Publications
Glioblastomas harbor diverse cell populations, including rare glioblastoma stem cells (GSCs) that drive tumorigenesis. To characterize functional diversity within this population, we performed single-cell RNA sequencing on >69,000 GSCs cultured from the tumors of 26 patients. We observed a high degree of inter- and intra-GSC transcriptional heterogeneity that could not be fully explained by DNA somatic alterations. Instead, we found that GSCs mapped along a transcriptional gradient spanning two cellular states reminiscent of normal neural development and inflammatory wound response. Genome-wide CRISPR-Cas9 dropout screens independently recapitulated this observation, with each state characterized by unique essential genes. Further single-cell RNA sequencing of >56,000 malignant cells from primary tumors found that the majority organize along an orthogonal astrocyte maturation gradient yet retain expression of founder GSC transcriptional programs. We propose that glioblastomas grow out of a fundamental GSC-based neural wound response transcriptional program, which is a promising target for new therapy development.
Microphthalmia, anophthalmia, and coloboma (MAC) are a heterogeneous spectrum of anomalous eye development and degeneration with genetic and environmental etiologies. Structural and copy number variants of chromosome 13 have been implicated in MAC; however, the specific loci involved in disease pathogenesis have not been well-defined. Herein we report a newborn with syndromic degenerative anophthalmia and a complex de novo rearrangement of chromosome 13q. Long-read genome sequencing improved the resolution and clinical interpretation of a duplication-triplication/inversion-duplication (DUP-TRP/INV-DUP) and terminal deletion. Sequence features at the breakpoint junctions suggested microhomology-mediated break-induced replication (MMBIR) of the maternal chromosome as the origin. Comparing this rearrangement to previously reported copy number alterations in 13q, we refine a putative dosage-sensitive critical region for MAC that might provide new insights into its molecular etiology.
Objectives: Acute myeloid leukaemia (AML) is a disease of older adults, who are vulnerable to socioeconomic factors. We determined AML incidence in older adults and the impact of socioeconomic factors on outcomes.
Methods: We included 3024 AML patients (1996-2016) identified from a population-based registry.
Results: AML incidence in patients ≥60 years increased from 11.01 (2001-2005) to 12.76 (2011-2016) per 100,000 population. Amongst 879 patients ≥60 years in recent eras (2010-2016), rural residents (<100,000 population) were less likely to be assessed by a leukaemia specialist (39% rural, 47% urban, p=0.032); no difference was seen for lower (43%, quintile 1-3) versus higher (47%, quintile 4-5) incomes (p=0.235). Similar numbers received induction chemotherapy between residence (16% rural, 18% urban, p=0.578) and incomes (17% lower, 17% high, p=1.0). Differences between incomes were seen for hypomethylating agent treatment (14% low, 20% high, p=0.041); this was not seen for residence (13% rural, 18% urban, p=0.092). Amongst non-adverse karyotype patients ≥70 years, 2-year overall survival was worse for rural (5% rural, 12% urban, p=0.006) and lower income (6% low, 15% high, p=0.017) patients.
Conclusions: AML incidence in older adults is increasing, and outcomes are worse for older rural and low-income residents; these patients face treatment barriers.
Inflammation is associated with bone marrow failure syndromes, but how specific molecules impact the bone marrow microenvironment is not well elucidated. We report a novel role for the miR-145 target, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), in driving bone marrow failure. We show that TIRAP is overexpressed in various types of myelodysplastic syndromes (MDS) and suppresses all three major hematopoietic lineages. TIRAP expression promotes up-regulation of Ifnγ, leading to myelosuppression through Ifnγ-Ifnγr-mediated release of the alarmin, Hmgb1, which disrupts the bone marrow endothelial niche. Deletion of Ifnγ blocks Hmgb1 release and is sufficient to reverse the endothelial defect and restore myelopoiesis. Contrary to current dogma, TIRAP-activated Ifnγ-driven bone marrow suppression is independent of T cell function or pyroptosis. In the absence of Ifnγ, TIRAP drives myeloproliferation, implicating Ifnγ in suppressing the transformation of MDS to acute leukemia. These findings reveal novel, noncanonical roles of TIRAP, Hmgb1, and Ifnγ in the bone marrow microenvironment and provide insight into the pathophysiology of preleukemic syndromes.
