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Protein-protein interactions are indispensable physiological processes regulating several biological functions. Despite the availability of structural information on protein-protein complexes, deciphering their complex topology remains an outstanding challenge. Raf kinase inhibitory protein (RKIP) has gained substantial attention as a favorable molecular target for numerous pathologies including cancer and Alzheimer’s disease. RKIP interferes with the RAF/MEK/ERK signaling cascade by endogenously binding with C-Raf (Raf-1 kinase) and preventing its activation. In the current investigation, the binding of RKIP with C-Raf was explored by knowledge-based protein-protein docking web-servers including HADDOCK and ZDOCK and a consensus binding mode of C-Raf/RKIP structural complex was obtained. Molecular dynamics (MD) simulations were further performed in an explicit solvent to sample the conformations for when RKIP binds to C-Raf. Some of the conserved interface residues were mutated to alanine, phenylalanine and leucine and the impact of mutations was estimated by additional MD simulations and MM/PBSA analysis for the wild-type (WT) and constructed mutant complexes. Substantial decrease in binding free energy was observed for the mutant complexes as compared to the binding free energy of WT C-Raf/RKIP structural complex. Furthermore, a considerable increase in average backbone root mean square deviation and fluctuation was perceived for the mutant complexes. Moreover, per-residue energy contribution analysis of the equilibrated simulation trajectory by HawkDock and ANCHOR web-servers was conducted to characterize the key residues for the complex formation. One residue each from C-Raf (Arg398) and RKIP (Lys80) were identified as the druggable “hot spots” constituting the core of the binding interface and corroborated by additional long-time scale (300 ns) MD simulation of Arg398Ala mutant complex. A notable conformational change in Arg398Ala mutant occurred near the mutation site as compared to the equilibrated C-Raf/RKIP native state conformation and an essential hydrogen bonding interaction was lost. The thirteen binding sites assimilated from the overall analysis were mapped onto the complex as surface and divided into active and allosteric binding sites, depending on their location at the interface. The acquired information on the predicted 3D structural complex and the detected sites aid as promising targets in designing novel inhibitors to block the C-Raf/RKIP interaction.

Unusual lipid modification of K-Ras makes Ras-directed cancer therapy a challenging task. Aiming to disrupt electrostatic-driven protein-protein interactions (PPIs) of K-Ras with FTase and GGTase I, a series of bivalent dual inhibitors that recognize the active pocket and the common acidic surface of FTase and GGTase I were designed. The structure-activity-relationship study resulted in 8 b, in which a biphenyl-based peptidomimetic FTI-277 was attached to a guanidyl-containing gallate moiety through an alkyl linker. Cell-based evaluation demonstrated that 8 b exhibited substantial inhibition of K-Ras processing without apparent interference with Rap-1A processing. Fluorescent imaging showed that 8 b disrupts localization of K-Ras to the plasma membrane and impairs interaction with c-Raf, whereas only FTI-277 was found to be inactive. These results suggest that targeting the PPI interface of K-Ras may provide an alternative method of inhibiting K-Ras.

Two out of 41 non-small cell lung cancer patients enrolled in a clinical study were found with a somatic CRAF mutation in their tumor, namely CRAFP261A and CRAFP207S. To our knowledge, both mutations are novel in lung cancer and CRAFP261A has not been previously reported in cancer. Expression of CRAFP261A in HEK293T cells and BEAS-2B lung epithelial cells led to increased ERK pathway activation in a dimer-dependent manner, accompanied with loss of CRAF phosphorylation at the negative regulatory S259 residue. Moreover, stable expression of CRAFP261A in mouse embryonic fibroblasts and BEAS-2B cells led to anchorage-independent growth. Consistent with a previous report, we could not observe a gain-of-function with CRAFP207S. Type II but not type I RAF inhibitors suppressed the CRAFP261A-induced ERK pathway activity in BEAS-2B cells, and combinatorial treatment with type II RAF inhibitors and a MEK inhibitor led to a stronger ERK pathway inhibition and growth arrest. Our findings suggest that the acquisition of a CRAFP261A mutation can provide oncogenic properties to cells, and that such cells are sensitive to combined MEK and type II RAF inhibitors. CRAF mutations should be diagnostically and therapeutically explored in lung and perhaps other cancers. ispartof: ONCOGENE vol:38 issue:31 pages:5933-5941 ispartof: location:England status: published

