Robert Vanner

Cancer stem cells: an evolving concept

The cancer stem cell (CSC) concept derives from the fact that cancers are dysregulated tissue clones whose continued propagation is vested in a biologically distinct subset of cells that are typically rare. This idea is not new, but has recently gained prominence because of advances in defining normal tissue hierarchies, a greater appreciation of the multistep nature of oncogenesis and improved methods to propagate primary human cancers in immunodeficient mice. As a result we have obtained new insights into why the CSC concept is not universally applicable, as well as a new basis for understanding the complex evolution, phenotypic heterogeneity and therapeutic challenges of many human cancers.

Quiescent Sox2+ Cells Drive Hierarchical Growth and Relapse in Sonic Hedgehog Subgroup Medulloblastoma

Functional heterogeneity within tumors presents a significant therapeutic challenge. Here we show that quiescent, therapy-resistant Sox2(+) cells propagate sonic hedgehog subgroup medulloblastoma by a mechanism that mirrors a neurogenic program. Rare Sox2(+) cells produce rapidly cycling doublecortin(+) progenitors that, together with their postmitotic progeny expressing NeuN, comprise tumor bulk. Sox2(+) cells are enriched following anti-mitotic chemotherapy and Smoothened inhibition, creating a reservoir for tumor regrowth. Lineage traces from Sox2(+) cells increase following treatment, suggesting that this population is responsible for relapse. Targeting Sox2(+) cells with the antineoplastic mithramycin abrogated tumor growth. Addressing functional heterogeneity and eliminating Sox2(+) cells presents a promising therapeutic paradigm for treatment of sonic hedgehog subgroup medulloblastoma.

A Tumorigenic MLL-Homeobox Network in Human Glioblastoma Stem Cells

Glioblastoma growth is driven by cancer cells that have stem cell properties, but molecular determinants of their tumorigenic behavior are poorly defined. In cancer, altered activity of the epigenetic modifiers Polycomb and Trithorax complexes may contribute to the neoplastic phenotype. Here, we provide the first mechanistic insights into the role of the Trithorax protein mixed lineage leukemia (MLL) in maintaining cancer stem cell characteristics in human glioblastoma. We found that MLL directly activates the Homeobox gene HOXA10. In turn, HOXA10 activates a downstream Homeobox network and other genes previously characterized for their role in tumorigenesis. The MLL-Homeobox axis we identified significantly contributes to the tumorigenic potential of glioblastoma stem cells. Our studies suggest a role for MLL in contributing to the epigenetic heterogeneity between tumor-initiating and non-tumor-initiating cells in glioblastoma.

MLL5 Orchestrates a Cancer Self-Renewal State by Repressing the Histone Variant H3.3 and Globally Reorganizing Chromatin

Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy

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.

ASCL1 Reorganizes Chromatin to Direct Neuronal Fate and Suppress Tumorigenicity of Glioblastoma Stem Cells

Glioblastomas exhibit a hierarchical cellular organization, suggesting that they are driven by neoplastic stem cells that retain partial yet abnormal differentiation potential. Here, we show that a large subset of patient-derived glioblastoma stem cells (GSCs) express high levels of Achaete-scute homolog 1 (ASCL1), a proneural transcription factor involved in normal neurogenesis. ASCL1hi GSCs exhibit a latent capacity for terminal neuronal differentiation in response to inhibition of Notch signaling, whereas ASCL1lo GSCs do not. Increasing ASCL1 levels in ASCL1lo GSCs restores neuronal lineage potential, promotes terminal differentiation, and attenuates tumorigenicity. ASCL1 mediates these effects by functioning as a pioneer factor at closed chromatin, opening new sites to activate a neurogenic gene expression program. Directing GSCs toward terminal differentiation may provide therapeutic applications for a subset of GBM patients and strongly supports efforts to restore differentiation potential in GBM and other cancers.

