Downstream Substrates Research Articles

NEDD4 E3 Ligases: Functions and Mechanisms in Bone and Tooth.

Protein ubiquitination is a precisely controlled enzymatic cascade reaction belonging to the post-translational modification of proteins. In this process, E3 ligases catalyze the binding of ubiquitin (Ub) to protein substrates and define specificity. The neuronally expressed developmentally down-regulated 4 (NEDD4) subfamily, belonging to the homology to E6APC terminus (HECT) class of E3 ligases, has recently emerged as an essential determinant of multiple cellular processes in different tissues, including bone and tooth. Here, we place special emphasis on the regulatory role of the NEDD4 subfamily in the molecular and cell biology of osteogenesis. We elucidate in detail the specific roles, downstream substrates, and upstream regulatory mechanisms of the NEDD4 subfamily. Further, we provide an overview of the involvement of E3 ligases and deubiquitinases in the development, repair, and regeneration of another mineralized tissue—tooth.

Differential roles and regulation of the protein kinases PAK4, PAK5 and PAK6 in melanoma cells.

The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations; PAK6 and PAK4 have fewer; and PAK4 is often amplified. To help interpret these genomic data, initially we compared the cellular regulation of the sister kinases and their roles in melanoma cells. In common with many ohnologue protein kinases, PAK4, PAK5 and PAK6 each have two 14-3-3-binding phosphosites of which phosphoSer99 is conserved. PAK4 localises to the leading edge of cells in response to phorbol ester-stimulated binding of 14-3-3 to phosphoSer99 and phosphoSer181, which are phosphorylated by two different PKCs or PKDs. These phosphorylations of PAK4 are essential for its phorbol ester-stimulated phosphorylation of downstream substrates. In contrast, 14-3-3 interacts with PAK5 in response to phorbol ester-stimulated phosphorylation of Ser99 and epidermal growth factor-stimulated phosphorylation of Ser288; whereas PAK6 docks onto 14-3-3 and is prevented from localising to cell–cell junctions when Ser133 is phosphorylated in response to cAMP-elevating agents via PKA and insulin-like growth factor 1 via PKB/Akt. Silencing of PAK4 impairs viability, migration and invasive behaviour of melanoma cells carrying BRAFV600E or NRASQ61K mutations. These defects are rescued by ectopic expression of PAK4, more so by a 14-3-3-binding deficient PAK4, and barely by PAK5 or PAK6. Together these genomic, biochemical and cellular data suggest that the oncogenic properties of PAK4 are regulated by PKC–PKD signalling in melanoma, while PAK5 and PAK6 are dispensable in this cancer.

Spectroscopic studies of 7,8-diacetoxy-4-methylcoumarin and 7,8-dipentynoyl-4-methylcoumarin binding with calreticulin.

Calreticulin (CRT) is a protein found mainly in the endoplasmic reticulum (ER) that maintains calcium levels and controls protein folding, but has recently been found at the cell surface, cytoplasm, and in the extracellular matrix. CRT participates in multiple physiological processes such as gene expression, the immune response, and cancer. Calreticulin has been shown to autoacetylate with the binding of preferred ligand 7,8-diacetoxy-4-methylcoumarin (DAMC). This project aims to develop a chemical biology approach to investigate importance of CRT acylating abilities on its nonendoplasmic reticulum functions by targeting the downstream substrates of CRT acetylation. Our goal was to use CRT to transfer a pentynoyl tag (using a novel ligand, DPeMC) to its substrates, which can then be used as a handle for protein identification. Molecular modelling using available data in the literature was used to approximate the binding interface between CRT and the acylation ligands. Molecular Operating Environment (MOE) software was used to perform sequence alignment, simulated annealing, positional refinement, and blind docking of acylated coumarins with the CRT model. Docking studies pointed to the P domain as the most thermodynamically and sterically favourable region for acylated coumarin binding with tryptophan residue 200 within the active site on CRT. Absorption and fluorescence spectra of all coumarin compounds in ethanol:PBS (1:9v/v) solution were investigated. Stern-Volmer quenching constant (KSV ), binding constant (K), and number of binding sites (n) of each coumarin compound with CRT was determined. Our studies demonstrated that acyl coumarin compounds bind to CRT using a dynamic quenching mechanism, bind to a single binding site on the P domain, and that the protein-ligand interaction is spontaneous and exothermic.