Purpose Goserelin is a drug used for chemical castration. In a rat model, we investigated whether surgical and chemical castration affected memory ability through the protein kinase A (PKA)/cyclic adenosine monophosphate response elementbinding protein (CREB)/brain-derived neurotrophic factor (BDNF) and c-Raf/mitogen-activated protein kinases-extracellular signal-regulated kinases (MEK)/extracellular signal-regulated kinases (ERK) pathways in the hippocampus. Methods Orchiectomy was performed for surgical castration and goserelin acetate was subcutaneously transplanted into the anterior abdominal wall for chemical castration. Immunohistochemistry was done to quantify neurogenesis. To assess the involvement of the PKA/CREB/BDNF and c-Raf/MEK/ERK pathways in the memory process, western blots were used. Results The orchiectomy group and the goserelin group showed less neurogenesis and impaired short-term and spatial memory. Phosphorylation of PKA/CREB/BDNF and phosphorylation of c-Raf/MEK/ERK decreased in the orchiectomy and goserelin groups. Conclusion Short-term memory and spatial memory were affected by surgical and chemical castration via the PKA/CREB/BDNF and c-Raf/MEK/ERK signaling pathways.

Significance Noonan syndrome belongs to a group of related developmental disorders termed the RASopathies, and both the M-Ras GTPase and its effector Shoc2 are signaling proteins mutated in specific subtypes of this disorder. Previously, the M-Ras/Shoc2 complex has been shown to act as a positive regulator of growth factor-mediated ERK cascade activation, and, here, we demonstrate a function for M-Ras/Shoc2/ERK cascade signaling in the dynamic control of cell–cell adhesion that is required for collective cell migration. Given the essential role of collective cell migration in human embryogenesis and organ development, our elucidation of M-Ras/Shoc2 function in this highly regulated process may provide insight regarding the cellular basis of defects associated with Noonan syndrome and other RASopathies., Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell–cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.

Diacylglycerol kinase (DGK) η translocates from the cytoplasm to punctate vehicles via osmotic shock. Apoptosis signal-regulating kinase (ASK) 3 (MAP kinase kinase kinase (MAPKKK) 15) is also reported to respond to osmotic shock. Therefore, in the present study, we examined the subcellular localization of DGKη and ASK3 expressed in COS-7 cells under osmotic stress. We found that DGKη was almost completely colocalized with ASK3 in punctate structures in response to osmotic shock. In contrast, DGKδ, which is closely related to DGKη structurally, was not colocalized with ASK3, and DGKη failed to colocalize with another MAPKKK, C-Raf, even under osmotic stress. The structures in which DGKη and ASK3 localized were not stained with stress granule makers. Notably, DGKη strongly interacted with ASK3 in an osmotic shock-dependent manner. These results indicate that DGKη and ASK3 undergo osmotic shock-dependent colocalization and associate with each other in specialized structures.

Over the last three decades, the scaffold proteins prohibitins-1 and -2 (PHB1/2) have emerged as key signaling proteins regulating a myriad of signaling pathways in health and diseases. Small molecules targeting PHBs display promising effects against cancers, osteoporosis, inflammatory, cardiac and neurodegenerative diseases. This review provides an updated overview of the various classes of PHB ligands, with an emphasis on their mechanism of action and therapeutic potential. We also describe how these ligands have been used to explore PHB signaling in different physiological and pathological settings. journal article review 2020 Sep 2020 02 15 imported

The goal of this study was to develop a potential druggable target for lung injury after SABR through the small animal model. Utilising the model, a radiation dose of 70 Gy or 90 Gy was focally (small volume) delivered to the left lung of mice. The highly expressed phosphorylation form of C-Raf was discovered through a protein array experiment, with the protein being extracted from the area of radiated mouse lung tissue, and was confirmed by IHC and western blot. C-Raf activation, along with morphological change and EMT (Epithelial to Mesenchymal Transition) marker expression, was observed after radiation to the mouse type II alveolar cell line MLE-12. C-Raf inhibitor GW5074 was able to reverse the EMT in cells effectively, and was found to be dependent on Twist1 expression. In the animal experiment, pretreatment of GW5074 alleviated EMT and lung injury after 70 Gy radiation was focally delivered to the lung of mice. Conclusively, these results demonstrate that C-Raf inhibitor GW5074 inhibits high-dose small-volume radiation-induced EMT via the C-Raf/Twist1 signalling pathway in mice. Therefore, pharmacological C-Raf inhibitors may be used effectively as inhibitors of SABR-induced lung fibrosis.

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., Graphical Abstract, In Brief Ozkan-Dagliyan et al. apply a chemical screen and identify concurrent inhibition of RAF and ERK as the most potent combination that causes low-dose apoptotic death of KRAS mutant pancreatic cancer. This vertical inhibition combination is resistant to loss of negative feedback mechanisms that reactivate ERK and potently suppresses diverse ERK-dependent processes.

