Brian S. White

Clonal diversity of recurrently mutated genes in myelodysplastic syndromes

Recent studies suggest that most cases of myelodysplastic syndrome (MDS) are clonally heterogeneous, with a founding clone and multiple subclones. It is not known whether specific gene mutations typically occur in founding clones or subclones. We screened a panel of 94 candidate genes in a cohort of 157 patients with MDS or secondary acute myeloid leukemia (sAML). This included 150 cases with samples obtained at MDS diagnosis and 15 cases with samples obtained at sAML transformation (8 were also analyzed at the MDS stage). We performed whole-genome sequencing (WGS) to define the clonal architecture in eight sAML genomes and identified the range of variant allele frequencies (VAFs) for founding clone mutations. At least one mutation or cytogenetic abnormality was detected in 83% of the 150 MDS patients and 17 genes were significantly mutated (false discovery rate ⩽0.05). Individual genes and patient samples displayed a wide range of VAFs for recurrently mutated genes, indicating that no single gene is exclusively mutated in the founding clone. The VAFs of recurrently mutated genes did not fully recapitulate the clonal architecture defined by WGS, suggesting that comprehensive sequencing may be required to accurately assess the clonal status of recurrently mutated genes in MDS.

SciClone: Inferring clonal architecture and tracking the spatial and temporal patterns of tumor evolution

The sensitivity of massively-parallel sequencing has confirmed that most cancers are oligoclonal, with subpopulations of neoplastic cells harboring distinct mutations. A fine resolution view of this clonal architecture provides insight into tumor heterogeneity, evolution, and treatment response, all of which may have clinical implications. Single tumor analysis already contributes to understanding these phenomena. However, cryptic subclones are frequently revealed by additional patient samples (e.g., collected at relapse or following treatment), indicating that accurately characterizing a tumor requires analyzing multiple samples from the same patient. To address this need, we present SciClone, a computational method that identifies the number and genetic composition of subclones by analyzing the variant allele frequencies of somatic mutations. We use it to detect subclones in acute myeloid leukemia and breast cancer samples that, though present at disease onset, are not evident from a single primary tumor sample. By doing so, we can track tumor evolution and identify the spatial origins of cells resisting therapy.

SIRT1 activators suppress inflammatory responses through promotion of p65 deacetylation and inhibition of NF-κB activity.

Chronic inflammation is a major contributing factor in the pathogenesis of many age-associated diseases. One central protein that regulates inflammation is NF-κB, the activity of which is modulated by post-translational modifications as well as by association with co-activator and co-repressor proteins. SIRT1, an NAD+-dependent protein deacetylase, has been shown to suppress NF-κB signaling through deacetylation of the p65 subunit of NF-κB resulting in the reduction of the inflammatory responses mediated by this transcription factor. The role of SIRT1 in the regulation of NF-κB provides the necessary validation for the development of pharmacological strategies for activating SIRT1 as an approach for the development of a new class of anti-inflammatory therapeutics. We report herein the development of a quantitative assay to assess compound effects on acetylated p65 protein in the cell. We demonstrate that small molecule activators of SIRT1 (STACs) enhance deacetylation of cellular p65 protein, which results in the suppression of TNFα-induced NF-κB transcriptional activation and reduction of LPS-stimulated TNFα secretion in a SIRT1-dependent manner. In an acute mouse model of LPS-induced inflammation, the STAC SRTCX1003 decreased the production of the proinflammatory cytokines TNFα and IL-12. Our studies indicate that increasing SIRT1-mediated NF-κB deacetylation using small molecule activating compounds is a novel approach to the development of a new class of therapeutic anti-inflammatory agents.

Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo

Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ∼ 11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.

Clonal architecture of secondary acute myeloid leukemia defined by single-cell sequencing.

Next-generation sequencing has been used to infer the clonality of heterogeneous tumor samples. These analyses yield specific predictions—the population frequency of individual clones, their genetic composition, and their evolutionary relationships—which we set out to test by sequencing individual cells from three subjects diagnosed with secondary acute myeloid leukemia, each of whom had been previously characterized by whole genome sequencing of unfractionated tumor samples. Single-cell mutation profiling strongly supported the clonal architecture implied by the analysis of bulk material. In addition, it resolved the clonal assignment of single nucleotide variants that had been initially ambiguous and identified areas of previously unappreciated complexity. Accordingly, we find that many of the key assumptions underlying the analysis of tumor clonality by deep sequencing of unfractionated material are valid. Furthermore, we illustrate a single-cell sequencing strategy for interrogating the clonal relationships among known variants that is cost-effective, scalable, and adaptable to the analysis of both hematopoietic and solid tumors, or any heterogeneous population of cells.