Role of caspase-3-cleaved/activated PAK2 in brusatol-triggered apoptosis of human lung cancer A549 cells.

Brusatol, a major quassinoid extract of Bruceae fructus, is an important bioactive component with antineoplastic capacity. Several beneficial pharmacological and biological properties of brusatol have been uncovered to date, including anti-inflammatory, anticolitis, antimalarial, and anticancer activities. To confer anticancer benefits, brusatol is reported to effectively inhibit the Nrf2-mediated antioxidant response and trigger apoptotic signaling. In this study, we investigated the regulatory mechanisms underlying apoptotic processes in brusatol-treated A549 cells in detail. Our experiments showed that brusatol induces cell death through intracellular ROS-triggered mitochondria-dependent apoptotic events and does not involve necrosis. Mechanistically, p21-activated protein kinase 2 (PAK2) was cleaved by caspase-3 to generate an activated p34 fragment involved in brusatol-induced apoptosis of A549 cells. Notably, PAK2 knockdown led to downregulation of caspase-3-mediated PAK2 activity, in turn, effectively attenuating brusatol-induced apoptosis, highlighting a crucial role of caspase-3-activated PAK2 in this process. Moreover, knockdown of PAK2 resulted in significant inhibition of c-Jun N-terminal kinase (JNK) activity in brusatol-treated A549 cells, clearly suggesting that JNK serves as a downstream substrate of caspase-3-cleaved/activated PAK2 in the apoptotic cascade. SP600125, a specific JNK inhibitor, significantly suppressed brusatol-induced JNK activity but only partially prevented apoptosis, implying that JNK serves as only one of a number of substrates for PAK2 in the brusatol-triggered apoptotic cascade. Based on the collective results, we propose a signaling cascade model for brusatol-induced apoptosis in human A549 cells involving ROS, caspases, PAK2, and JNK.

ZINC40099027 promotes monolayer circular defect closure by a novel pathway involving cytosolic activation of focal adhesion kinase and downstream paxillin and ERK1/2.

ZINC40099027 (ZN27) is a specific focal adhesion kinase (FAK) activator that promotes murine mucosal wound closure after ischemic or NSAID-induced injury. Diverse motogenic pathways involve FAK, but the direct consequences of pure FAK activation have not been studied, and how ZN27-induced FAK activation stimulates wound closure remained unclear. We investigated signaling and focal adhesion (FA) turnover after FAK activation by ZN27 in Caco-2 cells, confirming key results in CCD841 cells. ZN27 increased Caco-2 FAK-Y-397, FAK-Y-576/7, paxillin-Y-118, and ERK 1/2 phosphorylation and decreased FAK-Y-925 phosphorylation, without altering FAK-Y-861, p38, Jnk, or Akt phosphorylation. ZN27 increased FAK-paxillin interaction while decreasing FAK-Grb2 association. ZN27 increased membrane-associated FAK-Y-397 and FAK-Y-576/7 phosphorylation and paxillin-Y-118 and ERK 1/2 phosphorylation but decreased FAK-Y-925 phosphorylation without altering Src or Grb2. Moreover, ZN27 increased the fluorescence intensity of GFP-FAK and pFAK-Y397 in FAs and increased the total number of FAs but reduced their size in GFP-FAK-transfected Caco-2 cells, consistent with increased FA turnover. In contrast, FAK-Y397F transfection prevented ZN27 effects on FAK size and number and FAK and pFAK fluorescent intensity in FAs. We confirmed the proposed FAK/paxillin/ERK pathway using PP2 and U0126 to block Src and MEK1/2 in Caco-2 and CCD841 cells. These results suggest that ZN27 promotes intestinal epithelial monolayer defect closure by stimulating autophosphorylation of FAK in the cytosol, distinct from classical models of FAK activation in the FA. Phosphorylated FAK translocates to the membrane, where its downstream substrates paxillin and ERK are phosphorylated, leading to FA turnover and human intestinal epithelial cell migration.