Background Colorectal cancer (CRC) is one of the most common malignancies, and it’s expected that the CRC burden will substantially increase in the next two decades. New biomarkers for targeted treatment and associated molecular mechanism of tumorigenesis remain to be explored. In this study, we investigated whether PDCD6 plays an oncogenic role in colorectal cancer and its underlying mechanism. Methods Programmed cell death protein 6 (PDCD6) expression in CRC samples were analyzed by immunohistochemistry and immunofluorescence. The prognosis between PDCD6 and clinical features were analyzed. The roles of PDCD6 in cellular proliferation and tumor growth were measured by using CCK8, colony formation, and tumor xenograft in nude mice. RNA-sequence (RNA-seq), Mass Spectrum (MS), Co-Immunoprecipitation (Co-IP) and Western blot were utilized to investigate the mechanism of tumor progression. Immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) were performed to determine the correlation of PDCD6 and MAPK pathway. Results Higher expression levels of PDCD6 in tumor tissues were associated with a poorer prognosis in patients with CRC. Furthermore, PDCD6 increased cell proliferation in vitro and tumor growth in vivo. Mechanistically, RNA-seq showed that PDCD6 could affect the activation of the MAPK signaling pathway. PDCD6 interacted with c-Raf, resulting in the activation of downstream c-Raf/MEK/ERK pathway and the upregulation of core cell proliferation genes such as MYC and JUN. Conclusions These findings reveal the oncogenic effect of PDCD6 in CRC by activating c-Raf/MEK/ERK pathway and indicate that PDCD6 might be a potential prognostic indicator and therapeutic target for patients with colorectal cancer.

Graphical abstract, Proximally located in the membrane, oncogenic Ras dimers (or nanoclusters) can recruit and promote Raf dimerization and MAPK (Raf/MEK/ERK) signaling. Among Ras isoforms, KRas4B is the most frequently mutated. Recent data on the binary KRas4B–Raf-1 complex suggested that Raf-1 CRD not only executes membrane anchorage, but also supports the high-affinity interaction of Raf-1 RBD with KRas4B catalytic domain. For a detailed mechanistic picture of Raf activation at the membrane, we employ explicit MD simulations of the quaternary KRas4B–Raf-1 complex. The complex contains two active GTP-bound KRas4B proteins forming a dimer through the allosteric lobe interface and two tandem RBD-CRD segments of Raf-1 interacting with the effector lobes at both ends of the KRas4B dimer. We show that Raf-1 RBD-CRD supports stable KRas4B dimer at preferred interface and orientation at the membrane, thereby cooperatively enhancing the affinity of the KRas4B–Raf-1 interaction. We propose that a Ras dimer at the membrane can increase the population of proximal Raf kinase domains, promoting kinase domain dimerization in the cytoplasm. Collectively, the dynamic Ras–Raf assembly promotes Raf activation not by allostery; instead, Ras activates Raf by shifting its ensemble toward kinase domain-accessible states through enhanced affinity at the membrane.

Osteosarcoma, which is the most prevalent malignant bone tumor, is responsible for the great majority of bone cancer-associated deaths because of its highly metastatic potential. Although tomatidine is suggested to serve as a chemosensitizer in multidrug-resistant tumors, the anti-metastatic effect of tomatidine in osteosarcoma is still unknown. Here, we tested the hypothesis that tomatidine suppresses migration and invasion, features that are associated with metastatic process in human osteosarcoma cells and also investigate its underlying pathway. Tomatidine, up to 100 &mu, M, without cytotoxicity, inhibited the invasion and migration capabilities of human osteosarcoma U2OS and HOS cells and repressed presenilin 1 (PS-1) expression of U2OS cells. After the knockdown of PS-1, U2OS and HOS cells&rsquo, biological behaviors of cellular invasion and migratory potential were significantly reduced. While tomatidine significantly decreased the phosphorylation of c-Raf, mitogen/extracellular signal-regulated kinase (MEK), and extracellular signal-regulated protein kinase (ERK)1/2 in U2OS cells, no obvious influences on p-Jun N-terminal kinase, p38, and Akt, including their phosphorylation, were observed. In ERK 1 silencing U2 OS cells, tomatidine further enhanced the decrease of their migratory potential and invasive activities. We conclude that both PS-1 derived from U2OS and HOS cells and the c-Raf&ndash, MEK&ndash, ERK pathway contribute to cellular invasion and migration and tomatidine could inhibit the phenomenons. These findings indicate that tomatidine might be a potential candidate for anti-metastasis treatment of human osteosarcoma.

