Dominant-negative Mutant Research Articles

Abstract B009: TEAD inhibition overcomes YAP1/TAZ-driven resistance to RAS inhibitors in KRASG12C-mutant cancers

Abstract Activated mutants of KRAS comprise the major oncogenic drivers in lung (LAC), colorectal (CRC), and pancreatic ductal (PDAC) adenocarcinoma. Recent success in covalently targeting one KRAS mutant (KRASG12C) led to FDA approval of the first anti-KRAS therapy (G12Ci). However, both primary and treatment-induced acquired resistance to G12Ci have been observed. While analyses of relapsed patients have identified reactivation of the key KRAS effector signaling network as a driver of resistance, the mechanisms in ~50% of patients are not known. To identify potential resistance mechanisms, we applied a CRISPR-Cas9 loss-of-function screen targeting the druggable genome. In addition to genes discovered recently in relapsed patients (e.g., PTEN, NF1), we also identified loss of multiple components of the Hippo tumor suppressor pathway as drivers of G12Ci resistance (NF2, LATS1/2, TAOK1/2 and STK3/4). We therefore determined if activation of the functionally related transcriptional co-activators, YAP1 and TAZ, normally inhibited by Hippo signaling, can drive resistance to G12Ci. We first determined that ectopic expression of constitutively activated YAP1/TAZ was sufficient to impair the anti-proliferative and pro-apoptotic effects of G12Ci treatment in KRASG12C-mutant LAC, CRC, and PDAC cell lines. Conversely, genetic suppression of YAP1/TAZ enhanced G12Ci sensitivity. YAP1/TAZ requires association with TEAD and other transcription factors to regulate transcription. We determined that YAP1/TAZ mutants deficient in TEAD binding failed to drive resistance to G12Ci treatment. Further supporting a role for TEAD, both overexpression of a TEAD dominant negative mutant and treatment with pan-TEAD pharmacological inhibitors phenocopied the effects of YAP1/TAZ genetic suppression and sensitized KRASG12C mutant cancer cells to G12Ci. Finally, transcriptional analyses support a model where YAP1/TAZ-TEAD overcomes KRASG12C addiction by restoring a subset of KRAS-dependent gene transcription. In summary, our observations support YAP1/TAZ-TEAD signaling as a novel driver of resistance to KRAS inhibition and support the use of TEAD inhibitors to enhance the anti-tumor efficacy of KRAS-targeted therapies. Citation Format: Alexander C. Edwards, Clint A. Stalnecker, Alexis J. Morales, Khalilah E. Taylor, Jill Hallin, Peter Olson, Tracy T. Tang, Lars Engstrom, Leonard Post, James G. Christensen, Adrienne D. Cox, Channing J. Der. TEAD inhibition overcomes YAP1/TAZ-driven resistance to RAS inhibitors in KRASG12C-mutant cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B009.

Rab4A controls the overexpression of CD38 and depletion of IL-2 in CD4+ T cells: Potential involvement in proinflammatory lineage development in systemic lupus erythematosus

Abstract Rab4A is a GTPase that is overexpressed in systemic lupus erythematosus (SLE) patients’ T cells and enhances cell surface receptor recycling. Increased expression of CD38 and loss of IL-2 production have independently been associated with proinflammatory T cell development in SLE. We investigated the impact of Rab4A on the expression of CD38 and the production of IL-2. Our lab created Rab4A-mutant Jurkat cell lines, containing GFP-expressing vector alone (control), doxycycline-inducible vectors that overexpress Rab4A (Rab4A hi) or the dominant-negative mutant Rab4A S27N(Rab4A DN). They were co-stimulated with anti-CD3 antibody and PMA. After 24 hours, cell surface markers and cytokines were analyzed by flow cytometry and protein levels by western blot. NAD +levels were measured by LC-MS/MS. In the Rab4A hicells compared to the control, CD38 expression was 2-fold upregulated (p=2.49×10 −13), intracellular production and secretion of IL-2 were significantly decreased (fold change=−0.566, p=1.16×10 −7and fold change= −0.481, p=0.0401, respectively), NAD +concentration was decreased (fold change=0.647, p=0.0039), while pSTAT3 levels were 2-fold increased (p=0.0318). CD38 is an NAD +hydrolase, which regulates Sirtuin-1 activity, a NAD +-dependent histone deacetylase that suppresses STAT3 activity. Elevated pSTAT3 levels may underlie diminished IL-2 production by binding to the promoter of FoxO1, which inhibits IL-2 production. The overexpression of Rab4A and CD38, increased pSTAT3, and diminished IL-2 production reflect changes observed in SLE patients. Our results suggest that increased expression of Rab4A may underlie the overexpression of CD38 and diminished secretion of IL-2 in SLE.