Targeting UBE2C for degradation by bioPROTACs based on bacterial E3 ligase

UBE2C (Ubiquitin conjugating enzyme E2 C), a key regulator of cell cycle progression, is a promising target for discovery of antitumor agents. However, it is challenging to develop inhibitors of UBE2C owing to its lack of “druggable” pockets. BioPROTACs (biological proteolysis targeting chimeras) are a kind of protein-based degraders by fusing an adaptor to a subunit of E3 ligase for ubiquitination and subsequent proteasome-dependent degradation of target protein. We report herein the design and biological evaluation of a UBE2C-targeting bioPROTAC based on the NEL (novel E3 ligase) domain of bacterial E3 ligase IpaH9.8 and the UBE2C-binding WHB (winged-helix B) domain of APC2 (anaphase promoting complex subunit 2). The in vitro ubiquitination test and Mass Spectrometry analysis showed that the bioPROTAC could transfer ubiquitin to surface exposed lysines on UBE2C and catalyzed the formation of polyubiquitin chains. In addition, the transient co-expression experiment showed that the bioPROTAC could promote proteasomal degradation of heterologous UBE2C and rescue its downstream substrates in mammalian cells.

Analysis of neuroendocrine clones in NSCLCs using an immuno-guided laser-capture microdissection-based approach

SummaryClonal evolution and lineage plasticity are key contributors to tumor heterogeneity and response to treatment in cancer. However, capturing signal transduction events in coexisting clones remains challenging from a technical perspective. In this study, we developed and tested a signal-transduction-based workflow to isolate and profile coexisting clones within a complex cellular system like non-small cell lung cancers (NSCLCs). Cooccurring clones were isolated under immunohistochemical guidance using laser-capture microdissection, and cell signaling activation portraits were measured using the reverse-phase protein microarray. To increase the translational potential of this work and capture druggable vulnerabilities within different clones, we measured expression/activation of a panel of key drug targets and downstream substrates of FDA-approved or investigational agents. We isolated intermixed clones, including poorly represented ones (<5% of cells), within the tumor microecology and identified molecular characteristics uniquely attributable to cancer cells that undergo lineage plasticity and neuroendocrine transdifferentiation in NSCLCs.

ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy.

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.

Dysregulation of SIRT3 SUMOylation Confers AML Chemoresistance via Controlling HES1-Dependent Fatty Acid Oxidation.

Sirtuin 3 (SIRT3) deacetylase is a key regulator for chemoresistance in acute myeloid leukemia (AML) cells due to its capability of modulating mitochondrial metabolism and reactive oxygen species (ROS). SIRT3 is de-SUMOylated by SUMO-specific peptidase 1 (SENP1), which enhances its deacetylase activity. Therefore, dysregulation of SIRT3 SUMOylation may lead to fortified chemoresistance in AML. Indeed, SIRT3 de-SUMOylation was induced by chemotherapeutic agents, which in turn, exacerbated resistance against chemotherapies in AML by activating SIRT3 via preventing its proteasome degradation. Furthermore, RNA-seq revealed that expression of a collection of genes was altered by SIRT3 de-SUMOylation including inhibition of transcription factor Hes Family BHLH Transcription Factor 1 (HES1), a downstream substrate of Notch1 signaling pathway, leading to increased fatty acids oxidation (FAO). Moreover, the SENP1 inhibitor momordin-Ic or HES1 overexpression synergized with cytarabine to eradicate AML cells in vitro and in xenograft mouse models. In summary, the current study revealed a novel role of SIRT3 SUMOylation in the regulation of chemoresistance in AML via HES1-dependent FAO and provided a rationale for SIRT3 SUMOylation and FAO targeted interventions to improve chemotherapies in AML.

Cupid's quiver: Integrating sensory cues in rodent mating systems.

