The mammalian circadian clock is based on a transcription-translation feedback loop (TTFL) in which CLOCK and BMAL1 proteins act as transcriptional activators of Cryptochrome and Period genes, which encode proteins that repress CLOCK-BMAL1 with a periodicity of~24 h. In this model, the mechanistic roles of CRY and PER are unclear. Here, we used a controlled targeting system to introduce CRY1 or PER2 into the nuclei of mouse cells with defined circadian genotypes to characterize the functions of CRY and PER. Our data show that CRY is the primary repressor in the TTFL: It binds to CLOCK-BMAL1 at the promoter and inhibits CLOCK-BMAL1-dependent transcription without dissociating the complex (''blocking''-type repression). PER alone has no effect on CLOCK-BMAL1-activated transcription. However, in the presence of CRY, nuclear entry of PER inhibits transcription by displacing CLOCK-BMAL1 from the promoter (''displacement''-type repression). In light of these findings, we propose a new model for the mammalian circadian clock in which the negative arm of the TTFL proceeds by two different mechanisms during the circadian cycle.

SUMMARY Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feed-forward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth.

Controlling protein activity with chemogenetics and optogenetics has proven to be powerful for testing hypotheses regarding protein function in rapid biological processes. Controlling proteins by splitting them and then rescuing their activity through inducible reassembly offers great potential to control diverse protein activities. Building split proteins has been difficult due to spontaneous assembly, difficulty in identifying appropriate split sites, and inefficient induction of effective reassembly. Here we present an automated approach to design effective split proteins regulated by a ligand or by light (SPELL). We develop a scoring function together with an engineered domain to enable reassembly of protein halves with high efficiency and with reduced spontaneous assembly. We demonstrate SPELL by applying it to proteins of various shapes and sizes in living cells. The SPELL server (spell.dokhlab.org) offers an automated prediction of split sites.

SUMMARY We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1 , and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncogene family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform-specific differences in signaling events that support neoplastic cell growth. Recognition that there are RAS mutation-specific differences has led to expectations that defining RAS mutation-selective vulnerabilities will lead to new therapies. However, critical issues remain that require resolution to facilitate the success of these efforts. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available RAS antibodies using a set of unique and innovative reagents and cell lines. We validated antibodies for each of the four RAS isoforms, and for G12D- or G12V-mutant RAS proteins, for Western blot but not for immunofluorescence (IF) or immunohistochemical (IHC) analyses. Our results may help ensure accurate interpretation of future RAS studies.

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Non-steroidal anti-inflammatory drugs (NSAIDs), which are widely used for the treatment of rheumatic arthritis, pain, and many different types of inflammatory disorders, cause serious gastrointestinal (GI) side effects. The free carboxylic acid group existing on their chemical structure is correlated with GI toxicity related with all routine NSAIDs. Replacing this functional group with the 1,3,4-oxadiazole bioisostere is a generally used strategy to obtain an anti-inflammatory agent devoid of GI side effects. In the present work, a novel group of 5-(3,4-dichlorophenyl)-1,3,4-oxadiazole-2(3H)-one Mannich bases were synthesized and characterized on the basis of IR, H NMR, and elemental analysis results. The target compounds were first tested for cytotoxicity to determine a non-toxic concentration for anti-inflammatory screening. Anti-inflammatory effects of the compounds were evaluated by in vitro lipopolysaccharide (LPS)-induced NO production and in vivo carrageenan footpad edema with ulcerogenic profile. In LPS-induced RAW 264.7 macrophages, most of the compounds showed inhibitory activity on nitrite production while compounds 5a, 5h, and 5j exhibited the best profiles by suppressing the NO production. To evaluate the in vivo anti-inflammatory potency of the compounds, the inflammatory response was quantified by increment in paw size in the carrageenan footpad edema assay. The anti-inflammatory data scoring showed that compounds 5a-d, 5g, and 5j, at the dose of 100 mg/kg, exhibited anti-inflammatory activity, which for compound 5g was comparable to that of the reference drug indomethacin with 53.9% and 55.5% inhibition in 60 and 120 min, respectively.

Mutational loss of CDKN2A (encoding p16INK4A) tumor suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, anti-apoptotic signaling, and MYC expression. Based on these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell cycle regulatory proteins centered on CDK2 activation, PI3K-AKT-mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small molecule inhibitor-based approaches. Additionally, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to PDAC patients.