SHOC2 and CRAF mediate ERK1/2 reactivation in mutant NRAS-mediated resistance to RAF inhibitor.

ERK1/2 signaling is frequently dysregulated in tumors through BRAF mutation. Targeting mutant BRAF with vemurafenib frequently elicits therapeutic responses; however, durable effects are often limited by ERK1/2 pathway reactivation via poorly defined mechanisms. We generated mutant BRAF(V600E) melanoma cells that exhibit resistance to PLX4720, the tool compound for vemurafenib, that co-expressed mutant (Q61K) NRAS. In these BRAF(V600E)/NRAS(Q61K) co-expressing cells, re-activation of the ERK1/2 pathway during PLX4720 treatment was dependent on NRAS. Expression of mutant NRAS in parental BRAF(V600) cells was sufficient to by-pass PLX4720 effects on ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in a manner dependent on the RAF binding site in NRAS. ERK1/2 activation in BRAF(V600E)/NRAS(Q61K) cells required CRAF only in the presence of PLX4720, indicating a switch in RAF isoform requirement. Both ERK1/2 activation and resistance to apoptosis of BRAF(V600E)/NRAS(Q61K) cells in the presence of PLX4720 was modulated by SHOC-2/Sur-8 expression, a RAS-RAF scaffold protein. These data show that NRAS mutations confer resistance to RAF inhibitors in mutant BRAF cells and alter RAF isoform and scaffold molecule requirements to re-activate the ERK1/2 pathway.

Hierarchical phosphorylation within the ankyrin repeat domain defines a phosphoregulatory loop that regulates Notch transcriptional activity.

The Notch signal transduction pathway mediates important cellular functions through direct cell-to-cell contact. Deregulation of Notch activity can lead to an altered cell proliferation and has been linked to many human cancers. Casein kinase 2 (CK2), a ubiquitous kinase, regulates several cellular processes by phosphorylating proteins involved in signal transduction, gene expression, and protein synthesis. In this report we identify Notch(ICD) as a novel target of phosphorylation by CK2. Using mapping and mutational studies, we identified serine 1901, located in the ankyrin domain of Notch, as the target amino acid. Interestingly, phosphorylation of serine 1901 by CK2 appears to generate a second phosphorylation site at threonine 1898. Furthermore, threonine 1898 phosphorylation only occurs when Notch forms a complex with Mastermind and CSL. Phosphorylation of both threonine 1898 and serine 1901 resulted in decreased binding of the Notch-Mastermind-CSL ternary complex to DNA and consequently lower transcriptional activity. These data indicate that the phosphorylation of serine 1901 and threonine 1898 negatively regulates Notch function by dissociating the complex from DNA. This study identifies a new component involved in regulation of Notch(ICD) transcriptional activity, reinforcing the notion that a precise and tight regulation is required for this essential signaling pathway.

Mechanisms of resistance to RAF inhibitors in melanoma.

The recent RAF inhibitor trial with PLX4032/RG7204 in late-stage mutant B-RAF melanoma patients has been lauded as a success story for personalized cancer therapy since short-term clinical responses were observed in the majority of patients. However, initial responses were followed by subsequent tumor re-growth, and a subset of patients showed intrinsic resistance. Bi-directional translational efforts are now essential to determine the mechanisms underlying acquired/secondary and intrinsic resistance to RAF inhibitors.

Assembly of a Notch transcriptional activation complex requires multimerization.

Notch transmembrane receptors direct essential cellular processes, such as proliferation and differentiation, through direct cell-to-cell interactions. Inappropriate release of the intracellular domain of Notch (N(ICD)) from the plasma membrane results in the accumulation of deregulated nuclear N(ICD) that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear N(ICD) forms a transcriptional activation complex by interacting with the coactivator protein Mastermind-like 1 and the DNA binding protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) to regulate target gene expression. Although it is well understood that N(ICD) forms a transcriptional activation complex, little is known about how the complex is assembled. In this study, we demonstrate that N(ICD) multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly, we demonstrate that the assembly is mediated by N(ICD) multimers interacting with Skip and Mastermind. These interactions form a preactivation complex that is then resolved by CSL to form the Notch transcriptional activation complex on DNA.