In many animal species, males and females exploit different mating strategies, display sex-typical behaviors, and use distinct systems to recognize ethologically relevant cues. Mate selection thus requires mutual recognition across diverse social interactions based on distinct sensory signals. These sex differences in courtship and mating behaviors correspond to differences in sensory systems and downstream neural substrates engaged to recognize and respond to courtship signals. In many rodents, males tend to rely heavily on volatile olfactory and pheromone cues, while females appear to be guided more by a combination of these chemosensory signals with acoustic cues in the form of ultrasonic vocalizations. The mechanisms by which chemical and acoustic cues are integrated to control behavior are understudied in mating but are known to be important in the control of maternal behaviors. Socially monogamous species constitute a behaviorally distinct group of rodents. In these species, anatomic differences between males and females outside the nervous system are less prominent than in species with non-monogamous mating systems, and both sexes engage in more symmetric social behaviors and form attachments. Nevertheless, despite the apparent similarities in behaviors displayed by monogamous males and females, the circuitry supporting social, mating, and attachment behaviors in these species is increasingly thought to differ between the sexes. Sex differences in sensory modalities most important for mate recognition in across species are of particular interest and present a wealth of questions yet to be answered. Here, we discuss how distinct sensory cues may be integrated to drive social and attachment behaviors in rodents, and the differing roles of specific sensory systems in eliciting displays of behavior by females or males.

Bioinformatics analysis of the role of aldolase A in tumor prognosis and immunity

Aldolase A (ALDOA) is an enzyme that plays an important role in glycolysis and gluconeogenesis, which is closely related to tumor metabolism. In this study, the overall roles of ALDOA in pan-cancer have been investigated from several aspects using databases and online analysis tools. Using the ONCOMINE database, the expression of ALDOA in various cancers was analyzed. The prognostic role of ALDOA was explored by PrognoScan, GEPIA, and Kaplan–Meier Plotter. The immune-related role of ALDOA and its downstream substrates was decided by TIMER, cBioPortal and String. Our data indicate that ALDOA expression level in lung adenocarcinoma, liver hepatocellular carcinoma, head and neck squamous cell carcinoma is higher than that in normal tissues. Increased expression of ALDOA often indicates a poor prognosis for patients. The correlation between ALDOA and immune infiltration among different tumors is very different. We also investigate the relationship between ALDOA and its upstream/downstream proteins. Our results showed that ALDOA could be used as a biomarker for the tumor prognosis, and could be correlated with the infiltrating levels of macrophages, CD4+ T cells and CD8+ T cells.

Regulatory significance of CULLIN2 in neuronal differentiation and regeneration

BackgroundScaffold proteins coordinate multiple signalling pathways by integrating various proteins but the role of these proteins in neuronal pathways remains to be elucidated. The present study focused to evaluate the expression of the scaffold protein CULLIN2 in neuronal cells. MethodsThe neuronal precursor cell line N2A was differentiated to neurons in-vitro with retinoic acid and biochemical assays were used to understand the gene expression profiling of CULLIN2. Moreover, neddylation inhibitor MLN4924 was used to inhibit the activity of CULLIN2 and the downstream substrates were validated. Finally, the role of CULLIN2 in nerve regeneration was evaluated in an in vivo zebrafish model. ResultsExperimental data showed that the neuronal cells N2A have lower expression of CULLIN2 compared to skin cell lines (HaCaT and A431) and inactivation with the neddylation inhibitor resulted in cell death. Furthermore differentiating the neural precursor cell line into neurons with retinoic acid enhanced the expression of CULLIN2. Examining downstream signalling molecules with the neddylation inhibitor illuminates that MLN4924 treatment influences the cytokine signalling cascade (JAK-STAT) in neuronal cells. Moreover, for the first time, we show that the ubiquitin ligase protein CULLIN2 is perturbed in neural regeneration. Expression profile of CULLIN2 was significantly decreased in response to a nerve injury in Zebra fish and as the nerve regenerates there is corresponding reduction in the mRNA levels. ConclusionDuring differentiation CULLIN2 is upregulated whereas during regeneration there is significant downregulation. Thus, our findings reveal a crucial role of the scaffold protein CULLIN2 in nerve differentiation and regeneration which can be vital for the treatment of nerve injury.