Background: Antibiotic resistance is a growing global health concern prompting researchers to seek alternatives to conventional antibiotics. Antimicrobial peptides (AMPs) are attracting attention again as therapeutic agents with promising utility in this domain, and using in silico methods to discover novel AMPs is a strategy that is gaining interest. Such methods can sift through large volumes of candidate sequences and reduce lab screening costs.
Results: Here we introduce AMPlify, an attentive deep learning model for AMP prediction, and demonstrate its utility in prioritizing peptide sequences derived from the Rana [Lithobates] catesbeiana (bullfrog) genome. We tested the bioactivity of our predicted peptides against a panel of bacterial species, including representatives from the World Health Organization's priority pathogens list. Four of our novel AMPs were active against multiple species of bacteria, including a multi-drug resistant isolate of carbapenemase-producing Escherichia coli.
Conclusions: We demonstrate the utility of deep learning based tools like AMPlify in our fight against antibiotic resistance. We expect such tools to play a significant role in discovering novel candidates of peptide-based alternatives to classical antibiotics.
Single-cell Strand-seq generates directional genomic information to study DNA repair, assemble genomes, and map structural variation onto chromosome-length haplotypes. We report a nanoliter-volume, one-pot (OP) Strand-seq library preparation protocol in which reagents are added cumulatively, DNA purification steps are avoided, and enzymes are inactivated with a thermolabile protease. OP-Strand-seq libraries capture 10%-25% of the genome from a single-cell with reduced costs and increased throughput.
Tumor biology may play an important role as an effective predictive biomarker that is complementary to functional imaging for metastatic hormone-sensitive prostate cancer.
Diffuse large B cell lymphoma (DLBCL) is successfully treated with combination immuno-chemotherapy, but relapse with resistant disease occurs in ~ 40% of patients. However, little is known regarding relapsed/refractory DLBCL (rrDLBCL) genetics and alternative therapies. Based on findings from other tumors, we hypothesized that RAS-MEK-ERK signaling would be upregulated in resistant tumors, potentially correlating with mutations in RAS, RAF, or associated proteins. We analyzed mutations and phospho-ERK levels in tumor samples from rrDLBCL patients. Unlike other tumor types, rrDLBCL is not mutated in any Ras or Raf family members, despite having increased expression of p-ERK. In paired biopsies comparing diagnostic and relapsed specimens, 33% of tumors gained p-ERK expression, suggesting a role in promoting survival. We did find mutations in several Ras-associating proteins, including GEFs, GAPs, and downstream effectors that could account for increased ERK activation. We further investigated mutations in one such protein, RASGRP4. In silico modeling indicated an increased interaction between H-Ras and mutant RASGRP4. In cell lines, mutant RASGRP4 increased basal p-ERK expression and lead to a growth advantage in colony forming assays when challenged with doxorubicin. Relapsed/refractory DLBCL is often associated with increased survival signals downstream of ERK, potentially corresponding with mutations in protein controlling RAS/MEK/ERK signaling.
Hormonal therapies for prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD). Clinical development for inhibitors that bind to the N-terminal domain (NTD) of AR has yielded ralaniten and its analogues. Ralaniten acetate is well tolerated in patients at 3600 mgs/day. Clinical trials are ongoing with a second-generation analogue of ralaniten. Binding sites on different AR domains could result in differential effects on AR-regulated gene expression. Here, we provide the first comparison between AR-NTD inhibitors and AR-LBD inhibitors on androgen-regulated gene expression in prostate cancer cells using cDNA arrays, GSEA, and RT-PCR. LBD inhibitors and NTD inhibitors largely overlapped in the profile of androgen-induced genes that they each inhibited. However, androgen also represses gene expression by various mechanisms, many of which involve protein-protein interactions. De-repression of the transcriptome of androgen-repressed genes showed profound variance between these two classes of inhibitors. In addition, these studies revealed a unique and strong induction of expression of the metallothionein family of genes by ralaniten by a mechanism independent of AR and dependent on MTF1, thereby suggesting this may be an off-target. Due to the relatively high doses that may be encountered clinically with AR-NTD inhibitors, identification of off-targets may provide insight into potential adverse events, contraindications, or poor efficacy.