Jonathan B. Weitzman

Cancer hallmarks are characterized by wide-scale changes in gene expression programs. Pioneering studies showed how viral oncogenes target regulatory pathways, but little is known about tumorigenic mechanisms of non-viral pathogens. Theileria annulata is an intracellular parasite (related to apicomplexa parasites causing malaria) which remarkably transforms bovine leukocytes, hijacking host signaling pathways to induce cancer phenotypes, akin to human leukemias. While some host genes contribute to the proliferative or invasive hallmark phenotypes, there is still limited comprehensive understanding of the impact of Theileria infection on host transcription and transformation. We performed a multi-omics meta-analysis to investigate the effect of Theileria infection on cancer hallmarks in bovine B lymphocytes. Combining transcriptomic, proteomic and epigenomic analysis across multiple datasets, we show that Theileria infection suppresses host immune pathways. Specifically, genes encoding innate and adaptive immune mediators are repressed in T. annulata -infected B cells (and in T. parva infected T cells), including downregulation of genes for Toll-like receptors (TLR), inflammasome components of the guanylate binding protein (GBP) family and major histocompatibility complex class (MHC) II genes. Treatment with distinct theilericidal drugs could partially rescue immune gene expression. Mechanistically, we describe alterations in the host epigenome, including loss of activating histone modifications (e.g., H3K18ac, H3K4me3, H3K27ac) on the promoters of repressed immune genes, and enrichment of silencing marks (H3K27me3) on promoters of the BOLA genes and the gene encoding CIITA, the master transcriptional regulator of MHC class II gene expression. Our results suggest that intracellular T . annulata and T. parva parasites could drive an immune evasion cancer hallmark in host lymphocytes by epigenetic silencing of genes for innate and adaptive immunity.

Intracellular pathogens develop elaborate mechanisms to survive within the hostile environments of host cells. Theileria parasites infect bovine leukocytes and cause devastating diseases in cattle in developing countries. Theileria spp. have evolved sophisticated strategies to hijack host leukocytes, inducing proliferative and invasive phenotypes characteristic of cell transformation. Intracellular Theileria parasites secrete proteins into the host cell and recruit host proteins to induce oncogenic signaling for parasite survival. It is unknown how Theileria parasites evade host cell defense mechanisms, such as autophagy, to survive within host cells. Here, we show that Theileria annulata parasites sequester the host eIF5A protein to their surface to escape elimination by autophagic processes. We identified a small-molecule compound that reduces parasite load by inducing autophagic flux in host leukocytes, thereby uncoupling Theileria parasite survival from host cell survival. We took a chemical genetics approach to show that this compound induced host autophagy mechanisms and the formation of autophagic structures via AMPK activation and the release of the host protein eIF5A which is sequestered at the parasite surface. The sequestration of host eIF5A to the parasite surface offers a strategy to escape elimination by autophagic mechanisms. These results show how intracellular pathogens can avoid host defense mechanisms and identify a new anti-Theileria drug that induces autophagy to target parasite removal.

Supplementary Tables 2-3 from A Genomic Map of p53 Binding Sites Identifies Novel p53 Targets Involved in an Apoptotic Network

Supplementary Tables 2-3 from A Genomic Map of p53 Binding Sites Identifies Novel p53 Targets Involved in an Apoptotic Network

Supplementary Table 1 from A Genomic Map of p53 Binding Sites Identifies Novel p53 Targets Involved in an Apoptotic Network

Supplementary Table 1 from A Genomic Map of p53 Binding Sites Identifies Novel p53 Targets Involved in an Apoptotic Network

The transcriptional activity of the p53 protein is central to its role in tumor suppression. Identification of the complete repertoire of p53-regulated genes is critical for dissecting the complexity of the p53 network. Although several different approaches have been used to characterize the p53 genetic program, we still lack a comprehensive molecular understanding of how p53 prevents cancer. Using a computational approach, we generated a genome-wide map of p53 binding sites (p53BS) to identify novel p53 target genes. We show that the presence of nearby p53BS can identify new proapoptotic members of the Bcl2 family. We show that p53 binds to p53BS identified in the BCL-G/BCL2L14 gene and that induction of this gene contributes to p53-mediated apoptosis. We found that p53 activates the COL18A1 gene encoding the precursor for the antiangiogenic factor endostatin. We also show that p53 up-regulates the MAP4K4 gene and activates the c-Jun NH2-terminal kinase (JNK) pathway to drive apoptosis. Thus, unbiased mapping of the genomic landscape of p53BS provides a systematic and complementary approach to identify novel factors and connections in the p53 genetic network. Our study illustrates how systematic genomic approaches can identify binding sites that are functionally relevant for a p53 transcriptional program. The genetic link among p53, antiangiogenic factors, and the JNK signaling pathway adds new dimensions to understanding p53 function in highly connected genetic networks.

Theileria parasites are responsible for devastating cattle diseases, causing major economic losses across Africa and Asia. Theileria spp. stand apart from other apicomplexa parasites by their ability to transform host leukocytes into immortalized, hyperproliferating, invasive cells that rapidly kill infected animals. The emergence of resistance to the theilericidal drug Buparvaquone raises the need for new anti-Theileria drugs. We developed a microscopy-based screen to reposition drugs from the open-access Medicines for Malaria Venture (MMV) Pathogen Box. We show that Trifloxystrobin (MMV688754) selectively kills lymphocytes or macrophages infected with Theileria annulata or Theileria parva parasites. Trifloxystrobin treatment reduced parasite load in vitro as effectively as Buparvaquone, with similar effects on host gene expression, cell proliferation and cell cycle. Trifloxystrobin also inhibited parasite differentiation to merozoites (merogony). Trifloxystrobin inhibition of parasite survival is independent of the parasite TaPin1 prolyl isomerase pathway. Furthermore, modeling studies predicted that Trifloxystrobin and Buparvaquone could interact distinctly with parasite Cytochrome B and we show that Trifloxystrobin was still effective against Buparvaquone-resistant cells harboring TaCytB mutations. Our study suggests that Trifloxystrobin could provide an effective alternative to Buparvaquone treatment and represents a promising candidate for future drug development against Theileria spp.

Lysine methylation on histone tails impacts genome regulation and cell fate determination in many developmental processes. Apicomplexa intracellular parasites cause major diseases and they have developed complex life cycles with fine-tuned differentiation events. Yet, apicomplexa genomes have few transcription factors and little is known about their epigenetic control systems. Tick-borne Theileria apicomplexa species have relatively small, compact genomes and a remarkable ability to transform leucocytes in their bovine hosts. Here we report enriched H3 lysine 18 monomethylation (H3K18me1) on the gene bodies of repressed genes in Theileria macroschizonts. Differentiation to merozoites (merogony) leads to decreased H3K18me1 in parasite nuclei. Pharmacological manipulation of H3K18 acetylation or methylation impacted parasite differentiation and expression of stage-specific genes. Finally, we identify a parasite SET-domain methyltransferase (TaSETup1) that can methylate H3K18 and represses gene expression. Thus, H3K18me1 emerges as an important epigenetic mark which controls gene expression and stage differentiation in Theileria parasites.