The splicing factor RBM17 drives leukemic stem cell maintenance by evading nonsense-mediated decay of pro-leukemic factors

Chemo-resistance in acute myeloid leukemia (AML) patients is driven by leukemic stem cells (LSCs) resulting in high rates of relapse and low overall survival. Here, we demonstrate that upregulation of the splicing factor, RBM17 preferentially marks and sustains LSCs and directly correlates with shorten patient survival. RBM17 knockdown in primary AML cells leads to myeloid differentiation and impaired colony formation and in vivo engraftment. Integrative multi-omics analyses show that RBM17 repression leads to inclusion of poison exons and production of nonsense-mediated decay (NMD)-sensitive transcripts for pro-leukemic factors and the translation initiation factor, EIF4A2. We show that EIF4A2 is enriched in LSCs and its inhibition impairs primary AML progenitor activity. Proteomic analysis of EIF4A2-depleted AML cells shows recapitulation of the RBM17 knockdown biological effects, including pronounced suppression of proteins involved in ribosome biogenesis. Overall, these results provide a rationale to target RBM17 and/or its downstream NMD-sensitive splicing substrates for AML treatment.

Abstract 5410: First, second, and third generation mTOR pathways inhibitors for treatment of glioblastoma

Abstract The Mechanistic Target of Rapamycin (mTOR), which functions in two distinct multiprotein complexes, mTORC1 and mTORC2, is a downstream signaling kinase in the PI3K/AKT pathway. mTOR is often deregulated in glioblastoma (GB), the most aggressive primary brain tumor in adults, due to the frequent loss of the tumor suppressor, PTEN. mTORC1 and mTORC2 control cell growth, proliferation, migration, survival, and stem cell regulation in response to nutrients and growth factors. Nutrient-sensitive mTORC1 regulates protein synthesis through the downstream substrate, 4EBP1 (also called EIF4EBP1), p70S6K1, and p70S6K2. On the other hand, growth factor-sensitive mTORC2 is associated with Rapamycin-insensitivity. The presence of stem cells within the tumor mass and the surrounding areas precludes the possibility of the elimination of GB. Furthermore, the substrate of activated AKT, the proline-rich AKT substrate of 40 kDa (PRAS40), is a surrogate marker of activated AKT in response to mTOR inhibition therapy in GB patients that regulates mTORC1. The aim of this study is to evaluate the efficacy of first (Rapamycin (RAPA)), second (Torin1, Torin2, and XL388), and third (Rapalink-1) mTOR inhibitors to suppress cell growth, dissemination, overcome drug resistance of GB cells, and alter self-renewal properties of GB stem cells. Results demonstrated: a significant number of tumors showed an increased expression of pAKTSer473 and pmTORSer2448, suggesting an overactivation of the mTOR pathway; co-expression of mTOR and stem cell marker, nestin, was evident in GB tumors, implying its role in stem cell regulation; Torin2, but not Torin1 or XL388, suppressed mTORC1 activity completely as shown by the reduced expression of its pS6KSer235/236 in a dose-dependent manner; Torin1, Torin2, and XL388 inhibited the phosphorylation of 4E-BP1 and PRAS40; Torin1, Torin2, and XL388 suppressed dephosphorylation of pAKTSer473; functional analysis revealed that RAPA, Torin1, Torin2, XL388, and Rapalink-1 effectively suppressed proliferation and S-phase entry; RAPA, Torin1, Torin2 and Rapalink-1 inhibited GB cell migration, while XL388 was ineffective; RAPA, Torin1, Torin2, and Rapalink-1 suppressed the self-renewal of GB stem cells and inhibited their proliferation, XL388 remained ineffective; drug resistance analysis revealed that Torin2 was most effective in eradicating GB tumor cells. These results suggested that RAPA, Torin1, Torin2, and Rapalink-1, but not XL388, are useful in suppressing the mTORC1 and mTORC2 activities, thereby inhibiting GB cell proliferation, dissemination, and inhibiting stem cell self-renewal. Therefore, the use of these mTOR inhibitors presents a promising treatment strategy for Glioblastoma. Citation Format: Jose F. Dominguez, Haylen Rosberger, Paul Garell, Chirag D. Gandhi, Meena Jhanwar-Uniyal. First, second, and third generation mTOR pathways inhibitors for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5410.

