About eight percent of all human tumors (including 50% of melanomas) carry gain-of-function mutations in the BRAF oncogene. Mutated BRAF and subsequent hyperactivation of the MAPK signaling pathway ...has motivated the use of MAPK-targeted therapies for these tumors. Despite great promise, however, MAPK-targeted therapies in BRAF-mutant tumors are limited by the emergence of drug resistance. Mechanisms of resistance include genetic, non-genetic and epigenetic alterations. Epigenetic plasticity, often modulated by histone-modifying enzymes and gene regulation, can influence a tumor cell’s BRAF dependency and therefore, response to therapy. In this review, focusing primarily on class 1 BRAF-mutant cells, we will highlight recent work on the contribution of epigenetic mechanisms to inter- and intratumor cell heterogeneity in MAPK-targeted therapy response.
Hyperactivation of the MAPK signaling pathway motivates the clinical use of MAPK inhibitors for BRAF-mutant melanomas. Heterogeneity in differentiation state due to epigenetic plasticity, however, ...results in cell-to-cell variability in the state of MAPK dependency, diminishing the efficacy of MAPK inhibitors. To identify key regulators of such variability, we screen 276 epigenetic-modifying compounds, individually or combined with MAPK inhibitors, across genetically diverse and isogenic populations of melanoma cells. Following single-cell analysis and multivariate modeling, we identify three classes of epigenetic inhibitors that target distinct epigenetic states associated with either one of the lysine-specific histone demethylases Kdm1a or Kdm4b, or BET bromodomain proteins. While melanocytes remain insensitive, the anti-tumor efficacy of each inhibitor is predicted based on melanoma cells' differentiation state and MAPK activity. Our systems pharmacology approach highlights a path toward identifying actionable epigenetic factors that extend the BRAF oncogene addiction paradigm on the basis of tumor cell differentiation state.
Upon mild liver injury, new hepatocytes originate from preexisting hepatocytes. However, if hepatocyte proliferation is impaired, a manifestation of severe liver injury, biliary epithelial cells ...(BECs) contribute to new hepatocytes through BEC dedifferentiation into liver progenitor cells (LPCs), also termed oval cells or hepatoblast‐like cells (HB‐LCs), and subsequent differentiation into hepatocytes. Despite the identification of several factors regulating BEC dedifferentiation and activation, little is known about factors involved in the regulation of LPC differentiation into hepatocytes during liver regeneration. Using a zebrafish model of near‐complete hepatocyte ablation, we show that bone morphogenetic protein (Bmp) signaling is required for BEC conversion to hepatocytes, particularly for LPC differentiation into hepatocytes. We found that severe liver injury led to the up‐regulation of genes involved in Bmp signaling, including smad5, tbx2b, and id2a, in the liver. Bmp suppression did not block BEC dedifferentiation into HB‐LCs; however, the differentiation of HB‐LCs into hepatocytes was impaired due to the maintenance of HB‐LCs in an undifferentiated state. Later Bmp suppression did not affect HB‐LC differentiation but increased BEC number through proliferation. Notably, smad5, tbx2b, and id2a mutants exhibited similar liver regeneration defects as those observed in Bmp‐suppressed livers. Moreover, BMP2 addition promoted the differentiation of a murine LPC line into hepatocytes in vitro. Conclusions: Bmp signaling regulates BEC‐driven liver regeneration through smad5, tbx2b, and id2a: it regulates HB‐LC differentiation into hepatocytes through tbx2b and BEC proliferation through id2a; our findings provide insights into promoting innate liver regeneration as a novel therapy. (Hepatology 2017;66:1616–1630).
During aging, there is a progressive loss of volume and function in skeletal muscle that impacts mobility and quality of life. The repair of skeletal muscle is regulated by tissue-resident stem cells ...called satellite cells (or muscle stem cells MuSCs), but in aging, MuSCs decrease in numbers and regenerative capacity. The transcriptional networks and epigenetic changes that confer diminished regenerative function in MuSCs as a result of natural aging are only partially understood. Herein, we use an integrative genomics approach to profile MuSCs from young and aged animals before and after injury. Integration of these datasets reveals aging impacts multiple regulatory changes through significant differences in gene expression, metabolic flux, chromatin accessibility, and patterns of transcription factor (TF) binding activities. Collectively, these datasets facilitate a deeper understanding of the regulation tissue-resident stem cells use during aging and healing.