The RNA export factor TbMex67 connects transcription and RNA export in Trypanosoma brucei and sets boundaries for RNA polymerase I.

TbMex67 is the major mRNA export factor known to date in trypanosomes, forming part of the docking platform within the nuclear pore. To explore its role in co-transcriptional mRNA export, recently reported in Trypanosoma brucei, pulse labelling of nascent RNAs with 5-ethynyl uridine (5-EU) was performed with cells depleted of TbMex67 and complemented with a dominant-negative mutant (TbMex67-DN). RNA polymerase (Pol) II transcription was unaffected, but the procyclin loci, which encode mRNAs transcribed by Pol I from internal sites on chromosomes 6 and 10, showed increased levels of 5-EU incorporation. This was due to Pol I readthrough transcription, which proceeded beyond the procyclin and procyclin-associated genes up to the Pol II transcription start site on the opposite strand. Complementation by TbMex67-DN also increased Pol I-dependent formation of R-loops and γ-histone 2A foci. The DN mutant exhibited reduced nuclear localisation and binding to chromatin compared to wild-type TbMex67. Together with its interaction with chromatin remodelling factor TbRRM1 and Pol II, and transcription-dependent association of Pol II with nucleoporins, our findings support a role for TbMex67 in connecting transcription and export in T. brucei. In addition, TbMex67 stalls readthrough by Pol I in specific contexts, thereby limiting R-loop formation and replication stress.

Abstract LB034: EWSR1, Ewing sarcoma breakpoint region 1, maintains centromere identity by binding to centromeric R-loops

Abstract The centromere DNA-kinetochore complex is the specialized chromatin structure that mediates chromosome attachment to microtubules and is required for high-fidelity chromosome transmission. CENP-A is the centromere-specific histone H3 variant that epigenetically determines centromere position on the chromosome to ensure kinetochore assembly. Despite its importance, the precise mechanism of maintenance of CENP-A, i.e., the maintenance of centromere identity, remains obscure. In this study, we found that EWSR1 (Ewing Sarcoma Breakpoint Region 1) is required for CENP-A deposition at centromeres by binding to centromeric RNA. We determined that the CENP-A binding domain of EWSR1 is the SYGQ2 region within the prion-like domain, which plays an important role in phase separation for chromatin remodeling. Our data suggest that EWSR1 guards CENP-A in centromeric chromatins by binding to centromeric RNA. This is a novel mechanism to maintain the position of the centromere on the chromosome i.e., centromere identify. As EWSR1-FLI1 is the oncogenic fusion protein in Ewing sarcoma and as it inhibits EWSR1 function as a dominant negative mutant, it contributes to chromosome instability in Ewing sarcoma, which may lead to therapy resistance and metastasis. Citation Format: Risa Kitagawa, Argentina Becker, Peter Houghton, Katsumi Kitagawa. EWSR1, Ewing sarcoma breakpoint region 1, maintains centromere identity by binding to centromeric R-loops [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB034.

Homogenous TP53mut-associated tumor biology across mutation and cancer types revealed by transcriptome analysis

TP53 is the most frequently mutated gene in human cancer. While no TP53-targeting drugs have been approved in the USA or Europe so far, preclinical and clinical studies are underway to investigate targeting of specific or all TP53 mutations, for example, by restoration of the functionality of mutated TP53 (TP53mut) or protecting wildtype TP53 (TP53wt) from negative regulation. We performed a comprehensive mRNA expression analysis in 24 cancer types of TCGA to extract (i) a consensus expression signature shared across TP53 mutation types and cancer types, (ii) differential gene expression patterns between tumors harboring different TP53 mutation types such as loss of function, gain of function or dominant-negative mutations, and (iii) cancer-type-specific patterns of gene expression and immune infiltration. Analysis of mutational hotspots revealed both similarities across cancer types and cancer type-specific hotspots. Underlying ubiquitous and cancer type-specific mutational processes with the associated mutational signatures contributed to explaining this observation. Virtually no genes were differentially expressed between tumors harboring different TP53 mutation types, while hundreds of genes were over- and underexpressed in TP53mut compared to TP53wt tumors. A consensus list included 178 genes that were overexpressed and 32 genes that were underexpressed in the TP53mut tumors of at least 16 of the investigated 24 cancer types. In an association analysis of immune infiltration with TP53 mutations in 32 cancer subtypes, decreased immune infiltration was observed in six subtypes, increased infiltration in two subtypes, a mixed pattern of decreased and increased immune cell populations in four subtypes, while immune infiltration was not associated with TP53 status in 20 subtypes. The analysis of a large cohort of human tumors complements results from experimental studies and supports the view that TP53 mutations should be further evaluated as predictive markers for immunotherapy and targeted therapies.