Abstract 6188: Targeted inhibition of the E3 ligase SCFSkp2/Cks1 has survival benefit and antitumor activity in osteosarcoma

Abstract Background: Osteosarcoma (OS) is a highly aggressive bone malignancy for which treatment has remained unchanged for years. Our previous studies indicate that the F-box protein SKP2 is overexpressed in OS and acts as a proto-oncogene. We recently reported that abolishing the interaction between SKP2 and its downstream substrate p27 using a Thr187Ala knock-in mutation leads to impaired tumor growth in an RB1/TP53 deficient transgenic OS mouse model. These data suggest that inhibiting the function of SKP2 may yield therapeutic benefits in OS. However, the precise mechanisms by which SKP2 promotes tumorigenesis in OS are not fully understood. Purposes: To determine the role of SCFSkp2/Cks1 in OS tumorigenesis and as a potential therapeutic target. Methods: We generated a genetically engineered mouse model of OS with a double knockout of Rb1 and p53 only within cells of the osteoblastic lineage (Osx1-Cre;Rb1lox/lox;Trp53lox/lox, DKO). The SKP2-knockout OS mouse model was further developed in the DKO background (Osx1-Cre;Rb1lox/lox;Trp53lox/lox; Skp2-/-, TKO). Mice of both genotypes were monitored for tumor growth, overall survival, and in vivo drug response. Early passaged OS cells and OS organoid culture was derived from mice tumors and used for in vitro analysis. Western Blot and IHC were used for protein level determination. RT-qPCR and RNA-seq were used for transcriptional level determination. Two small-molecule SCFSkp2/Cks1 inhibitors (C1 and Pevonedistat) were used for in vivo and in vitro testing. Results: All genotypes were born at the expected ratios and developmentally normal. DKO animals developed OS tumors with nearly full penetrance. SKP2 deletion significantly prolongs the overall survival (p&amp;lt;0.0001) and reduced the average tumor occurrence in OS mice (p&amp;lt;0.001). The in vivo tumor proliferation was also significantly delayed in the SKP2 deletion OS tumorigenesis (p=0.007). A histological analysis comparing tumors from two groups showed a decrease in proliferation (PCNA) and an increase in pro-apoptotic markers (TUNEL,etc) expression in SKP2-deleted OS. RT-qPCR and RNA-seq revealed upregulation of E2F1 downstream apoptotic-related target genes in Skp2-/- OS tumors. The transcriptional level analysis also revealed inhibition of cancer stemness markers and pathways in SKP2 deleted tumors and further proved by functional analyses. Two SCFSkp2/Cks1 inhibitors not only showed anti-tumoral activities both in vivo (p&amp;lt;0.05) and in organoid culture, but also synergistic with chemotherapy agents (ZIP synergy Score:9.34±1.24). Conclusions: Using genetic and pharmacologic approaches, we show that inhibiting SKP2 leads to significant antitumor activities in a clinically relevant OS mouse model. The strong survival benefit highlights SKP2 as a downstream actionable target in TP53/RB1-deficient OS, for which the targeted therapies have been disappointing to date. Citation Format: Jichuan Wang, Alexander Ferrena, Ranxin Zhang, Swapnil Singh, Hongling Zhao, Deyou Zheng, Hasibagan Borjihan, Waleed Al-Harden, Janet Tingling, Rui Yang, David S. Geller, Bang H. Hoang. Targeted inhibition of the E3 ligase SCFSkp2/Cks1 has survival benefit and antitumor activity in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6188.

