Insights into the cell of origin, pathogenesis, and translational potential of canine peripheral T-cell lymphoma

Abstract

Peripheral T-cell lymphoma (PTCL) refers to a heterogenous group of T-cell neoplasms in humans and dogs with short survival times and poor treatment responses. The clinical features and immunophenotype of PTCL in dogs closely resemble PTCL, not otherwise specified (PTCL-NOS), the most common and most poorly understood subtype of PTCL in humans. This has led to interest in their potential as a preclinical model for this disease. Because these are naturally occurring tumors in dogs, they offer some unique advantages as a preclinical model compared to traditional xenograft and genetically engineered mouse models, including the ability to study these cancers in an immunocompetent host with a more comparable tumor microenvironment, the potential to investigate the genetic origins of these tumors in hosts with similar genetic diversity to humans, and the consideration of environmental risk factors, since humans and dogs largely share the same environments. One remaining barrier to this proposed canine model is our limited understanding of how the molecular features of canine PTCL compare to human PTCL-NOS. In humans, gene expression and mutational profiling studies have unveiled prognostically significant molecular subtypes of PTCL-NOS, identified enriched oncogenic signaling pathways, recognized mutations in key tumor suppressor genes and oncogenes, and discovered large structural variants such as fusion genes that contribute to the pathogenesis of PTCL-NOS and represent opportunities for novel targeted therapies. However, similar gene expression and mutational profiling studies in canine PTCL are lacking. We hypothesized that canine PTCL would exhibit a similar global gene expression profile to PTCL-NOS, would be enriched for the same oncogenic signaling pathways, and would share some of the most common gene mutations of human PTCL-NOS. We tested this hypothesis by performing bulk RNA-sequencing, differential gene expression analysis, and variant analysis on canine PTCL, normal canine nodal lymphocytes, and normal canine thymocytes. We discovered that the gene expression profile of the most common CD3+CD5+/-CD4+CD8– immunophenotype of canine PTCL closely resembles human PTCL-NOS—particularly the GATA3-expressing subtype associated with an inferior prognosis—and canine PTCL often exhibits mutations in the same genes commonly mutated in human PTCL-NOS, including DNMT3A, TP53, TET2, and PTEN. The cell of origin of human PTCL-NOS remains a subject of ongoing speculation. The observation that subsets of human PTCL-NOS upregulate either TBX21 or GATA3—transcription factors controlling the differentiation of naïve T cells to mature T helper 1 (Th1) or T helper 2 (Th2) cells, respectively—led to suggestions that these tumors are derived from their respective normal mature T-helper cell counterparts. However, more recent studies have demonstrated that GATA3 is an oncogene upregulated by a variety of neoplasms, including immature precursor T-cell neoplasms and even non-hematopoietic neoplasms, and its expression in GATA3-PTCL is associated with promotion of general T-cell survival and proliferation rather than Th2 differentiation. Additionally, mature T cells have been shown to be highly resistant to oncogenic transformation, suggesting that even seemingly mature T-cell neoplasms may actually arise from earlier precursors. Given the similarities between human PTCL-NOS and canine CD4+ PTCL, investigations into the cell of origin in dogs may offer additional insight into the cell of origin in human PTCL-NOS. One previous study identified decreased surface expression of CD25 and MHC class II in canine PTCL, molecules normally associated with T-cell maturity and activation, suggesting that canine PTCL may arise from an earlier T-cell precursor. Recently, an RNA-sequencing study comparing the gene expression profile of canine T-zone lymphoma, a more indolent form of T-cell lymphoma in dogs, to gene signatures of human and murine naïve and activated T-cell subsets successfully identified the cell of origin for this disease as a mature, previously activated αβ T cell. Therefore, we sought to employ similar methods to test our hypothesis that the cell of origin of canine CD4+ PTCL is a thymic precursor cell. Additionally, to help overcome some of the limitations of using publicly available data in other species, we sought to develop a single-cell transcriptomic atlas of normal canine thymic and lymph node tissue to identify key changing genes throughout T-cell development and differentiation in dogs and evaluate those expression signatures in canine CD4+ PTCL. We found that canine CD4+ PTCL upregulated several markers of immaturity, including CD34, KIT, DNTT, and CCR9. Canine CD4+ PTCL was enriched for gene signatures associated with immature murine and human thymocytes compared to more mature T-cell subsets. Finally, based on data derived from our single-cell atlas of normal thymus and lymph node, canine CD4+ PTCL tended to upregulate genes expressed early in canine thymocytes and downregulate genes expressed as canine T cells progressed to more mature stages of development. Taken together, these findings support an immature thymic precursor cell of origin for canine CD4+ PTCL. For a subset of human PTCL-NOS, a type of mutation known as fusion genes play a role in tumorigenesis and offer an opportunity for targeted therapies. These mutations are characterized by the juxtaposition of two previously independent genes, which can then be transcribed and potentially translated together to oncogenic effect. In small subsets of human PTCL-NOS, the presence of a fusion gene drives malignant transformation through upregulation of specific oncogenic signaling pathways, and inhibitors of these pathways have shown promise as an alternative treatment option in these tumors. Despite their potential as actionable drivers in many hematologic cancers, fusion genes have not been previously investigated or described as a feature of canine PTCL. We hypothesized that recurrent fusion genes could be identified in canine CD4+ PTCL, and that some would correlate with enrichment for particular oncogenic pathways. To test this hypothesis, we utilized fusion calling algorithms for bulk RNA-sequencing data and long-read DNA sequencing techniques to investigate expression of fusion mRNA transcripts and the presence of large chromosomal aberrations, respectively, in canine CD4+ PTCL. We identified 13 recurrent fusion genes at the mRNA level, 3 of which were tumor-specific and not detected in dogs without lymphoma. 2 of these 3 PTCL-specific fusions, TOX2-LMO4 and PER1-EIF5A, were associated with enrichment for TNF and NF-ĸB signaling and greater enrichment for elements of the tumor microenvironment, respectively, suggesting possible mechanisms by which these fusions could contribute to CD4+ PTCL. Long-read sequencing additionally revealed 3 fusion genes in CD4+ PTCL DNA that were also predicted in bulk RNA-seq data: STAG2-SH2D1A, PLEKHA5-AEBP2, and SERPINB5-ENSCAFG00000031329. The evidence of concurrent chromosomal aberrations and fusion transcript expression for these candidates make them stronger contenders for potential driver mutations that should be explored in future studies. Taken together, the work in this dissertation supports that canine CD4+ PTCL is a naturally occurring neoplasm in dogs that arises from a thymic precursor cell of origin and closely resembles the GATA3-expressing subtype of human PTCL-NOS. Additionally, similar to human PTCL-NOS, recurrent fusion genes are a feature of canine CD4+ PTCL that may arise from a combination of structural chromosomal variants and alternative RNA splicing events, and these fusions may contribute to the pathogenesis of CD4+ PTCL through activation of certain oncogenic signaling pathways. The shared features between canine CD4+ PTCL and human PTCL-NOS represent potential opportunities to explore novel therapies in the dog that may be translated to the human disease.