Lysophosphatidic acid receptor LPA1 trafficking and interaction with Rab proteins, as evidenced by Förster resonance energy transfer

LPA1 internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA1 receptors and distinct eGFP-tagged Rab proteins. Lysophosphatidic acid (LPA)-induced internalization was rapid and decreased afterward: phorbol myristate acetate (PMA) action was slower and sustained. LPA stimulated LPA1-Rab5 interaction rapidly but transiently, whereas PMA action was rapid but sustained. Expression of a Rab5 dominant-negative mutant blocked LPA1-Rab5 interaction and receptor internalization. LPA-induced LPA1-Rab9 interaction was only observed at 60 min, and LPA1-Rab7 interaction after 5 min with LPA and after 60 min with PMA. LPA triggered immediate but transient rapid recycling (i.e., LPA1-Rab4 interaction), whereas PMA action was slower but sustained. Agonist-induced slow recycling (LPA1-Rab11 interaction) increased at 15 min and remained at this level, whereas PMA action showed early and late peaks. Our results indicate that LPA1 receptor internalization varies with the stimuli.

Osteoblastic RAR Inhibition Causes VAD-Like Craniofacial Skeletal Deformity

Retinoid signaling disorders cause craniofacial deformity, among which infants with maternal vitamin A deficiency (VAD) exhibited malformation of the eye, nose, palate, and parietal and jaw bone. Previous research uncovered the pathogenesis of eye defect and cleft palate of VAD in mice, but the studies on craniofacial skeletal deformity met obstacles, and the cell/lineage and underlying mechanism remain unclear. The retinoic acid receptor (RAR) is the key transcription factor in retinoid signaling, but individual knockout cannot simulate pathway inhibition. Here, we conditionally expressed dominant-negative RARα mutation (dnRARα) in osteoblasts to specifically inhibit the transcription activity of RAR in mice, which mimics the craniofacial deformities caused by VAD in clinical cases: hypomineralization of cranial bones, mandibular deformity, and clavicular hypoplasia. Furthermore, we performed 3-dimensional reconstruction based on micro–computed tomography and confirmed the abnormalities in the shape, size, and ossification of craniofacial bones due to osteoblastic RAR inhibition. Histological analysis indicated that inhibition of RAR in osteoblasts impaired both bone formation and bone resorption, which was confirmed by transcriptome sequencing of the calvaria. Furthermore, mechanism investigation showed that inhibition of RAR in osteoblasts directly decreased osteoblast differentiation in a cell-autonomous manner by impairing osteogenic gene transcription and also inhibited osteoclast differentiation via osteoblast–osteoclast crosstalk by impairing Rankl transcription. In summary, osteoblastic RAR activity is critical to craniofacial skeletal development, and its dysfunction leads to skeletal deformities mimicking VAD craniofacial defects, providing a new insight for VAD pathogenesis.

Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells

Iron overload is associated with various pathological changes which contribute to metabolic syndrome, many of which have been proposed to occur via damaging tissue through an excessive amount of reactive oxygen species (ROS) production. In this study, we established a model of iron overload in L6 skeletal muscle cells and observed that iron enhanced cytochrome c release from depolarized mitochondria, assayed by immunofluorescent colocalization of cytochrome c with Tom20 and the use of JC-1, respectively. This subsequently elevated apoptosis, determined via use of a caspase-3/7 activatable fluorescent probe and western blotting for cleaved caspase-3. Using CellROX deep red and mBBr, we observed that iron increased generation of reactive oxygen species (ROS), and that pretreatment with the superoxide dismutase mimetic MnTBAP reduced ROS production and attenuated iron-induced intrinsic apoptosis and cell death. Furthermore, using MitoSox Red we observed that iron enhanced mROS and the mitochondria-targeted anti-oxidant SKQ1 reduced iron-induced ROS generation and cell death. Western blotting for LC3-II and P62 levels as well as immunofluorescent detection of autophagy flux with LC3B and P62 co-localization indicated that iron acutely (2–8 h) activated and later (12–24 h) attenuated autophagic flux. We used autophagy-deficient cell models generated by overexpressing a dominant-negative Atg5 mutant or CRISPR-mediated ATG7 knock out to test the functional significance of autophagy and observed that autophagy-deficiency exacerbated iron-induced ROS production and apoptosis. In conclusion, our study showed that high iron levels promoted ROS production, blunted the self-protective autophagy response and led to cell death in L6 skeletal muscle cells.