Abstract 2662: Bi-steric mTORC1 inhibitor RMC-6272 synergizes with immune checkpoint inhibitors to induce sustained regression of MYC-driven hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths in the world. Current treatments for HCC have limited efficacy. First-line HCC therapy atezolizumab with bevacizumab increased patient 6-month PFS from 37% to 55% relative to sorafenib, a multi-kinase inhibitor currently serving as a standard-of-care treatment. Although immunotherapies provide survival benefits, reported patient response and disease control rates were 27% and 74%, respectively, highlighting the need for novel therapeutic agents that can re-establish responsiveness to immunotherapies and enhance their therapeutic effects. Revolution Medicines has developed a class of selective mTORC1 inhibitors, termed ‘bi-steric’, which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-6272 is a representative bi-steric inhibitor that exhibits potent and selective (&amp;gt;30-fold) inhibition of mTORC1 over mTORC2. Several lines of evidence suggest that inhibition of the mTORC1 pathway may result in synthetic lethality of MYC-driven HCC. Thus, we hypothesized that MYC-driven HCC tumors are sensitive to mTORC1 inhibition and p-4EBP1 suppression. A single dose of RMC-6272 induced HCC regression in 80% of the Lap-tTA/Tet-O-MYC mouse model of MYC-driven HCC, whereas sorafenib did not cause tumor regression. Mechanistically, RMC-6272 inhibited the phosphorylation of mTORC1 downstream substrates S6k and 4EBP1 and depleted MYC protein levels. Given that the MYC oncogene enables immune evasion in HCC, we hypothesized that by reducing MYC, RMC-6272 can re-establish anti-tumor immune surveillance and responsiveness to α-PD-1 immunotherapy. Indeed, RMC-6272 induced tumor recruitment of dendritic cells, T cells, B cells, and natural killer (NK) cells, producing similar anti-tumor immunity as observed upon MYC genetic inactivation. Moreover, combining RMC-6272 with α-PD-1 resulted in deeper tumor regression and a more durable response as compared to either RMC-6272 or α-PD-1 alone in the MYC-driven HCC mouse model. By immunohistochemical staining and analysis, RMC-6272 in combination with α-PD-1, but not either agent alone, promoted tumor recruitment of CD4+ T cells and NK cells, immune cell degranulation, and the release of perforins and granzymes in MYC-driven HCC tumors. Our results show that the bi-steric mTORC1 inhibitor RMC-6272 synergizes with α-PD-1 to induce immune activation and the release of perforins and granzymes, resulting in sustained tumor regression in a MYC-driven HCC mouse model. The bi-steric mTORC1 inhibitor RMC-5552 is the first clinical candidate of this class and clinical testing is ongoing (NCT04774952). These preclinical data highlight a prospective anti-cancer therapeutic opportunity for mTORC1 selective inhibitors in combination with immune checkpoint inhibitors in MYC-driven cancers. Citation Format: Wadie D. Mahauad-Fernandez, Yu C. Yang, Ian Lai, Jangho Park, Lilian Yao, James W. Evans, Jacqueline A. Smith, Mallika Singh, Dean W. Felsher. Bi-steric mTORC1 inhibitor RMC-6272 synergizes with immune checkpoint inhibitors to induce sustained regression of MYC-driven hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2662.

Abstract LB032: CXCR7 drives prostate cancer growth through AURKA activation

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer among males in the United States. The mainstay treatment for metastatic PCa patients is androgen-deprivation therapies, including high-affinity androgen receptor antagonists, such as enzalutamide. Unfortunately, most patients develop resistance and relapse with castration-resistant PCa (CRPC). We have previously reported up-regulation of CXCR7, an atypical chemokine receptor, in CRPC. CXCR7 interacts with beta-arrestin (ARRB2), a cytoplasmic scaffold protein, and translocates to the cytoplasm to induce signal transduction and PCa progression. However, much remains to be known regarding CXCR7-driven downstream signaling and its therapeutic targeting in advanced PCa. To unveil CXCR7-downstream signaling, we utilized RNA-seq to profile gene expression. Gene ontology analyses revealed remarkable enrichment of CXCR7-regulated genes in G2/M cell cycle progression. Comprehensive phospho-proteomics analysis of CXCR7-knockdown PCa cells showed markedly reduced phosphorylation of AURKA substrates. AURKA, a serine/threonine-protein kinase, is a key regulator of mitosis and G2/M progression and is frequently upregulated in CRPC and the late-stage neuroendocrine PCa (NEPC). Activated AURKA experiences autophosphorylation and phosphorylates downstream substrates, e.g., TACC3, that mediate proper cell division. Immunoblotting confirmed that CXCR7 depletion reduces phosphorylation of AURKA and TACC3. Co-immunoprecipitation revealed protein-protein interaction between CXCR7 and AURKA, which is mediated by ARRB2. Accordingly, ARRB2-knockdown similarly inhibits AURKA phosphorylation and arrests cell proliferation. Immunofluorescence coupled with confocal imaging demonstrated that CXCR7 is packed into clathrin-coated vesicles and locates in the plasma membrane, cytoplasm, and perinuclear Golgi complex, which surrounds the centrosomal AURKA and ARRB2. We found that CXCR7 translocates to the Golgi complex along microtubule-dependent trafficking. Finally, we found that pharmacological inhibitors of AURKA abolish CXCR7-driven PCa cell proliferation in vitro and tumor growth in vivo. Conclusions: Our results describe for the first time that the CXCR7-ARRB2 complex interacts with AURKA and promotes its activation. Our data suggest that AURKA-targeting is a promising therapeutic strategy for CRPC and NEPC with CXCR7 up-regulation. Citation Format: Galina Gritsina, Zhuoyuan Lin, Ka Wing Fong, Xiaodong Lu, Changsheng Zhao, Jindan Yu. CXCR7 drives prostate cancer growth through AURKA activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB032.