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•Chromatin enzymes that mediate heterochromatin packaging vary in aging•Distinct activity of one-carbon and retinol pathways observed in aged satellite cells•Changes in transcription factor binding in aged satellite cells post-injury
The number and regenerative capacity of tissue-resident stem muscle cells are attenuated with age. Shcherbina et al. profile MuSCs from young and aged animals pre- and post-injury, discovering that aging impacts regulatory changes through differences in gene expression, metabolic flux, chromatin accessibility, and transcription factor binding.
Cell-to-cell variability generates subpopulations of drug-tolerant cells that diminish the efficacy of cancer drugs. Efficacious combination therapies are thus needed to block drug-tolerant cells via ...minimizing the impact of heterogeneity. Probabilistic models such as Bliss independence have been developed to evaluate drug interactions and their combination efficacy based on probabilities of specific actions mediated by drugs individually and in combination. In practice, however, these models are often applied to conventional dose-response curves in which a normalized parameter with a value between zero and one, generally referred to as fraction of cells affected (fa), is used to evaluate the efficacy of drugs and their combined interactions. We use basic probability theory, computer simulations, time-lapse live cell microscopy, and single-cell analysis to show that fa metrics may bias our assessment of drug efficacy and combination effectiveness. This bias may be corrected when dynamic probabilities of drug-induced phenotypic events, i.e. induction of cell death and inhibition of division, at a single-cell level are used as metrics to assess drug efficacy. Probabilistic phenotype metrics offer the following three benefits. First, in contrast to the commonly used fa metrics, they directly represent probabilities of drug action in a cell population. Therefore, they deconvolve differential degrees of drug effect on tumor cell killing versus inhibition of cell division, which may not be correlated for many drugs. Second, they increase the sensitivity of short-term drug response assays to cell-to-cell heterogeneities and the presence of drug-tolerant subpopulations. Third, their probabilistic nature allows them to be used directly in unbiased evaluation of synergistic efficacy in drug combinations using probabilistic models such as Bliss independence. Altogether, we envision that probabilistic analysis of single-cell phenotypes complements currently available assays via improving our understanding of heterogeneity in drug response, thereby facilitating the discovery of more efficacious combination therapies to block drug-tolerant cells.
Malformations of the intrahepatic biliary structure cause cholestasis, a liver pathology that corresponds to poor bile flow, which leads to inflammation, fibrosis, and cirrhosis. Although the ...specification of biliary epithelial cells (BECs) that line the bile ducts is fairly well understood, the molecular mechanisms underlying intrahepatic biliary morphogenesis remain largely unknown. Wnt/β‐catenin signaling plays multiple roles in liver biology; however, its role in intrahepatic biliary morphogenesis remains unclear. Using pharmacological and genetic tools that allow one to manipulate Wnt/β‐catenin signaling, we show that in zebrafish both suppression and overactivation of Wnt/β‐catenin signaling impaired intrahepatic biliary morphogenesis. Hepatocytes, but not BECs, exhibited Wnt/β‐catenin activity; and the global suppression of Wnt/β‐catenin signaling reduced Notch activity in BECs. Hepatocyte‐specific suppression of Wnt/β‐catenin signaling also reduced Notch activity in BECs, indicating a cell nonautonomous role for Wnt/β‐catenin signaling in regulating hepatic Notch activity. Reducing Notch activity to the same level as that observed in Wnt‐suppressed livers also impaired biliary morphogenesis. Intriguingly, expression of the Notch ligand genes jag1b and jag2b in hepatocytes was reduced in Wnt‐suppressed livers and enhanced in Wnt‐overactivated livers, revealing their regulation by Wnt/β‐catenin signaling. Importantly, restoring Notch activity rescued the biliary defects observed in Wnt‐suppressed livers. Conclusion: Wnt/β‐catenin signaling cell nonautonomously controls Notch activity in BECs by regulating the expression of Notch ligand genes in hepatocytes, thereby regulating biliary morphogenesis. (Hepatology 2018;67:2352‐2366).
