Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds.

BRAF inhibitors can delay the progression of metastatic melanoma, but resistance usually emerges, leading to relapse. Drugs simultaneously targeting two or more pathways essential for cancer growth could slow or prevent the development of resistant clones. Here, we identified pyridinyl imidazole compounds SB202190, SB203580, and SB590885 as dual inhibitors of critical proliferative pathways in human melanoma cells bearing the V600E activating mutation of BRAF kinase. We found that the drugs simultaneously disrupt the BRAF V600E-driven extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activity and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in melanoma cells. Pyridinyl imidazole compounds directly inhibit BRAF V600E kinase. Moreover, they interfere with the endolysosomal compartment, promoting the accumulation of large acidic vacuole-like vesicles and dynamic changes in mTOR signaling. A transient increase in mTORC1 activity is followed by the enrichment of the Ragulator complex protein p18/LAMTOR1 at contact sites of large vesicles and delocalization of mTOR from the lysosomes. The induced disruption of the endolysosomal pathway not only disrupts mTORC1 signaling, but also renders melanoma cells sensitive to endoplasmic reticulum (ER) stress. Our findings identify new activities of pharmacologically relevant small molecule compounds and provide a biological rationale for the development of anti-melanoma therapeutics based on the pyridinyl imidazole core.

Inactivation of PLK4-STIL Module Prevents Self-Renewal and Triggers p53-Dependent Differentiation in Human Pluripotent Stem Cells.

Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs.

An Efficient Method for Generation of Knockout Human Embryonic Stem Cells Using CRISPR/Cas9 System.

Human embryonic stem cells (hESCs) represent a promising tool to study functions of genes during development, to model diseases, and to even develop therapies when combined with gene editing techniques such as CRISPR/CRISPR-associated protein-9 nuclease (Cas9) system. However, the process of disruption of gene expression by generation of null alleles is often inefficient and tedious. To circumvent these limitations, we developed a simple and efficient protocol to permanently downregulate expression of a gene of interest in hESCs using CRISPR/Cas9. We selected p53 for our proof of concept experiments. The methodology is based on series of hESC transfection, which leads to efficient downregulation of p53 expression even in polyclonal population (p53 Low cells), here proven by a loss of regulation of the expression of p53 target gene, microRNA miR-34a. We demonstrate that our approach achieves over 80% efficiency in generating hESC clonal sublines that do not express p53 protein. Importantly, we document by a set of functional experiments that such genetically modified hESCs do retain typical stem cells characteristics. In summary, we provide a simple and robust protocol to efficiently target expression of gene of interest in hESCs that can be useful for laboratories aiming to employ gene editing in their hESC applications/protocols.