The ARID1B/BAF250b subunit of the human SWI/SNF chromatin remodeling complex is a canonical nuclear tumor suppressor. We employed in silico prediction, intracellular fluorescence and cellular fractionation based subcellular localization analyses to identify the ARID1B nuclear localization signal. A cytoplasm-restricted ARID1B-NLS mutant was significantly compromised in its canonical transcription activation and tumor suppressive functions, as expected. Surprisingly however, cytoplasmic localization appeared to induce a gain of oncogenic function in ARID1B as evidenced from several cell line and mouse xenograft based assays. Mechanistically, cytoplasm-localized ARID1B could bind c-RAF and PPP1CA causing stimulation of RAF-ERK signaling and β-catenin transcription activity. ARID1B harboring NLS mutations derived from tumor samples also exhibited aberrant cytoplasmic localization and acquired a neo-morphic oncogenic function via activation of RAF-ERK signaling. Further, immunohistochemistry on a tissue microarray revealed significant correlation of ARID1B cytoplasmic localization with increased levels of active forms of ERK1/2 and β-catenin as well as with advanced tumor stage and lymph node positivity in human primary pancreatic tumor tissues. ARID1B therefore promotes oncogenesis through cytoplasm-based gain of function mechanisms in addition to dysregulation in the nucleus.

We recently reported that (23R, 24E)-23-acetoxymangiferonic acid (23R-AMA), a cycloartane triterpenoid isolated by activity-guided separation from a methanol extract of Garcinia sp. bark, inhibited melanin production via inhibition of tyrosinase (TYR) expression in the B16-F10 melanoma cell line. Since 23R-AMA also inhibited microphthalmia-associated transcription factor (MITF) expression, an upstream factor of TYR, these features of 23R-AMA were thought to be appropriate for development of whitening cosmetics. However, 23R-AMA exhibited growth inhibition other than inhibition of melanin production in B16-F10 cells. Therefore, we investigated biological activities of 23R-AMA in detail, focused on its application as an anti-melanoma compound. In this study, we demonstrated that 23R-AMA inhibited cell proliferation and basic FGF (bFGF)-induced migration in B16-F10 cells. Furthermore, 23R-AMA promoted ser45/thr41 phosphorylation of β-catenin and suppressed its intranuclear accumulation, which was suggested to be related to inhibition of MITF expression. The transcriptional activity of MITF is known to be regulated by phosphorylation via activated ERK. Further investigation revealed that 23R-AMA inhibited phosphorylation of c-Raf, MEK-1, and ERK, and also that of upstream molecules including FAK and c-Src. These results suggested that 23R-AMA inhibited growth and migration of B16-F10 melanoma by regulating both MITF expression and its activity. The activities of 23R-AMA reported in this study are new aspects of cycloartane triterpenoids. Electronic supplementary material The online version of this article (10.1007/s11418-018-1233-7) contains supplementary material, which is available to authorized users.

The mechanism through which oncogenic Ras activates its effectors is vastly important to resolve. If allostery is at play, then targeting allosteric pathways could help in quelling activation of MAPK (Raf/MEK/ERK) and PI3K (PI3K/Akt/mTOR) cell proliferation pathways. On the face of it, allosteric activation is reasonable: Ras binding perturbs the conformational ensembles of its effectors. Here, however, we suggest that at least for Raf, PI3K, and NORE1A (RASSF5), that is unlikely. Raf's long disordered linker dampens effective allosteric activation. Instead, we suggest that the high-affinity Ras–Raf binding relieves Raf's autoinhibition, shifting Raf's ensemble from the inactive to the nanocluster-mediated dimerized active state, as Ras also does for NORE1A. PI3K is recruited and allosterically activated by RTK (e.g., EGFR) at the membrane. Ras restrains PI3K's distribution and active site orientation. It stabilizes and facilitates PIP2 binding at the active site and increases the PI3K residence time at the membrane. Thus, RTKs allosterically activate PI3Kα; however, merging their action with Ras accomplishes full activation. Here we review their activation mechanisms in this light and draw attention to implications for their pharmacology.

Invasion and intrahepatic metastasis are major factors of poor prognosis in patients with hepatocellular carcinoma (HCC). In this study, we show that increased Src homolog and collagen homolog 3 (Shc3) expression in malignant HCC cell lines associate with HCC invasion and metastasis. Shc3 (N-Shc) was significantly upregulated in tumors of 33 HCC patient samples as compared with adjacent normal tissues. Further analysis of 52 HCC patient samples showed that Shc3 expression correlated with microvascular invasion, cancer staging, and poor prognosis. Shc3 interacted with major vault protein, resulting in activation of MEK1/2 and ERK1/2 independently of Shc1 and c-Raf; this interaction consequently induced epithelial–mesenchymal transition and promoted HCC cell proliferation and metastasis. The observed increase in Shc3 levels was due to demethylation of its upstream promoter, which allowed c-Jun binding. In turn, Shc3 expression promoted c-Jun phosphorylation in a positive feedback loop. Analysis of metastasis using a tumor xenograft mouse model further confirmed the role of Shc3 in vivo. Taken together, our results indicate the importance of Shc3 in HCC progression and identify Shc3 as a novel biomarker and potential therapeutic target in HCC. Significance: Ectopic expression of Shc3 forms a complex with MVP/MEK/ERK to potentiate ERK activation and plays an important role in sorafinib resistance in HCC. Cancer Res; 78(9); 2219–32. ©2018 AACR.