Upon mild liver injury, new hepatocytes originate from pre-existing hepatocytes. However, if hepatocyte proliferation is impaired, a manifestation of severe liver injury, biliary epithelial cells ...(BECs) contribute to new hepatocytes through BEC dedifferentiation into liver progenitor cells (LPCs), also termed oval cells or hepatoblast-like cells (HB-LCs), and subsequent differentiation into hepatocytes. Despite the identification of several factors regulating BEC dedifferentiation and activation, little is known about factors involved in the regulation of LPC differentiation into hepatocytes during liver regeneration. Using a zebrafish model of near-complete hepatocyte ablation, we here show that Bmp signaling is required for BEC conversion to hepatocytes, particularly for LPC differentiation into hepatocytes. We found that severe liver injury led to the upregulation of genes involved in Bmp signaling, including
smad5
,
tbx2b
, and
id2a
, in the liver. Bmp suppression did not block BEC dedifferentiation into HB-LCs; however, the differentiation of HB-LCs into hepatocytes was impaired due to the maintenance of HB-LCs in an undifferentiated state. Later Bmp suppression did not affect HB-LC differentiation, but increased BEC number through proliferation. Notably,
smad5
,
tbx2b
, and
id2a
mutants exhibited similar liver regeneration defects as those observed in Bmp-suppressed livers. Moreover, BMP2 addition promoted the differentiation of a murine LPC cell line into hepatocytes
in vitro. Conclusions:
Bmp signaling regulates BEC-driven liver regeneration via
smad5
,
tbx2b
and
id2a
: it regulates HB-LC differentiation into hepatocytes via
tbx2b
and BEC proliferation via
id2a
. Our findings provide insights into promoting innate liver regeneration as a novel therapy.
After liver injury, regeneration manifests as either (1) hepatocytes proliferating to restore the lost hepatocyte mass or (2) if hepatocyte proliferation is compromised, biliary epithelial cells ...(BECs) dedifferentiating into liver progenitor cells (LPCs), which subsequently differentiate
into hepatocytes. Following pharmacogenetic ablation of hepatocytes in Tg(fabp10a:CFP-NTR) zebrafish, resulting in severe liver injury, signal transducer and activator of transcription 3 (Stat3) and its target gene and negative regulator, socs3a, were upregulated in regenerating
livers. Using either Stat3 inhibitors, JSI-124 and S3I-201, or stat3 zebrafish mutants, we investigated the role of Stat3 in LPC-driven liver regeneration. Although Stat3 suppression reduced the size of regenerating livers, BEC dedifferentiation into LPCs was unaffected. However, regenerating
livers displayed a delay in LPC-to-hepatocyte differentiation and a significant reduction in the number of BECs. While no difference in cell death was detected, Stat3 inhibition significantly reduced LPC proliferation. Notably, stat3 mutants phenocopied the effects of Stat3 chemical
inhibitors, although the mutant phenotype was incompletely penetrant. Intriguingly, a subset of socs3a mutants also displayed a lower number of BECs in regenerating livers. We conclude that the Stat3/Socs3a pathway is necessary for the proper timing of LPC-to-hepatocyte differentiation and establishing the proper number of BECs during LPC-driven liver regeneration.
Chronic liver disease encompasses the steady destruction of hepatic tissue over time, resulting in the replacement of healthy liver tissue with damaged fibrotic and cirrhotic tissue. Among other ...symptoms, cirrhosis manifests itself with regenerative hepatic nodules and portal hypertension, triggering a loss of liver functionality and poor quality of life. Moreover, in the United States, cirrhosis remains the 12th leading cause of death, incurring healthcare costs of billions of dollars. Despite the astronomical costs and limited palliative care, the only effective treatment for cirrhosis is liver transplantation. The demand for liver transplants, however, far exceeds the availability of donor livers, underpinning the need for harnessing the liver’s innate regenerative capacity. Using zebrafish as a model organism, the main aim of this dissertation is to elucidate the underlying mechanisms of liver progenitor cell (LPC)-driven liver regeneration as well as liver development, since events involved in the former process can be recapitulated in the latter. Our lab previously characterized the biliary epithelial cell (BEC)-driven liver regeneration in which after extensive hepatocyte ablation, BECs dedifferentiate into LPCs and subsequently, the LPCs can redifferentiate into mature hepatocytes/BECs. During this regenerative process, our RNAseq analysis showed an upregulation of genes mediating the Bone Morphogenetic Protein (BMP) pathway, including a BMP downstream target gene, Inhibitor of DNA binding 2a (Id2a). Using loss- and gain-of-function approaches, we show Id2a to be an important regulator of hepatic outgrowth during liver development as well as an important mediator of BEC repopulation in LPCdriven liver regeneration. In addition, as LPC activation is often accompanied by an inflammatory cell response, our second focus was on the role of Signal Transducer and Activator of Transcription 3 (Stat3) in the LPC-driven liver regeneration model. By chemically inhibiting Stat3, we show Stat3 signaling regulates not only the differentiation of LPCs into hepatocytes, but also the proliferation of BECs during LPC-driven liver regeneration.
The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In ...hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.
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