The utility of in vitro human disease models is mainly dependent on the availability and functional maturity of tissue-specific cell types. We have previously screened for and identified small ...molecules that can enhance hepatocyte function in vitro. Here, we characterize the functional effects of one of the hits, FH1, on primary human hepatocytes in vitro, and also in vivo on primary hepatocytes in a zebrafish model. Furthermore, we conducted an analogue screen to establish the structure–activity relationship of FH1. We performed affinity-purification proteomics that identified NQO2 to be a potential binding target for this small molecule, revealing a possible link between inflammatory signaling and hepatocellular function in zebrafish and human hepatocyte model systems.
Although Apc mutation is widely considered an initiating event in colorectal cancer, little is known about the earliest stages of tumorigenesis following sporadic Apc loss. Therefore, we have ...utilized a novel mouse model that facilitates the sporadic inactivation of Apc via frameshift reversion of Cre in single, isolated cells and subsequently tracks the fates of Apc-deficient intestinal cells. Our results suggest that consistent with Apc being a 'gatekeeper', loss of Apc early in life during intestinal growth leads to adenomas or increased crypt fission, manifested by fields of mutant but otherwise normal-appearing crypts. In contrast, Apc loss occurring later in life has minimal consequences, with mutant crypts being less prone to either increased crypt fission or adenoma formation. Using the stem cell-specific Lgr5-CreER mouse, we generated different sized fields of Apc-deficient crypts via independent recombination events and found that field size correlates with progression to adenoma. To evaluate this early stage prior to adenoma formation as a therapeutic target, we examined the chemopreventive effects of sulindac on Apc-deficient occult crypt fission. We found that sulindac treatment started early in life inhibits the morphologically occult spread of Apc-deficient crypts and thus reduces adenoma numbers. Taken together these results suggest that: (i) earlier Apc loss promotes increased crypt fission, (ii) a field of Apc-deficient crypts, which can form via occult crypt fission or independent neighboring events, is an important intermediate between loss of Apc and adenoma formation and (iii) normal-appearing Apc-deficient crypts are potential unappreciated targets for cancer screening and chemoprevention.
In recent years, many mouse models have been developed to mark and trace the fate of adult cell populations using fluorescent proteins. High-resolution visualization of such fluorescent markers in ...their physiological setting is thus an important aspect of adult stem cell research. Here we describe a protocol to produce sections (150-200 μm) of near-native tissue with optimal tissue and cellular morphology by avoiding artifacts inherent in standard freezing or embedding procedures. The activity of genetically expressed fluorescent proteins is maintained, thereby enabling high-resolution three-dimensional (3D) reconstructions of fluorescent structures in virtually all types of tissues. The procedure allows immunofluorescence labeling of proteins to depths up to 50 μm, as well as a chemical 'Click-iT' reaction to detect DNA-intercalating analogs such as ethynyl deoxyuridine (EdU). Generation of near-native sections ready for imaging analysis takes approximately 2-3 h. Postsectioning processes, such as antibody labeling or EdU detection, take up to 10 h.
The Wnt signaling pathway was originally uncovered as one of the prototype developmental signaling cascades in invertebrates as well as in vertebrates. The first indication that Wnt signaling also ...plays a role in the adult animal came from the study of the intestine of Tcf-4 (Tcf7L2) knockout mice. The gastrointestinal epithelium continuously self-renews over the lifetime of an organism and is, in fact, the most rapidly self-renewing tissue of the mammalian body. Recent studies indicate that Wnt signaling plays a central role in the biology of gastrointestinal stem cells. Furthermore, mutational activation of the Wnt cascade is the principle cause of colon cancer.
The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. In this work, we have developed a method to photocage ...the activity of the guide RNA called “CRISPR‐plus” (CRISPR‐precise light‐mediated unveiling of sgRNAs). The photoactivation capability of our CRISPR‐plus method is compatible with the simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic investigations.
Die lichtgesteuerte Kontrolle von CRISPR gelang durch die Hybridisierung einer einzelnen chimären Guide‐RNA (sgRNA) mit einem komplementären Oligonucleotid, das photospaltbare Gruppen enthält (schützendes Oligonucleotid). Diese geschützte sgRNA (p‐sgRNA) bleibt inaktiv und blockiert somit die CRISPR‐Aktivität, bis das schützende Oligonucleotid unter Bestrahlung abgespalten wird.