Association of Raf kinase with activated Ras triggers downstream signaling cascades toward regulating transcription in the cells’ nucleus. Dysregulation of Ras–Raf signaling stimulates cancers. We investigate the C-Raf RBD and CRD regions when bound to oncogenic K-Ras4B at the membrane. All-atom molecular dynamics simulations suggest that the membrane plays an integral role in regulating the configurational ensemble of the complex. Remarkably, the complex samples a few states dynamically, reflecting a competition between C-Raf CRD- and K-Ras4B- membrane interactions. This competition arises because the interaction between the RBD and K-Ras is strong while the linker between the RBD and CRD is short. Such a mechanism maintains a modest binding for the overall complex at the membrane and is expected to facilitate fast signaling processes. Competition of protein–membrane contacts is likely a common mechanism for other multiprotein complexes, if not multidomain proteins at membranes., As two counterparts of a protein complex, K-Ras4B and C-Raf CRD interact with membrane in a competitive manner due to topological restriction, presenting two major dynamic membrane-associated states.

Differentiation therapy is used in cancer treatment. Epidemiologic studies showed that higher vitamin D levels are associated with reduced cancer risks. However, the therapeutic doses needed for differentiation are accompanied by hypercalcemia and intolerable pathological sequelae. In the present work we evaluated if RRD-251, a small-molecule, can enhance vitamin D3-induced differentiation of leukemic cells, in the hope of decreasing the needed vitamin D3-dose. We demonstrate that RRD-251 enhances vitamin D3-induced differentiation of leukemic cells, the enrichment of the c-Raf kinase in the nucleus, the binding of nuclear c-Raf to GSK-3, increased phosphorylation of GSK-3 ser 21/9 inhibitory sites, and the binding of GSK-3 to VDR, where GSK-3 inhibition is known to enhance transcriptional activation by VDR. Enhancement of D3-induced p-GSK-3 ser 21/9 by RRD-251 was associated with enhanced Akt-GSK-3 binding, Akt being a known GSK-3 inhibitor, and diminished Erk1/2 binding. Diminishing Erk interaction with GSK-3 was associated with enhanced interaction with Vav1, a known driver of myeloid differentiation. This is redolent of an ATRA/c-Raf/GSK-3/RARα axis we previously reported, although the phosphorylation effects to enhance transcriptional activation on RARα vs VDR diverge. Taken together this indicates potential therapeutic significance for a c-Raf/GSK-3/VDR or RARα axis for leukemic myelo-monocytic differentiation.

Background: According to the National Cancer Institute, B-cell acute lymphoblastic leukemia (B-ALL) is the most common cancer of children and adolescents (ALL, NCI, PDQ, accessed 8/2/2021). Recently, obesity has been identified as a risk factor which is associated with poor survival outcomes (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012) which is concerning due to the obesity rates in children and adolescents having tripled since the 1970's (Ogden et al., 2006; Ogden et al., 2020). Indeed, survival rates in obese pediatric patients with B-ALL can decline by as much as 30% relative to outcomes observed in lean patients, with obese patients more commonly presenting with treatment-related adverse events (Butturini et al., 2007; Eissa et al., 2017; Ethier et al., 2012). A hallmark of obesity is the accumulation of adipocytes, an endocrine cell type which can promote chemoresistance (Ehsanipour et al., 2013; Sheng et al., 2016; Mittelman., 2021). The mechanistic understanding of how adipocytes promote chemoresistance in B-ALL is still under investigation and further insight into this relationship could lead to the rational design of effective therapeutic strategies for obese patients with limited treatment options. Methods: A cytokine/chemokine array was performed on adipocyte and stromal cell secretomes to identify potential adipocyte-secreted inflammatory mediators, which may promote chemoresistance in human B-ALL cells. Once candidate cytokines were identified, we performed in vitro assays to measure how the addition or neutralization of proteins of interest impacted the proliferation, activation of signaling pathways, steady-state mitochondrial protein levels, and survival of human B-ALL cells in the absence or presence of chemotherapy treatment. Additionally, we mined publicly available databases to determine how protein-coding genes of interest were associated with patient survival. Furthermore, we have used the diet-induced murine model of obesity to determine how targeting candidate cytokines impact B-ALL pathogenesis. Results: We have made the novel finding that interleukin-9 (IL-9) levels are higher in adipose-rich microenvironments and activates pro-survival pathways that promote chemoresistance in human B-ALL cells. We have found that obese mice lacking IL-9 are more resistant to B-ALL development due to significant increases in survival outcomes compared to lean mice transplanted with B-ALL cells. Furthermore, we have discovered that human B-ALL cells upregulate the interleukin-9 receptors (IL-9R) when exposed to the adipocyte secretome. This potential feedback loop may increase the responsiveness of leukemia cells to local IL-9 levels. These observations were supported by our data mining results, which revealed that IL-9R gene expression levels were higher in more aggressive subtypes of B-ALL, including Ph-like B-ALL. When human B-ALL cells were treated with recombinant IL-9 (rIL-9), chemoresistance to methotrexate and doxorubicin was observed. Mechanistically, rIL-9 treatment of human B-ALL cells also downregulated the protein expression of the pro-apoptotic mitochondrial-associated protein Bim and pro-proliferative protein Raf. In all, our experiments have identified IL-9 as an adipocyte-enriched cytokine, which promotes pan-chemoresistance in human B-ALL cells. Furthermore, we have shown that this effect maybe mediated in part by suppressing the protein of expression of pro-apoptotic and proliferative proteins. Conclusions: To our knowledge, our results represent the first reports of IL-9 mediated chemoresistance in human B-ALL and the first to demonstrate that IL-9 regulates the protein homeostasis of anti- and pro-apoptotic mitochondrial proteins. In ongoing studies, we are conducting in vitro and murine studies with parental and IL-9R-deficient B-ALL cells to determine how B-ALL pathogenesis and chemosensitivity are impacted. Subsequent studies will be conducted in lean and obese mice transplanted with B-ALL cells who receive chemotherapy treatment alone or in combination with IL-9 neutralizing antibody administration. Disclosures Lee: PureTech Health: Research Funding. Henry: PureTech Health: Research Funding.

