Oncogenic signaling in the adult Drosophila prostate-like accessory gland leads to activation of a conserved pro-tumorigenic program, in the absence of proliferation.

Drosophila models for tumorigenesis and metastasis have revealed conserved mechanisms of signaling that are also involved in mammalian cancer. Many of these models use the proliferating tissues of the larval stages of Drosophila development, when tissues are highly mitotically active, or stem cells are abundant. Fewer Drosophila tumorigenesis models use adult animals to initiate tumor formation when many tissues are largely terminally differentiated and postmitotic. The Drosophila accessory glands are prostate-like tissues and a model for some aspects of prostate tumorigenesis using this tissue has been explored. In this model, oncogenic signaling was induced during the proliferative stage of accessory gland development, raising the question of how oncogenic activity would impact the terminally differentiated and postmitotic adult tissue. Here, we show that oncogenic signaling in the adult Drosophila accessory gland leads to activation of a conserved pro-tumorigenic program, similar to that observed in mitotic larval tissues, but in the absence of proliferation. Oncogenic signaling in the adult postmitotic gland leads to tissue hyperplasia with nuclear anaplasia and aneuploidy through endoreduplication, which increases polyploidy and occasionally results in non-mitotic neoplastic-like extrusions. We compare gene expression changes in our Drosophila model with that of endocycling prostate cancer cells induced by chemotherapy, which potentially mediate tumor recurrence after treatment. Similar signaling pathways are activated in the Drosophila gland and endocycling cancer cells, suggesting the adult accessory glands provide a useful model for aspects of prostate cancer progression that do not involve cellular proliferation.

Enhancer-AAVs allow genetic access to oligodendrocytes and diverse populations of astrocytes across species.

Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and our astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.

Interplay between Cell Death and Cell Proliferation Reveals New Strategies for Cancer Therapy.

Cell division and cell death are fundamental processes governing growth and development across the tree of life. This relationship represents an evolutionary link between cell cycle and cell death programs that is present in all cells. Cancer is characterized by aberrant regulation of both, leading to unchecked proliferation and replicative immortality. Conventional anti-cancer therapeutic strategies take advantage of the proliferative dependency of cancer yet, in doing so, are triggering apoptosis, a death pathway to which cancer is inherently resistant. A thorough understanding of how therapeutics kill cancer cells is needed to develop novel, more durable treatment strategies. While cancer evolves cell-intrinsic resistance to physiological cell death pathways, there are opportunities for cell cycle agnostic forms of cell death, for example, necroptosis or ferroptosis. Furthermore, cell cycle independent death programs are immunogenic, potentially licensing host immunity for additional antitumor activity. Identifying cell cycle independent vulnerabilities of cancer is critical for developing alternative strategies that can overcome therapeutic resistance.

Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex.

Viral genetic tools that target specific brain cell types could transform basic neuroscience and targeted gene therapy. Here, we use comparative open chromatin analysis to identify thousands of human-neocortical-subclass-specific putative enhancers from across the genome to control gene expression in adeno-associated virus (AAV) vectors. The cellular specificity of reporter expression from enhancer-AAVs is established by molecular profiling after systemic AAV delivery in mouse. Over 30% of enhancer-AAVs produce specific expression in the targeted subclass, including both excitatory and inhibitory subclasses. We present a collection of Parvalbumin (PVALB) enhancer-AAVs that show highly enriched expression not only in cortical PVALB cells but also in some subcortical PVALB populations. Five vectors maintain PVALB-enriched expression in primate neocortex. These results demonstrate how genome-wide open chromatin data mining and cross-species AAV validation can be used to create the next generation of non-species-restricted viral genetic tools.

Targeted Delivery of Cytotoxic NAMPT Inhibitors Using Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are a therapeutic modality that enables the targeted delivery of cytotoxic drugs to cancer cells. Identification of active payloads with unique mechanisms of action is a key aim of research efforts in the field. Herein, we report the development of inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) as a novel payload for ADC technology. NAMPT is a component of a salvage biosynthetic pathway for NAD, and inhibition of this enzyme results in disruption of primary cellular metabolism leading to cell death. Through derivatization of the prototypical NAMPT inhibitor FK-866, we identified potent analogues with chemical functionality that enables the synthesis of hydrophilic enzyme-cleavable drug linkers. The resulting ADCs displayed NAD depletion in both cell-based assays and tumor xenografts. Antitumor efficacy is demonstrated in five mouse xenograft models using ADCs directed to indication-specific antigens. In rat toxicology models, a nonbinding control ADC was tolerated at >10-fold the typical efficacious dose used in xenografts. Moderate, reversible hematologic effects were observed with ADCs in rats, but there was no evidence for the retinal and cardiac toxicities reported for small-molecule inhibitors. These findings introduce NAMPT inhibitors as active and well-tolerated payloads for ADCs with promise to improve the therapeutic window of NAMPT inhibition and enable application in clinical settings.