Human Biology Publications

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Notch blockade overcomes endothelial cell-mediated resistance of FLT3/ITD-positive AML progenitors to AC220 treatment

Leukemia

2020 Brandon Hadland; Eric Holland; Quy Le; Soheil Meshinchi

N/A

HPV16 induces penile intraepithelial neoplasia and squamous cell carcinoma in transgenic mice: first mouse model for HPV-related penile cancer

J Pathol

2020 Funda Vakar-Lopez; Peter Nelson

Penile cancer is an under-studied disease that occurs more commonly in developing countries and 30-50% of cases show high-risk human papillomavirus (HPV) infection. Therapeutic advances are slow, largely due to the absence of animal models for translational research. Here, we report the first mouse model for HPV-related penile cancer. Ten-week-old mice expressing all the HPV16 early genes under control of the cytokeratin 14 (Krt14) gene promoter and matched wild-type controls were exposed topically to dimethylbenz(o)anthracene (DMBA) or vehicle for 16 weeks. At 30 weeks of age mice were sacrificed for histological analysis. Expression of Ki67, cytokeratin 14 and of the HPV16 oncogenes E6 and E7 was confirmed using immunohistochemistry and quantitative PCR, respectively. HPV16-transgenic mice developed intraepithelial lesions including condylomas and penile intraepithelial neoplasia (PeIN). Lesions expressed cytokeratin 14 and the HPV16 oncogenes E6 and E7 and showed deregulated cell proliferation, demonstrated by Ki67-positive supra-basal cells. HPV16-transgenic mice exposed to DMBA showed increased penile intraepithelial neoplasia (PeIN) incidence and squamous cell carcinoma. Malignant lesions showed varied histological features closely resembling those of HPV-associated human penile cancers. Wild-type mice showed no malignant or pre-malignant lesions even when exposed to DMBA. These observations provide the first experimental evidence to support the etiological role of HPV16 in penile carcinogenesis. Importantly, this is the first mouse model to recapitulate key steps of HPV-related penile carcinogenesis and to reproduce morphological and molecular features of human penile cancer, providing a unique in vivo tool for studying its biology and advancing basic and translational research. This article is protected by copyright. All rights reserved.

Selective Translation of Cell Fate Regulators Mediates Tolerance to Broad Oncogenic Stress

Cell Stem Cell

2020 Slobodan Beronja; Alexandre Germanos; Madeline Larkin; Yi Cai; Sonali Arora; Megan Kufeld; Samantha Schuster; Andrew Hsieh

Human skin tolerates a surprisingly high burden of oncogenic lesions. Although adult epidermis can suppress the expansion of individual mutant clones, the mechanisms behind tolerance to oncogene activation across broader regions of tissue are unclear. Here, we uncover a dynamic translational mechanism that coordinates oncogenic HRAS-induced hyperproliferation with loss of progenitor self-renewal to restrain aberrant growth and tumorigenesis. We identify translation initiator eIF2B5 as a central co-regulator of HRAS proliferation and cell fate choice. By coupling in vivo ribosome profiling with genetic screening, we provide direct evidence that oncogene-induced loss of progenitor self-renewal is driven by eIF2B5-mediated translation of ubiquitination genes. Ubiquitin ligase FBXO32 specifically inhibits epidermal renewal without affecting overall proliferation, thus restraining HRAS-driven tumorigenesis while maintaining normal tissue growth. Thus, oncogene-driven translation is not necessarily inherently tumor promoting but instead can manage widespread oncogenic stress by steering progenitor fate to prolong normal tissue growth.