The mitogen-activated protein kinase (MAPK) signaling pathway is a three-tiered kinase cascade where mitogen-activated protein kinase kinase kinases (MAP3Ks) lead to the activation of mitogen-activated protein kinase kinases (MAP2K), and ultimately MAPK proteins. MAPK signaling can promote a diverse set of biological outcomes, ranging from cell death to proliferation. There are multiple mechanisms which govern MAPK output, such as the duration and strength of the signal, cellular localization to upstream and downstream binding partners, pathway crosstalk and the binding to scaffold and adaptor molecules. This review will focus on scaffold and adaptor proteins that bind to and regulate apoptosis signal-regulating kinase 1 (ASK1), a MAP3K protein with a critical role in mediating stress response pathways.

// Rodica P. Bunaciu 1 , Robert J. MacDonald 1 , Feng Gao 1, 4, 5 , Lynn M. Johnson 3 , Jeffrey D. Varner 4 , Xin Wang 5 , Sarah Nataraj 2 , Monica L. Guzman 2 and Andrew Yen 1 1 Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA 2 Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medical College, New York, NY, USA 3 Cornell Statistical Unit, Cornell University, Ithaca, NY, USA 4 Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA 5 Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China Correspondence to: Andrew Yen, email: ay13@cornell.edu , rpb78@cornell.edu Keywords: retinoic acid; c-Raf; c-Cbl; AML; precision oncology Received: October 16, 2017 Accepted: December 09, 2017 Published: December 23, 2017 ABSTRACT Acute myeloid leukemia (AML) has high mortality rates, perhaps reflecting a lack of understanding of the molecular diversity in various subtypes and a lack of known actionable targets. There are currently 12 open clinical trials for AML using combination therapeutic modalities including all- trans retinoic acid (RA). Mutant nucleophosmin-1, proposed as a possible marker for RA response, is the criterion for recruiting patients in three active RA phase 3 clinical trials. We tested the ability of RA alone or in combination with either bosutinib (B) or 6-formylindolo(3,2-b) carbazole (F) to induce conversion of 12 de novo AML samples toward a more differentiated phenotype. We assessed levels of expression of cell surface markers associated with differentiation, aldehyde dehydrogenase activity, and glucose uptake activity. Colony formation capacity was reduced with the combined treatment of RA and B or F, and correlated with modulation of a c-Cbl/Lyn/c-Raf-centered signalsome. Combination treatment was in most cases more effective than RA alone. Based on their responses to the treatments, some primary leukemic samples cluster closer to HL-60 cells than to other primary samples, suggesting that they may represent a hitherto undefined AML subtype that is potentially responsive to RA in a combination differentiation therapy.

The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.

Prolonged exposure to high concentrations of glutamate leads to cell type specific glutathione depletion and resulting oxidative stress, known as oxytosis. As a result of glutathione depletion, accumulation of reactive oxygen species and Ca2+ influx are increased; however, the specific target of oxytosis has yet to be identified. In the present study, we focused on the effect of glutamate-induced oxidative stress on the extracellular-regulated protein kinase (ERK) pathway using the murine hippocampal HT22 cell line. Although the contribution of the ERK pathway to glutamate-induced oxytosis in HT22 cells is controversial, Western blot analysis revealed that glutamate caused down-regulation of mitogen-activated protein kinase kinase kinase (c-Raf) and a resulting decrease in the phosphorylation of c-Raf, as well as of mitogen-activated protein kinase kinase1/2 (MEK1/2) and ERK1/2, downstream components of the c-Raf/MEK/ERK pathway. Furthermore, neuroprotective (arylthio)cyclopentenone prostaglandins prevented glutamate-induced c-Raf down-regulation and consequently maintained the basal activity of c-Raf and its downstream signaling components. A pull-down assay using biotin-labeled cyclopentenone prostaglandins revealed that they preferentially bound to c-Raf relative to other signaling molecules of the ERK pathway, including Ras, MEK1/2, and ERK. These results suggest that neuroprotective (arylthio)cyclopentenone prostaglandins directly bind to c-Raf protein and protect cells from down-regulation of the c-Raf protein itself, resulting in neuroprotection against oxidative stress.

