Browsing by Subject "Tumor immunology"

The APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family of cytidine deaminases is one of the most common endogenous sources of single base substitution mutations in human cancer. Accordingly, APOBEC enzymes represent a major source of intratumor genetic heterogeneity and have been associated with immunotherapy response in diverse cancer types. However, the consequences of APOBEC mutagenesis on tumor progression in vivo are not well understood. To address this, I developed several murine tumor models with inducible APOBEC3B expression and studied the contribution of APOBEC activity to tumor evolution and immunogenicity. First, I explored the effects of APOBEC activity on tumor relapse using a murine model of mammary tumor recurrence. APOBEC activity led to a significant acceleration in tumor recurrence following the strong selective pressure of oncogenic driver signaling loss. Recurrent APOBEC tumors had undifferentiated histological features and large, irregularly shaped nuclei containing defects like micronuclei, multinucleation, and chromatin bridges. I found that recurrent APOBEC tumors amplified the therapy resistance-associated oncogene, c-Met, on circular extrachromosomal DNA, likely driving the proliferation of the recurrent cancer cells. Second, because APOBEC mutational signatures are enriched in the majority of HER2-positive breast cancer patients, I used a syngeneic HER2-driven mammary tumor model to study the effects of APOBEC activity on the tumor immune microenvironment. I found that APOBEC activity induced an antitumor adaptive immune response and CD4+ T cell-mediated tumor growth inhibition. While polyclonal APOBEC tumors had a moderate growth defect, clonal APOBEC tumors were almost completely rejected by the immune system, suggesting that APOBEC-mediated genetic heterogeneity limits the antitumor adaptive immune response. In human breast cancers, the relationship between APOBEC mutagenesis and immunogenicity varied by breast cancer subtype and the frequency of subclonal mutations. Consistent with the observed immune infiltration in murine APOBEC tumors, APOBEC activity sensitized HER2-driven breast tumors to checkpoint inhibition. This work provides a mechanistic basis for the sensitivity of APOBEC tumors to checkpoint inhibitors and suggests a rationale for using APOBEC mutational signatures and clonality as biomarkers predicting immunotherapy response in HER2-positive breast cancers. In conclusion, I’ve identified a novel role for APOBEC activity in generating chromosomal instability, consisting of mitotic errors, oncogene amplification, and extrachromosomal DNA formation to promote tumor recurrence. Moreover, APOBEC activity also stimulated an antitumor adaptive immune response and sensitized tumors to immunotherapy.

The histologic subtypes of malignant glial neoplasms range from anaplastic astrocytoma to the most deadly World Health Organization (WHO) Grade IV glioblastoma (GBM), the most common primary brain tumor in adults. Over the past 40 years, only modest advancements in the treatment of GBM tumors have been reached. Current therapies are predominantly for palliative endpoints rather than curative, although some treatment modalities have been shown to extend survival in particular cases. Patients undergoing current standard of care therapy, including surgical resection, radiation therapy, and chemotherapy, have a median survival of 12-15 months, with less than 25% of patients surviving up to two years and fewer than 10% surviving up to five years. A variety of factors contribute to standard treatment failure, including highly invasive tumor grade at the time of diagnosis, the intrinsic resistance of glioma cells to radiation therapy, the frequent impracticality of maximal tumor resection of eloquent cortical structures, and the fragile intolerance of healthy brain for cytotoxic therapies. Treatment with immunotherapy is a potential answer to the aforementioned problems, as the immune system can be harnessed and educated to license rather potent antitumor responses in a highly specific and safe fashion. One of the most promising vehicles for immunotherapy is the use of dendritic cells, which are professional antigen-presenting cells that are highly effective in the processing of foreign antigens and the education of soon-to-be activated T cells against established tumors. The work outlined in this dissertation encompasses the potential of dendritic cell therapy, the current limitations of reaching full efficacy with this platform, and the recent efforts employed to overcome such barriers. This work spans the characterization and preclinical testing of utilizing protein antigens such as tetanus-diphtheria toxoid to pre-condition the injection site prior to dendritic cell vaccination against established tumors expressing tumor-specific antigens.

Chapter 1 comprises an overview of the current standard therapies for malignant brain tumors. Chapters 2 and 3 provide a review of immunotherapy for malignant gliomas in the setting of preclinical animal models and discuss issues relevant to the efficacy of dendritic cell vaccines for targeting of GBM. Chapters 4 provides the rationale, methodology, and results of research to improve the lymph node homing and immunogenicity of tumor antigen-specific dendritic cell vaccines in mouse models and in patients with newly diagnosed GBM. Chapter 5 delineates the interactions discovered through efforts in Chapter 4 that comprise protein antigen-specific CD4+ T cell responses to induced chemokines and how these interactions result in increased dendritic cell migration and antitumor responses. Lastly, Chapter 6 discusses the future utility of migration of DC vaccines as a surrogate for antitumor responses and clinical outcomes.