High-throughput, microscope-based sorting to dissect cellular heterogeneity

Mol Syst Biol

2020 Raymond Monnat; Douglas Fowler; Emily Hatch; Cole Trapnell

Microscopy is a powerful tool for characterizing complex cellular phenotypes, but linking these phenotypes to genotype or RNA expression at scale remains challenging. Here, we present Visual Cell Sorting, a method that physically separates hundreds of thousands of live cells based on their visual phenotype. Automated imaging and phenotypic analysis directs selective illumination of Dendra2, a photoconvertible fluorescent protein expressed in live cells; these photoactivated cells are then isolated using fluorescence-activated cell sorting. First, we use Visual Cell Sorting to assess hundreds of nuclear localization sequence variants in a pooled format, identifying variants that improve nuclear localization and enabling annotation of nuclear localization sequences in thousands of human proteins. Second, we recover cells that retain normal nuclear morphologies after paclitaxel treatment, and then derive their single-cell transcriptomes to identify pathways associated with paclitaxel resistance in cancers. Unlike alternative methods, Visual Cell Sorting depends on inexpensive reagents and commercially available hardware. As such, it can be readily deployed to uncover the relationships between visual cellular phenotypes and internal states, including genotypes and gene expression programs.

Retrocopying expands functional repertoire of APOBEC3 antiviral proteins in primates

Elife

2020 Michael Emerman; Harmit Malik; Richard McLaughlin

Host-virus arms races are inherently asymmetric; viruses evolve much more rapidly than host genomes. Thus, there is high interest in discovering mechanisms by which host genomes keep pace with rapidly evolving viruses. One family of restriction factors, the APOBEC3 (A3) cytidine deaminases, has undergone positive selection and expansion via segmental gene duplication and recombination. Here, we show that new copies of A3 genes have also been created in primates by reverse transcriptase-encoding elements like LINE-1 or endogenous retroviruses via a process termed retrocopying. First, we discovered that all simian primate genomes retain the remnants of an ancient A3 retrocopy: A3I. Furthermore, we found that some New World monkeys encode up to ten additional APOBEC3G (A3G) retrocopies. Some of these A3G retrocopies are transcribed in a variety of tissues and able to restrict retroviruses. Our findings suggest that host genomes co-opt retroelement activity in the germline to create new host restriction factors as another means to keep pace with the rapid evolution of viruses. (163).

Pervasive promoter hypermethylation of silenced TERT alleles in human cancers

Cell Oncol (Dordr)

2020 Michael Haffner

BACKGROUND: In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of TERT gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating TERT gene expression in cancer cells is as yet not fully understood. METHODS: Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines, including primary, immortalized and cancer cell types, as well as in control and reference samples. RESULTS: In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally found that hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. CONCLUSIONS: Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to attenuation of TERT activation in cancer cells. This type of fine tuning of TERT expression may account for the modest activation of TERT expression in most cancers.

Targeting RET Kinase in Neuroendocrine Prostate Cancer

Mol Cancer Res

2020 Ilsa Coleman; Peter Nelson; Nathan Lau; John Lee; Eva Corey; Colm Morrissey

The increased treatment of metastatic castration resistant prostate cancer (mCRPC) with second-generation anti-androgen therapies (ADT) has coincided with a greater incidence of lethal, aggressive variant prostate cancer (AVPC) tumors that have lost dependence on androgen receptor (AR) signaling. These AR independent tumors may also transdifferentiate to express neuroendocrine lineage markers and are termed neuroendocrine prostate cancer (NEPC). Recent evidence suggests kinase signaling may be an important driver of NEPC. To identify targetable kinases in NEPC, we performed global phosphoproteomics comparing several AR independent to AR dependent prostate cancer cell lines and identified multiple altered signaling pathways, including enrichment of RET kinase activity in the AR independent cell lines. Clinical NEPC patient samples and NEPC patient derived xenografts displayed upregulated RET transcript and RET pathway activity. Genetic knockdown or pharmacological inhibition of RET kinase in multiple mouse and human models of NEPC dramatically reduced tumor growth and decreased cell viability. Our results suggest that targeting RET in NEPC tumors with high RET expression could be an effective treatment option. Currently, there are limited treatment options for patients with aggressive neuroendocrine prostate cancer and none are curative. Implications: Identification of aberrantly expressed RET kinase as a driver of tumor growth in multiple models of NEPC provides a significant rationale for testing the clinical application of RET inhibitors in AVPC patients.