The differentiation of endometrial stromal cells into decidual cells, termed decidualization, is an integral step in the establishment of pregnancy. The mitogen-activated protein kinase homolog, WNK lysine deficient protein kinase 1 (WNK1), is activated downstream of epidermal growth factor receptor during decidualization. Primary human endometrial stromal cells (HESCs) were subjected to small interfering RNA knockdown of WNK1 followed by in vitro decidualization. This abrogated expression of the decidual marker genes, insulin like growth factor binding protein 1 (IGFBP1) and prolactin (PRL), and prevented adoption of decidual cell morphology. Analysis of the WNK1-dependent transcriptome by RNA-Seq demonstrated that WNK1 regulates the expression of 1858 genes during decidualization. Gene ontology and upstream regulator pathway analysis showed that WNK1 regulates cell migration, differentiation, and proliferation. WNK1 was required for many of the gene expression changes that drive decidualization, including the induction of the inflammatory cytokines, C-C motif chemokine ligand 8 (CCL8), interleukin 1 beta (IL1B), and interleukin 15 (IL15), and the repression of transforming growth factor-beta (TGF-beta) pathway genes, including early growth response 2 (EGR2), SMAD family member 3 (SMAD3), integrin subunit alpha 2 (ITGA2), integrin subunit alpha 4 (ITGA4), and integrin subunit beta 3 (ITGB3). In addition to abrogating decidualization, WNK1 knockdown decreased the migration and proliferation of HESCs. Furthermore, mitogen-activated protein kinase 7 (MAPK7), a known downstream target of WNK1, was activated during decidualization in a WNK1-dependent manner. Small interfering RNA knockdown of MAPK7 demonstrated that MAPK7 regulates a subset of WNK1-regulated genes and controls the migration and proliferation of HESCs. These results indicate that WNK1 and MAPK7 promote migration and proliferation during decidualization and regulate the expression of inflammatory cytokines and TGF-beta pathway genes in HESCs.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a key role in mediating non-homologous end joining (NHEJ), a major repair pathway for DNA double-strand breaks (DSBs). The activation, function and dynamics of DNA-PKcs is regulated largely by its reversible phosphorylation at numerous residues, many of which are targeted by DNA-PKcs itself. Interestingly, these DNA-PKcs phosphorylation sites function in a distinct, and sometimes opposing manner, suggesting that they are differentially regulated via complex actions of both kinases and phosphatases. In this study we identified several phosphatase subunits as potential DSB-associated proteins. In particular, protein phosphatase 1 (PP1) is recruited to a DSB-mimicking substrate in Xenopus egg extracts and sites of laser microirradiation in human cells. Depletion of PP1 impairs NHEJ in both Xenopus egg extracts and human cells. PP1 binds multiple motifs of DNA-PKcs, regulates DNA-PKcs phosphorylation, and is required for DNA-PKcs activation after DNA damage. Interestingly, phosphatase 1 nuclear targeting subunit (PNUTS), an inhibitory regulator of PP1, is also recruited to DNA damage sites to promote NHEJ. PNUTS associates with the DNA-PK complex and is required for DNA-PKcs phosphorylation at Ser-2056 and Thr-2609. Thus, PNUTS and PP1 together fine-tune the dynamic phosphorylation of DNA-PKcs after DNA damage to mediate NHEJ.

To construct a highly expressed and active MEK1R4F (a constitutively active form of mitogen-activated protein kinase kinase 1) by fusion of soluble adenylate kinase (Adk) tag, resulting in Adk-MEK1R4F protein suitable for preparation of phosphorylated ERK. We fused the Adk to the N-terminus of MEK1R4F through overlapping PCR. The expression of Adk-MEK1R4F fusion protein increased ~10-fold in Escherichia coli, and was purified to 95% via two purification steps including Ni–NTA and Q Sepharose fast flow (QFF) chromatography. The purified Adk-MEK1R4F protein was functional for ERK phosphorylation and could use ADP in addition to ATP. The Adk-MEK1R4F had higher catalytic activity than regular MEK1R4F both in vitro and in cell-free extracts system. Adenylate kinase was used as a soluble tag to facilitate MEK1R4F protein expression and its application in large-scale phosphorylated ERK1/2 preparation and purification.