Time-resolved phosphoproteome and proteome analysis reveals kinase signaling on master transcription factors during myogenesis

SummaryMyogenesis is governed by signaling networks that are tightly regulated in a time-dependent manner. Although different protein kinases have been identified, knowledge of the global signaling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict the substrates that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signaling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles in myogenesis. Lastly, we identified and validated the transcription factor nuclear factor 1 X-type (NFIX) as a novel MAPK1/3 substrate and demonstrated the functional impact of NFIX phosphorylation on myogenesis. Altogether, these data characterize the dynamics, interactions, and downstream control of kinase signaling networks during myogenesis on a global scale.

SIRT6 mediates multidimensional modulation to maintain organism homeostasis.

As a member of the silent information regulators (sirtuins) family, SIRT6 can regulate a variety of biological processes, including DNA repair, glucose and lipid metabolism, oxidative stress and lifespan, and so forth. SIRT6 maintains organism homeostasis in a variety of phenotypes by mediating epigenetic regulation and posttranslational modification of functional proteins. In this review, we outline the structural basis of SIRT6 enzyme activity and its mechanism of maintaining organism homeostasis in a variety of phenotypes, with an emphasis on the upstream that regulates SIRT6 expression and the downstream substrates. And how SIRT6 achieves multidimensional coordination to maintain organism homeostasis and even extend lifespan. We try to understand the regulatory mechanism of SIRT6 in different phenotypes from the perspective of protein interaction.

Cdk5-mediated JIP1 phosphorylation regulates axonal outgrowth through Notch1 inhibition

BackgroundActivated Cdk5 regulates a number of processes during nervous system formation, including neuronal differentiation, growth cone stabilization, and axonal growth. Cdk5 phosphorylates its downstream substrates located in axonal growth cones, where the highly expressed c-Jun N-terminal kinase (JNK)-interacting protein1 (JIP1) has been implicated as another important regulator of axonal growth. In addition, stringent control of the level of intracellular domain of Notch1 (Notch1-IC) plays a regulatory role in axonal outgrowth during neuronal differentiation. However, whether Cdk5-JIP1-Notch1 cooperate to regulate axonal outgrowth, and the mechanism of such joint contribution to this pathway, is presently unknown, and here we explore their potential interaction.ResultsOur interactome screen identified JIP1 as an interactor of p35, a Cdk5 activator, and we sought to explore the relationship between Cdk5 and JIP1 on the regulation of axonal outgrowth. We demonstrate that JIP1 phosphorylated by Cdk5 at Thr205 enhances axonal outgrowth and a phosphomimic JIP1 rescues the axonal outgrowth defects in JIP1−/− and p35−/− neurons. Axonal outgrowth defects caused by the specific increase of Notch1 in JIP1−/− neurons are rescued by Numb-mediated inhibition of Notch1. Finally, we demonstrate that Cdk5 phosphorylation of JIP1 further amplifies the phosphorylation status of yet another Cdk5 substrate E3-ubiquitin ligase Itch, resulting in increased Notch1 ubiquitination.ConclusionsOur findings identify a potentially critical signaling axis involving Cdk5-JIP1-Itch-Notch1, which plays an important role in the regulation of CNS development. Future investigation into the way this pathway integrates with additional pathways regulating axonal growth will further our knowledge of normal central nervous system development and pathological conditions.