U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing.

We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of the patients with de novo myelodysplastic syndrome (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity-binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis.

Owl: next generation system monitoring

As microarchitectural and system complexity grows, comprehending system behavior becomes increasingly difficult, and often requires obtaining and sifting through voluminous event traces or coordinating results from multiple, non-localized sources. Owl is a proposed framework that overcomes limitations faced by traditional performance counters and monitoring facilities in dealing with such complexity by pervasively deploying programmable monitoring elements throughout a system. The design exploits reconfigurable or programmable logic to realize hardware monitors located at event sources, such as memory buses. These monitors run and writeback results autonomously with respect to the CPU, mitigating the system impact of interrupt-driven monitoring or the need to communicate irrelevant events to higher levels of the system. The monitors are designed to snoop any kind of system transaction, e.g., within the core, on a bus, across the wire, or within I/O devices

Defining NELF-E RNA Binding in HIV-1 and Promoter-Proximal Pause Regions

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

Stem cell dynamics in mouse hair follicles: A story from cell division counting and single cell lineage tracing

Understanding tissue stem cells behavior is a prerequisite for elucidating the mechanisms that govern their self-renewal and differentiation. Previously, we provided single cell lineage tracing and proliferation history data (based on H2B-GFP label dilution over time) in mouse hair follicles. We proposed a population deterministic model with symmetric stem cell fate decisions throughout life. Here we provide data suggesting that in hair follicle stem cells the self-renewing divisions within the niche (bulge) are symmetric with respect to localization of daughter cells near the basement membrane, an important niche component. In contrast, when cells migrate from the niche to the differentiating zone where they become short-lived progenitors, their daughter cells can orient themselves asymmetrically relative to the basement membrane. Furthermore, we document the dynamic re-localization of cells within the bulge to accommodate the hair follicle morphological changes through hair cycle. In addition, we provide a method to compute the change in number of cells generated by division from H2B-GFP pulse-chase data, and to estimate the minimum cell loss encountered when the fold change can be experimentally determined. We computed a minimum of 42% of bulge cell loss during one hair cycle, a massive rate of loss previously unrecognized. Finally, we showed that a multipotent population of cells found at the junction zone between hair follicle and epidermis, known to express Lrig1, cycle more rapidly than some other hair follicle compartments.

Runx1 and p21 synergistically limit the extent of hair follicle stem cell quiescence in vivo

Mechanisms of tissue stem cell (SC) quiescence control are important for normal homeostasis and for preventing cancer. Cyclin-dependent kinase inhibitors (CDKis) are known inhibitors of cell cycle progression. We document CDKis expression in vivo during hair follicle stem cell (HFSC) homeostasis and find p21 (cyclin-dependent kinase inhibitor 1a, Cdkn1a), p57, and p15 up-regulated at quiescence onset. p21 appears important for HFSC timely onset of quiescence. Conversely, we find that Runx1 (runt related transcription factor 1), which is known for promoting HFSC proliferation, represses p21, p27, p57, and p15 transcription in HFSC in vivo. Intriguingly, in cell culture, tumors, and normal homeostasis, Runx1 and p21 interplay modulates proliferation in opposing directions under the different conditions. Unexpectedly, Runx1 and p21 synergistically limit the extent of HFSC quiescence in vivo, which antagonizes the role of p21 as a cell cycle inhibitor. Importantly, we find in cultured keratinocytes that Runx1 and p21 bind to the p15 promoter and synergistically repress p15 mRNA transcription, thereby restraining cell cycle arrest. This documents a surprising ability of a CDKi (p21) to act as a direct transcriptional repressor of another CDKi (p15). We unveil a robust in vivo mechanism that enforces quiescence of HFSCs, and a context-dependent role of a CDKi (p21) to limit quiescence of SCs, potentially by directly down-regulating mRNA levels of (an)other CDKi(s).