The interference from endogenous signaling enzymes represents a major hurdle for building orthogonal signaling cascade inside cells, particularly among the close related isoforms within an enzyme family. Here we employed the genetically-encoded chemical decaging strategy to build orthogonal-activated kinase isoforms, with the endogenous counterparts temporally disabled by an extracellular-delivered bacterial effector. This approach eliminated the potential interference from other kinase isoforms as well as the endogenous kinases, which allowed the specific, gain-of-function report of mitogen activated protein kinase kinase 1 (MEK1) activity as opposed to MEK2 with high temporal resolution. Our study dissected the distinct enzymatic activity, feedback regulation and signal outputs between these close related kinase isoforms.

Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.

Protein kinase A (PKA) has recently been shown to mimic the actions of follicle-stimulating hormone (FSH) by activating signaling pathways that promote granulosa cell (GC) differentiation, such as phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). We sought to elucidate the mechanism by which PKA, a Ser/Thr kinase, intersected the PI3K/AKT and MAPK/ERK pathways that are canonically activated by receptor tyrosine kinases (RTKs). Our results show that for both of these pathways, the RTK is active in the absence of FSH yet signaling down the pathways to commence transcriptional responses requires FSH-stimulated PKA activation. For both pathways, PKA initiates signaling by regulating the activity of a protein phosphatase (PP). For the PI3K/AKT pathway, PKA activates the Ser/Thr PP1 complexed with the insulinlike growth factor 1 receptor (IGF-1R) and insulin receptor substrate 1 (IRS1) to dephosphorylate Ser residues on IRS1, authorizing phosphorylation of IRS1 by the IGF-1R to activate PI3K. Treatment of GCs with FSH and exogenous IGF-1 initiates synergistic IRS1 Tyr phosphorylation and resulting gene activation. The mechanism by which PKA activates PI3K is conserved in preovulatory GCs, MCF7 breast cancer cells, and FRTL thyroid cells. For the MAPK/ERK pathway, PKA promotes inactivation of the MAPK phosphatase (MKP) dual specificity phosphatase (DUSP) MKP3/DUSP6 to permit MEK-phosphorylated ERK to accumulate downstream of the epidermal growth factor receptor. Thus, for the two central signaling pathways that regulate gene expression in GCs, FSH via PKA intersects canonical RTK-regulated signaling by modulating the activity of PPs.

Stimulation of transient receptor potential M3 (TRPM3) cation channels with pregnenolone sulfate induces an influx of Ca2+ ions into the cells and a rise in the intracellular Ca2+ concentration, leading to the activation of the activator protein-1 (AP-1) transcription factor. Here, we show that expression of a constitutively active mutant of the Ca2+ /calmodulin-dependent protein phosphatase calcineurin attenuated pregnenolone sulfate-induced AP-1 activation in TRPM3-expressing cells. Likewise, expression of the regulatory B subunit of calcineurin reduced AP-1 activity in the cells following stimulation of TRPM3 channels. MAP kinase phosphatase-1 has been shown to attenuate TRPM3-mediated AP-1 activation. Here, we show that pregnenolone sulfate-induced stimulation of TRPM3 triggers the phosphorylation and activation of the MAP kinase extracellular signal-regulated protein kinase (ERK1/2). Pharmacological and genetic experiments revealed that stimulation of ERK1/2 is essential for the activation of AP-1 in cells expressing stimulated TRPM3 channels. ERK1/2 is required for the activation of the transcription factor c-Jun, a key component of the AP-1 transcription factor, and regulates c-Fos promoter activity. In addition, we identified c-Jun N-terminal protein kinase (JNK1/2) as a second signal transducer of activated TRPM3 channels. Together, the data show that calcineurin and the protein kinases ERK1/2 and JNK1/2 are important regulators within the signaling cascade connecting TRPM3 channel stimulation with increased AP-1-regulated transcription. J. Cell. Biochem. 118: 2409-2419, 2017. © 2017 Wiley Periodicals, Inc.

Protein kinases are central players in the regulation of cell cycle and signalling pathways. Their catalytic activities are strictly regulated through post-translational modifications and protein-protein interactions that control switching between inactive and active states. These states have been studied extensively using protein crystallography, although the dynamic nature of protein kinases makes it difficult to capture all relevant states. Here, we describe two recent structures of Aurora-A kinase that trap its active and inactive states. In both cases, Aurora-A is trapped through interaction with a synthetic protein, either a single-domain antibody that inhibits the kinase or a hydrocarbon-stapled peptide that activates the kinase. These structures show how the distinct synthetic proteins target the same allosteric pocket with opposing effects on activity. These studies pave the way for the development of tools to probe these allosteric mechanisms in cells.