Naama Kanarek

Regulation of NF‐κB by ubiquitination and degradation of the IκBs

Summary:  The nuclear factor‐κB (NF‐κB) signaling pathway is a busy ground for the action of the ubiquitin–proteasome system; many of the signaling steps are coordinated by protein ubiquitination. The end point of this pathway is to induce transcription, and to this end, there is a need to overcome a major obstacle, a set of inhibitors (IκBs) that bind NF‐κB and prohibit either the nuclear entry or the DNA binding of the transcription factor. Two major signaling steps are required for the elimination of the inhibitors: activation of the IκB kinase (IKK) and degradation of the phosphorylated inhibitors. IKK activation and IκB degradation involve different ubiquitination modes; the latter is mediated by a specific E3 ubiquitin ligase SCFβ‐TrCP. The F‐box component of this E3, β‐TrCP, recognizes the IκB degron formed following phosphorylation by IKK and thus couples IκB phosphorylation to ubiquitination. SCFβ‐TrCP‐mediated IκB ubiquitination and degradation is a very efficient process, often resulting in complete degradation of the key inhibitor IκBα within a few minutes of cell stimulation. In vivo ablation of β‐TrCP results in accumulation of all the IκBs and complete NF‐κB inhibition. As many details of IκB‐β‐TrCP interaction have been worked out, the development of β‐TrCP inhibitors might be a feasible therapeutic approach for NF‐κB‐associated human disease. However, we may still need to advance our understanding of the mechanism of IκB degradation as well as of the diverse functions of β‐TrCP in vivo.

Human and Mouse VEGFA-Amplified Hepatocellular Carcinomas Are Highly Sensitive to Sorafenib Treatment

UNLABELLED Death rates from hepatocellular carcinoma (HCC) are steadily increasing, yet therapeutic options for advanced HCC are limited. We identify a subset of mouse and human HCCs harboring VEGFA genomic amplification, displaying distinct biologic characteristics. Unlike common tumor amplifications, this one seems to work via heterotypic paracrine interactions; stromal VEGF receptors (VEGFR), responding to tumor VEGF-A, produce hepatocyte growth factor (HGF) that reciprocally affects tumor cells. VEGF-A inhibition results in HGF downregulation and reduced proliferation, specifically in amplicon-positive mouse HCCs. Sorafenib-the first-line drug in advanced HCC-targets multiple kinases, including VEGFRs, but has only an overall mild beneficial effect. We found that VEGFA amplification specifies mouse and human HCCs that are distinctly sensitive to sorafenib. FISH analysis of a retrospective patient cohort showed markedly improved survival of sorafenib-treated patients with VEGFA-amplified HCCs, suggesting that VEGFA amplification is a potential biomarker for HCC response to VEGF-A-blocking drugs. SIGNIFICANCE Using a mouse model of inflammation-driven cancer, we identified a subclass of HCC carrying VEGFA amplification, which is particularly sensitive to VEGF-A inhibition. We found that a similar amplification in human HCC identifies patients who favorably responded to sorafenib-the first-line treatment of advanced HCC-which has an overall moderate therapeutic efficacy.

Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase

A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis-an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.

Spermatogenesis rescue in a mouse deficient for the ubiquitin ligase SCFβ-TrCP by single substrate depletion

beta-TrCP, the substrate recognition subunit of a Skp1-Cul1-F-box (SCF) ubiquitin ligase, is ubiquitously expressed from two distinct paralogs, targeting many regulatory proteins for proteasomal degradation. We generated inducible beta-TrCP hypomorphic mice and found that they are surprisingly healthy, yet have a severe testicular defect. We show that the two beta-TrCP paralogs have a nonredundant role in spermatogenesis. The testicular defect is tightly associated with cell adhesion failure within the seminiferous tubules and is fully reversible upon beta-TrCP restoration. Remarkably, testicular depletion of a single beta-TrCP substrate, Snail1, rescued the adhesion defect and restored spermatogenesis. Our studies highlight an unexpected functional reserve of this central E3, as well as a bottleneck in a specific tissue: a single substrate whose stabilization is incompatible with testicular differentiation.

Critical role for IL-1β in DNA damage-induced mucositis

Significance Deletion of the E3 β-TrCP in the mouse gut epithelium deregulates enterocyte cell cycle, induces a DNA damage response (DDR), and abolishes the epithelium barrier function, resulting in a lethal mucosal inflammation. Epithelial-derived IL-1β, likely induced by DDR independently of NF-κB, is a major culprit, and initiates the pathology by compromising epithelial tight junctions (TJs). Anti–IL-1β treatment secures the TJs and prevents the fulminant mucosal inflammation. IL-1β secretion accompanies human mucositis, a severe mucosal inflammatory reaction caused by chemoradiation therapy-induced DNA damage, which often results in treatment suspension. We propose that anti–IL-1β preventive treatment may ameliorate mucositis, as well as multiple disorders associated with epithelial barrier permeability, including burn injuries, head and neck trauma, alcoholic intoxication, and graft-vs.-host disease. β-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-κB inhibitor IκB. To appreciate tissue-specific roles of β-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1β as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1β is induced by DNA damage via an NF-κB–independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-κB, with failure to express the endogenous IL-1β receptor antagonist IL-1Ra upon four-allele loss. Antibody neutralization of IL-1β prevents epithelial tight junction dysfunction and alleviates mucositis in β-TrCP–deficient mice. IL-1β antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.

Segregation between acquisition and long-term memory in sensorimotor learning

It is widely accepted that learning first involves generating new memories and then consolidating them into long‐term memory. Thus learning is generally viewed as a single continuous process with two sequential stages; acquisition and consolidation. Here, we tested an alternative hypothesis proposing that acquisition and consolidation take place, at least partly, in parallel. Human subjects learned two visuomotor tasks. One task required moving a cursor under visuomotor rotation and the other required arbitrary association of colour to direction of movement. Subjects learned the two tasks in sequence, and were tested for acquisition of the second immediately after learning the first, and for retention of the first on the following day. The results show that learning one task led to proactive interference to acquisition of the second. However, this interference was not accompanied by retroactive interference to consolidation of the first task, indicating that acquisition and consolidation can be uncoupled.

Abstract 4988: Genome-wide CRISPR screen and metabolite profiling reveal a new mechanism of methotrexate sensitivity

The chemotherapeutic drug methotrexate (MTX) inhibits nucleotide biosynthesis and causes cell death by blocking DNA and RNA production. While MTX has had tremendous success as an anti-cancer treatment and is the subject of nearly a thousand ongoing clinical trials, we do not fully understand why certain cancers are more sensitive to it and why resistance emerges. To study these questions, we used a CRISPR-based genetic screen to identify genes that confer sensitivity to MTX. We identified an enzyme that plays a key role in a metabolic pathway that was not known to be associated with MTX sensitivity. Inhibition of the pathway, either by genetic loss of several genes in the pathway or through nutritional modulations, causes dramatic resistance to MTX in cultured cancer cells. Our results improved our understanding of the cellular response to MTX, and suggest that expression levels of enzymes in the pathway may serve as clinical predictor for MTX response in patients. Citation Format: Naama Kanarek, Elizaveta Freinkman, Jason Cantor, Monther Abu-Remaileh, Tim Wang, David M. Sabatini. Genome-wide CRISPR screen and metabolite profiling reveal a new mechanism of methotrexate sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4988. doi:10.1158/1538-7445.AM2017-4988