Cancer therapy has traditionally focused on eliminating fast-growing populations of cells. Yet, an increasing body of evidence suggests that small subpopulations of cancer cells can evade strong selective drug pressure by entering a ‘persister' state of negligible growth. This drug-tolerant state has been hypothesized to be part of an initial strategy towards eventual acquisition of bona fide drug-resistance mechanisms. However, the diversity of drug-resistance mechanisms that can expand from a persister bottleneck is unknown. Here we compare persister-derived, erlotinib-resistant colonies that arose from a single, EGFR-addicted lung cancer cell. We find, using a combination of large-scale drug screening and whole-exome sequencing, that our erlotinib-resistant colonies acquired diverse resistance mechanisms, including the most commonly observed clinical resistance mechanisms. Thus, the drug-tolerant persister state does not limit—and may even provide a latent reservoir of cells for—the emergence of heterogeneous drug-resistance mechanisms.
Drugs that mirror the cellular effects of starvation mimics are considered promising therapeutics for common metabolic disorders, such as obesity, liver steatosis, and for ageing. Starvation, or caloric restriction, is known to activate the transcription factor EB (TFEB), a master regulator of lipid metabolism and lysosomal biogenesis and function. Here, we report a nanotechnology-enabled high-throughput screen to identify small-molecule agonists of TFEB and discover three novel compounds that promote autophagolysosomal activity. The three lead compounds include the clinically approved drug, digoxin; the marine-derived natural product, ikarugamycin; and the synthetic compound, alexidine dihydrochloride, which is known to act on a mitochondrial target. Mode of action studies reveal that these compounds activate TFEB via three distinct Ca2+-dependent mechanisms. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles confers hepatoprotection against diet-induced steatosis in murine models and extends lifespan of Caenorhabditis elegans. These results support the therapeutic potential of small-molecule TFEB activators for the treatment of metabolic and age-related disorders.
Autophagy, a lysosomal degradation pathway, plays a crucial role in
cellular homeostasis, development, immunity, tumor suppression, metabolism,
prevention of neurodegeneration and lifespan extension. Thus, pharmacological
stimulation of autophagy may be an effective approach for preventing or treating
certain human diseases and/or aging. We sought to establish a method for
developing new chemical compounds that specifically induce autophagy. To do
this, we developed two assays to identify compounds that target a key regulatory
node of autophagy induction – specifically, the binding of Bcl-2 (a
negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the
Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation).
These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2
binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2
binding in vitro. We screened two different chemical compound
libraries, comprising ~300K compounds, to identify small molecules that
disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were
identified that directly inhibit Beclin 1/Bcl-2 interaction with an
IC50 in the micromolar range and increase autophagic flux. These
compounds do not demonstrate significant cytotoxicity and they exert selectivity
for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared to the
BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have
identified candidate molecules that serve as lead templates for developing
potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as
autophagy-inducing agents.
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