mouse p53G259A/G259A mutation was transcribed in mouse embryonic fibroblasts, and hyper-stabilized in untreated cells. Signaling was modestly deficient and did not result in a significant tumor burden in p53G259A/G259A mouse models.

Conclusions: Mouse p53E221D/E221D and human p53E224D mutations lead to splice variation and a biologically relevant p53 loss of function in vitro and in vivo. Mouse p53G259A/G259A and human p53G262A are deficient in transactivation, but maintenance of homeostasis is achieved through hyperstabilization of the mutant.