DNA hypermethylation within TERT promoter upregulates TERT expression in cancer

Replicative immortality is a hallmark of cancer cells governed by telomere maintenance. Approximately 90% of human cancers maintain their telomeres by activating telomerase, driven by the transcriptional upregulation of telomerase reverse transcriptase (TERT). Although TERT promoter mutations (TPMs) are a major cancer-associated genetic mechanism of TERT upregulation, many cancers exhibit TERT upregulation without TPMs. In this study, we describe the TERT hypermethylated oncological region (THOR), a 433-bp genomic region encompassing 52 CpG sites located immediately upstream of the TERT core promoter, as a cancer-associated epigenetic mechanism of TERT upregulation. Unmethylated THOR repressed TERT promoter activity regardless of TPM status, and hypermethylation of THOR counteracted this repressive function. THOR methylation analysis in 1,352 human tumors revealed frequent (>45%) cancer-associated DNA hypermethylation in 9 of 11 (82%) tumor types screened. Additionally, THOR hypermethylation, either independently or along with TPMs, accounted for how approximately 90% of human cancers can aberrantly activate telomerase. Thus, we propose that THOR hypermethylation is a prevalent telomerase-activating mechanism in cancer that can act independently of or in conjunction with TPMs, further supporting the utility of THOR hypermethylation as a prognostic biomarker.

Abstract A25: Evolving functional heterogeneity in B-acute lymphoblastic leukemia

Current cancer therapies are directed at molecular markers or dominant pathways present in the bulk of neoplastic cells. However, recent studies have identified many genetically distinct subclones co-existing within a single neoplasm. In over 50% of patients with relapsed acute lymphoblastic leukemia (ALL), the genetic clones present at relapse are not the dominant clone present at diagnosis, but have evolved from a minor or ancestral clone (Mullighan et al., Science, 2008). Previous work has shown that this subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells (L-IC) capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients of varying cytogenetics. Patient samples were subjected to whole exome sequencing (WES), SNP analysis and RNA sequencing. Diagnosis-specific, relapse-specific, and shared variants at both clonal and subclonal frequencies were identified. Limiting dilution analysis by transplantation of CD19+ leukemic blasts into 870 immune-deficient mice (xenografts) identified no significant trend in enrichment in L-IC frequency between paired patient samples with a median frequency of 1 in 2691. Despite similar frequencies of L-IC, functional differences within identically sourced patient xenografts were observed, including increased leukemic dissemination of relapse cells to distal sites such as the central nervous system (CNS), differences in engraftment levels and differences in immunophenotypes. Targeted-sequencing and copy number analysis of the xenografts, in comparison to the patient sample from which they were derived, has uncovered clonal variation and the unequivocal identification of minor subclones ancestral to the relapse in xenografts transplanted with the diagnostic sample from 8 patients. Some of these subclones are rare and were not captured through standard WES analysis of the patient samples, highlighting the value of xenografting to functionally identify and viably isolate subclones for further study. Interrogation of the therapeutic responses of the ‘relapse-like’ diagnosis subclones in secondary xenografts displayed differential resistance to standard chemotherapeutic agents (vincristine and L-asparaginase) pre-existing in the patient diagnosis samples prior to treatment. Furthermore, investigation of different sites of leukemic infiltration in the xenografts provided evidence of distinct clonal selection in the CNS, a known site of disease relapse, in comparison to the bone marrow. Using this data we can begin to draw the evolutionary paths to relapse. We have shown evidence that minor subclones at diagnosis, ancestral to the relapsing clone, possess functional advantages over other diagnostic clones. Overall, this work provides a substantial advance in connecting genetic diversity to functional consequences, thereby furthering our understanding of the heterogeneity identified in B-ALL and its contributions to therapy failure and disease recurrence. Citation Format: Stephanie M. Dobson, Robert Vanner, Esme Waanders, Olga I. Gan, Jessica McLeod, Ildiko Grandal, Debbie Payne-Turner, Michael Edmonson, Zhaohui Gu, Xioatu Ma, Yiping Fan, Pankaj Gupta, Sagi Abelson, Michael Rusch, Ying Shao, Scott Olsen, Geoffrey Neale, John Easton, Cynthia J. Guidos, Jayne S. Danska, Jinghui Zhang, Mark D. Minden, Charles G. Mullighan, John E. Dick. Evolving functional heterogeneity in B-acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-341.