Pro-senescence therapies are increasingly being considered for the treatment of cancer. Identifying additional targets to induce senescence in cancer cells could further enable such therapies. ...However, screening for targets whose suppression induces senescence on a genome-wide scale is challenging, as senescent cells become growth arrested, and senescence-associated features can take 1 to 2 weeks to develop. For a screen with a whole-genome CRISPR library, this would result in billions of undesirable proliferating cells by the time the senescent features emerge in the growth arrested cells. Here, we present a suicide switch system that allows genome-wide CRISPR screening in growth-arrested subpopulations by eliminating the proliferating cells during the screen through activation of a suicide switch in proliferating cells. Using this system, we identify in a genome-scale CRISPR screen several autophagy-related proteins as targets for senescence induction. We show that inhibiting macroautophagy with a small molecule ULK1 inhibitor can induce senescence in cancer cell lines of different origin. Finally, we show that combining ULK1 inhibition with the senolytic drug ABT-263 leads to apoptosis in a panel of cancer cell lines. IMPLICATIONS: Our suicide switch approach allows for genome-scale identification of pro-senescence targets, and can be adapted to simplify other screens depending on the nature of the promoter used to drive the switch.
The ability to remotely trigger CRISPR/Cas9 activity would enable new strategies to study cellular events with greater precision and complexity. We developed a method to photocage the activity of the ...guide RNA called ‘CRISPR-plus’ (CRISPR-precise light-mediated unveiling of sgRNAs). The photoactivatable capability of our CRISPR-plus method is compatible with simultaneous targeting of multiple DNA sequences and supports numerous modifications that can enable guide RNA labeling for use in imaging and mechanistic inquiries.
Turn ‘ON’ CRISPR with light:
CRISPR can be brought under the control of light simply by hybridizing a single chimeric guide RNAs (sgRNA) with its complementary oligonucleotide containing photocleavable groups (protector oligonucleotide). These protected sgRNA (p-sgRNA) remain inactive, blocking CRISPR activity, until the protector oligonucleotides are cleaved by a remote light trigger.
Senolytics, drugs that kill senescent cells, have been proposed to improve the response to pro-senescence cancer therapies; however, this remains challenging due to a lack of broadly acting senolytic ...drugs. Using CRISPR/Cas9-based genetic screens in different senescent cancer cell models, we identify loss of the death receptor inhibitor cFLIP as a common vulnerability of senescent cancer cells. Senescent cells are primed for apoptotic death by NF-κB-mediated upregulation of death receptor 5 (DR5) and its ligand TRAIL, but are protected from death by increased cFLIP expression. Activation of DR5 signaling by agonistic antibody, which can be enhanced further by suppression of cFLIP by BRD2 inhibition, leads to efficient killing of a variety of senescent cancer cells. Moreover, senescent cells sensitize adjacent non-senescent cells to killing by DR5 agonist through a bystander effect mediated by secretion of cytokines. We validate this 'one-two punch' cancer therapy by combining pro-senescence therapy with DR5 activation in different animal models.
Drug-tolerant persisters (DTPs) are a rare subpopulation of cells within a tumor that can survive therapy through nongenetic adaptive mechanisms to develop relapse and repopulate the tumor following ...drug withdrawal. Using a cancer cell line with an engineered suicide switch to kill proliferating cells, we perform both genetic screens and compound screens to identify the inhibition of bromodomain and extraterminal domain (BET) proteins as a selective vulnerability of DTPs. BET inhibitors are especially detrimental to DTPs that have reentered the cell cycle (DTEPs) in a broad spectrum of cancer types. Mechanistically, BET inhibition induces lethal levels of ROS through the suppression of redox-regulating genes highly expressed in DTPs, including GPX2, ALDH3A1, and MGST1. In vivo BET inhibitor treatment delays tumor relapse in both melanoma and lung cancer. Our study suggests that combining standard of care therapy with BET inhibitors to eliminate residual persister cells is a promising therapeutic strategy.
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•Unbiased genetic screen and compound screen identify BRD2 as a vulnerability of DTPs•BET inhibitors suppress outgrowth of DTPs in a broad spectrum of cancer types•BET inhibitors suppress DTEPs through transcriptional repression of GPX2/ALDH3A1/MGST1•BET inhibitors delay tumor relapse
Chen et al. uses high-throughput screening approaches to identify BRD2 inhibition as a vulnerability of drug-tolerant persisters (DTPs). They further demonstrate that BET inhibitors suppress the emergence of DTPs in multiple cancer cell lines in vitro and can forestall drug resistance through the eradication of DTPs in vivo.
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