BAF facilitates interphase nuclear membrane repair through recruitment of nuclear transmembrane proteins

Mol Biol Cell

2020 Emily Hatch; Alexandra Young; Amanda Gunn

Nuclear membrane rupture during interphase occurs in a variety of cell contexts, both healthy and pathological. Membrane ruptures can be rapidly repaired, but these mechanisms are still unclear. Here we show BAF, a nuclear envelope protein that shapes chromatin and recruits membrane proteins in mitosis, also facilitates nuclear membrane repair in interphase, in part through recruitment of the nuclear membrane proteins emerin and LEMD2 to rupture sites. Characterization of GFP-BAF accumulation at nuclear membrane rupture sites confirmed BAF is a fast, accurate, and persistent mark of nucleus rupture whose kinetics are partially dictated by membrane resealing. BAF depletion significantly delayed nuclear membrane repair, with a larger effect on longer ruptures. This phenotype could be rescued by GFP-BAF, but not by a BAF mutant lacking the LEM-protein binding domain. Depletion of the BAF interactors LEMD2 or emerin, and to a lesser extent lamin A/C, increased the duration of nucleus ruptures, consistent with LEM-protein binding being a key function of BAF during membrane repair. Overall our results suggest a model where BAF is critical for timely repair of large ruptures in the nuclear membrane, potentially by facilitating membrane attachment to the rupture site. [Media: see text].

Combined TP53 and RB1 Loss Promotes Prostate Cancer Resistance to a Spectrum of Therapeutics and Confers Vulnerability to Replication Stress

Cell Rep

2020 Peter Nelson; Michael Nyquist; Alexandra Corella; Colin Pritchard; Robert Montgomery; Roman Gulati; Gavin Ha; Ilsa Coleman; Colm Morrissey; Lisa Ang; Payel Chatterjee; Eva Corey; Navonil De Sarkar; Jared Lucas; Arja Kaipainen

Prostate cancers (PCs) with loss of the potent tumor suppressors TP53 and RB1 exhibit poor outcomes. TP53 and RB1 also influence cell plasticity and are frequently lost in PCs with neuroendocrine (NE) differentiation. Therapeutic strategies that address these aggressive variant PCs are urgently needed. Using deep genomic profiling of 410 metastatic biopsies, we determine the relationships between combined TP53 and RB1 loss and PC phenotypes. Notably, 40% of TP53/RB1-deficient tumors are classified as AR-active adenocarcinomas, indicating that NE differentiation is not an obligate consequence of TP53/RB1 inactivation. A gene expression signature reflecting TP53/RB1 loss is associated with diminished responses to AR antagonists and reduced survival. These tumors exhibit high proliferation rates and evidence of elevated DNA repair processes. While tumor cells lacking TP53/RB1 are highly resistant to all single-agent therapeutics tested, the combination of PARP and ATR inhibition is found to produce significant responses, reflecting a clinically exploitable vulnerability resulting from replication stress.

MAX Functions as a Tumor Suppressor and Rewires Metabolism in Small Cell Lung Cancer

Cancer Cell

2020 Haritha Mathsyaraja; Nan Wu; Arnaud Augert; Ryan Basom; Lucas Sullivan; Ali Ibrahim; David MacPherson; Brian Freie; Pei-Feng Cheng; Joseph Hiatt; Robert Eisenman; Michael Geuenich

Small cell lung cancer (SCLC) is a highly aggressive and lethal neoplasm. To identify candidate tumor suppressors we applied CRISPR/Cas9 gene inactivation screens to a cellular model of early-stage SCLC. Among the top hits was MAX, the obligate heterodimerization partner for MYC family proteins that is mutated in human SCLC. Max deletion increases growth and transformation in cells and dramatically accelerates SCLC progression in an Rb1/Trp53-deleted mouse model. In contrast, deletion of Max abrogates tumorigenesis in MYCL-overexpressing SCLC. Max deletion in SCLC resulted in derepression of metabolic genes involved in serine and one-carbon metabolism. By increasing serine biosynthesis, Max-deleted cells exhibit resistance to serine depletion. Thus, Max loss results in metabolic rewiring and context-specific tumor suppression.