This dissertation comprises original research as well as figures and illustrations from previously published material used to exemplify distinct concepts in immunotherapy for cancer. These published examples were reproduced with permission in accordance with journal and publisher policies described in the Appendix.

In summary, this work 1) identifies inefficient lymph node homing of peripherally administered dendritic cells as one of the glaring barriers to effective dendritic cell immunotherapy, 2) provides answers to overcome this limitation with the use of readily available pre-conditioning recall antigens, 3) has opened up a new line of investigation for interaction between recall responses and host chemokines to activate immune responses against a separate antigen, and 4) provides future prospects of utilizing chemokines as adjuvants for additional immunotherapies targeting aggressive tumors. Together, these studies hold great promise to improve the responses in patients with GBM.

Over half of breast cancer related deaths are due to recurrence five or more years after initial diagnosis and treatment. This latency suggests that a population of residual tumor cells can survive treatment and persist in a dormant state for many years. The role of the microenvironment in regulating the survival and proliferation of residual cells following therapy remains unexplored. Using a conditional mouse model for Her2-driven breast cancer, we identify interactions between residual tumor cells and their microenvironment as critical for promoting tumor recurrence. Her2 downregulation leads to an inflammatory program driven by TNFα/NFκB signaling, which promotes immune cell infiltration in regressing and residual tumors. The cytokine CCL5 is elevated following Her2 downregulation and remains high in residual tumors. CCL5 promotes tumor recurrence by recruiting CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors. Blocking this TNFα-CCL5-macrophage axis may be efficacious in preventing breast cancer recurrence.

In addition, it remains unclear how the clonal composition of tumors changes during tumor relapse. We used cellular barcoding to directly monitor clonal dynamics during tumor recurrence in a genetically engineered mouse model. We found that the clonal diversity of tumors progressively decreased during tumor regression, residual disease, and recurrence. Only a fraction of subclones survived oncogene withdrawal and persisted in residual tumors. The minimal residual disease phase itself was accompanied by a continued attrition of clones, suggesting an ongoing process of selection during dormancy. The reactivation of dormant residual cells into recurrent tumors followed several distinct evolutionary routes. Approximately half of the recurrent tumors exhibited a striking clonal dominance in which one or two subclones comprised the vast majority of the tumor. The majority of these clonal recurrent tumors exhibited evidence of de novo acquisition of Met amplification, and were sensitive to small-molecule Met inhibitors. A second group of recurrent tumors exhibited marked polyclonality, with thousands of subclones and a clonal architecture very similar to primary tumors. These polyclonal recurrent tumors were not sensitive to Met inhibitors, but were instead dependent upon an autocrine IL-6 – Stat3 pathway. These results suggest that the survival and reactivation of dormant tumors can proceed via multiple independent routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.

CD8+ T cells have potent anti-tumor responses. However, continuous stimulation by tumor cells causes these cells differentiate down an exhaustion pathway. This differentiation negatively impacts CD8+ T cell function and response to immunotherapies. One potential factor that influences the progression along this pathway is tumor necrosis factor (TNF). Using multiple tumor models and a chronic viral infection model, we determined that the TNF receptor (TNFR2) is significantly upregulated in tumor- infiltrating CD8+ T cells and CD8+ T cells in the spleen of virus-infected mice. Furthermore, TNFR2 correlated with other canonical markers of exhaustion. We identified that upregulation of TNFR2 was associated with loss of progenitor-like functions and acquisition of functions resembling terminally exhausted cells. To investigate the role of TNFR2 in this transition, we employed TNFR2 KO mice. While the frequency of the progenitor and terminally exhaustion populations were similar, the exhaustion- associated transcription factor TOX was significantly decreased in TNFR2 KO T cells. We demonstrate that the loss of TNFR2 is sufficient to increase effector functions of CD8+ T cells in tumor-draining lymph nodes, but not in the tumor. Loss of TNFR2 resulted in a CD8-mediated reduction in tumor growth in various subcutaneous tumor models. Finally, treatment with a TNFR2 antagonist in combination with an immune checkpoint inhibitor (aPD1) resulted in decreased tumor growth. Together, these data suggest that TNFR2 is a novel marker of exhaustion and blockade could be utilized as a novel treatment strategy.