Dr. Martin Hirst, PhD

Mechanistic studies in human cancer have relied heavily on cell lines and mouse models, but are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts; however, these are hampered by variable genetic background, inability to study early events, and practical issues with availability/reproducibility. We report here an efficient, reproducible model of T-cell leukemia in which lentiviral transduction of normal human cord blood yields aggressive leukemia that appears indistinguishable from natural disease. We utilize this synthetic model to uncover a role for oncogene-induced HOXB activation which is operative in leukemia cells-of-origin and persists in established tumors where it defines a novel subset of patients distinct from other known genetic subtypes and with poor clinical outcome. We show further that anterior HOXB genes are specifically activated in human T-ALL by an epigenetic mechanism and confer growth advantage in both pre-leukemia cells and established clones.

Acute leukemias are aggressive malignancies of developmentally arrested hematopoietic progenitors. We sought here to explore the possibility that changes in hematopoietic stem/progenitor cells during development might alter the biology of leukemias arising from this tissue compartment. Using a mouse model of acute T cell leukemia, we found that leukemias generated from fetal liver (FL) and adult bone marrow (BM) differed dramatically in their leukemia stem cell activity with FL leukemias showing markedly reduced serial transplantability as compared to BM leukemias. We present evidence that this difference is due to NOTCH1-driven autocrine IGF1 signaling, which is active in FL cells but restrained in BM cells by EZH2-dependent H3K27 trimethylation. Further, we confirmed this mechanism is operative in human disease and show that enforced IGF1 signaling effectively limits leukemia stem cell activity. These findings demonstrate that resurrecting dormant fetal programs in adult cells may represent an alternate therapeutic approach in human cancer.

RUNX1 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). The spectrum of RUNX1 mutations has led to the notion that it acts as a tumor suppressor in this context; however, other studies have placed RUNX1, along with transcription factors TAL1 and NOTCH1, as core drivers of an oncogenic transcriptional program. To reconcile these divergent roles, we knocked down RUNX1 in human T-ALL cell lines and deleted Runx1 or Cbfb in primary mouse T-cell leukemias. RUNX1 depletion consistently resulted in reduced cell proliferation and increased apoptosis. RUNX1 upregulated variable sets of target genes in each cell line, but consistently included a core set of oncogenic effectors including insulin-like growth factor 1 receptor (IGF1R) and NRAS. Our results support the conclusion that RUNX1 has a net positive effect on cell growth in the context of established T-ALL.

Prenatal alcohol exposure (PAE) can alter the development of neurobiological systems, leading to lasting neuroendocrine, neuroimmune, and neurobehavioral deficits. Although the etiology of this reprogramming remains unknown, emerging evidence suggests DNA methylation as a potential mediator and biomarker for the effects of PAE due to its responsiveness to environmental cues and relative stability over time. Here, we utilized a rat model of PAE to examine the DNA methylation profiles of rat hypothalami and leukocytes at four time points during early development to assess the genome-wide impact of PAE on the epigenome and identify potential biomarkers of PAE. Our model of PAE resulted in blood alcohol levels of ~80-150 mg/dl throughout the equivalent of the first two trimesters of human pregnancy. Hypothalami were analyzed on postnatal days (P) 1, 8, 15, 22 and leukocytes at P22 to compare central and peripheral markers. Genome-wide DNA methylation analysis was performed by methylated DNA immunoprecipitation followed by next-generation sequencing. PAE resulted in lasting changes to DNA methylation profiles across all four ages, with 118 differentially methylated regions (DMRs) displaying persistent alterations across the developmental period at a false-discovery rate (FDR) < 0.05. In addition, 299 DMRs showed the same direction of change in the hypothalamus and leukocytes of P22 pups at an FDR < 0.05, with some genes overlapping with the developmental profile findings. The majority of these DMRs were located in intergenic regions, which contained several computationally-predicted transcription factor binding sites. Differentially methylated genes were generally involved in immune function, epigenetic remodeling, metabolism, and hormonal signaling, as determined by gene ontology analyses. Persistent DNA methylation changes in the hypothalamus may be associated with the long-term physiological and neurobehavioral alterations in observed in PAE. Furthermore, correlations between epigenetic alterations in peripheral tissues and those in the brain will provide a foundation for the development of biomarkers of fetal alcohol spectrum disorder (FASD). Finally, findings from studies of PAE provide important insight into the etiology of neurodevelopmental and mental health disorders, as they share numerous phenotypes and comorbidities.

Increasing evidence of functional and transcriptional heterogeneity in phenotypically similar cells examined individually has prompted interest in obtaining parallel methylome data. We describe the development and application of such a protocol to index-sorted murine and human hematopoietic cells that are highly enriched in their content of functionally defined stem cells. Utilizing an optimized single-cell bisulfite sequencing protocol, we obtained quantitative DNA methylation measurements of up to 5.7 million CpGs in single hematopoietic cells. In parallel, we developed an analytical strategy (PDclust) to define single-cell DNA methylation states through pairwise comparisons of single-CpG methylation measurements. PDclust revealed that a single-cell epigenetic state can be described by a small (<1%) stochastically sampled fraction of CpGs and that these states are reflective of cell identity and state. Using relationships revealed by PDclust, we derive near complete methylomes for epigenetically distinct subpopulations of hematopoietic cells enriched for functional stem cell content.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.