CARD11 dominant negative mutation leads to altered human Natural Killer cell homeostasis

Dominant negative mutations in CARD11 have been reported in patients with immune dysregulation, severe atopic features, and variable T cell alterations. Data on Natural killer (NK) cells from affected patients are lacking. We report on a 12-year-old boy with severe atopic dermatitis, food induced anaphylaxis and hypogammaglobulinemia harbouring a novel de novo heterozygous variant c.169G > A; p.Glu57Lys in CARD11. The dominant negative effect of this mutation was confirmed on both CD4+ and CD8+. CTLA4+Foxp3+CD4+ Tregs were severely reduced. Patient’s NK cells showed reduced expression of NKp46, NKG2D and CD69. Patient’s CD56bright NK cells showed in vitro impaired production of IFN-γ. Steady state pS6 levels on patient’s NK cells were increased and remained elevated upon IL2 + IL12 + IL18 overnight stimulation. Overall, the effect of CARD11 mutation on mTORC1 differs between T and NK cells. These findings may explain the increased susceptibility to viral infections and the reduced immune surveillance in affected patients.

The AtERF19 gene regulates meristem activity and flower organ size in plants.

Ethylene-responsive factors (ERFs) have diverse functions in the regulation of various plant developmental processes. Here, we demonstrate the dual role of an Arabidopsis ERF gene, AtERF19, in regulating reproductive meristem activity and flower organ size through the regulation of genes involved in CLAVATA-WUSCHEL (CLV-WUS) and auxin signaling, respectively. We found that AtERF19 stimulated the formation of flower primordia and controlled the number of flowers produced by activating WUS and was negatively regulated by CLV3. 35S::AtERF19 expression resulted in significantly more flowers, whereas 35S::AtERF19 + SRDX dominant-negative mutants produced fewer flowers. In addition, AtERF19 also functioned to control flower organ size by promoting the division/expansion of the cells through activating Small Auxin Up RNA Gene 32 (SAUR32), which positively regulated MYB21/24 in the auxin signaling pathway. 35S::AtERF19 and 35S::SAUR32 resulted in similarly larger flowers, whereas 35S::AtERF19 + SRDX and 35S::SAUR32-RNAi mutants produced smaller flowers than the wild type. The functions of AtERF19 were confirmed by the production of similarly more and larger flowers in 35S::AtERF19 transgenic tobacco (Nicotiana benthamiana) and in transgenic Arabidopsis which ectopically expressed the orchid gene (Nicotiana benthamiana) PaERF19 than in wild-type plants. The finding that AtERF19 regulates genes involved in both CLV-WUS and auxin signaling during flower development significantly expands the current knowledge of the multifunctional evolution of ERF genes in plants. The results presented in this work indicate a dual role for the transcription factor AtERF19 in controlling the number of flowers produced and flower organ size through the regulation of genes involved in CLV-WUS and auxin signaling, respectively. Our findings expand the knowledge of the roles of ERF genes in the regulation of reproductive development.

Abstract 2607: Homogenous TP53mut-associated tumor biology across mutation and cancer types revealed by comprehensive mRNA expression analysis

Abstract TP53 mutations are the most common single gene alteration in human cancer with diagnostic and prognostic implications in some cancer types. While no TP53-targeting therapeutics have been approved in the USA or Europe yet, drugs tailored to specific TP53 mutations, restoring the functionality of mutated TP53 (TP53mut), and protecting TP53 from negative regulation are being explored in preclinical studies and clinical trials. We performed a comprehensive mRNA expression analysis in 24 cancer types of the TCGA to extract (i) a consensus expression signature shared across TP53 mutation types and cancer types, (ii) differential gene expression patterns between different TP53 mutation types such as LOF, GOF, as well as dominant-negative mutations, and (iii) cancer types specific gene expression patterns. Mutational hotspots showed a similar pattern across cancer types, but at the same time prevalence was significantly different between cancer types for about half of the most prevalent hotspots. Both can be explained in part by the mutational processes operational for example the clock-like process behind SBS1 that is operational across cancer types and aflatoxin exposure assocated with SBS24 that ist operational in liver hepatocellular carcinoma. Virtually no genes were differential between tumors harboring different types of TP53 mutations in none of the cancer types, while hundreds of genes were over- and underexpressed in TP53mut compared to TP53wt tumors. A consensus gene list of 178 common over- and 32 common underexpressed genes was shared between at least two-thirds of the 24 cancer types. Analysis of the immune tumor microenvironment revealed exclusively decreased immune cell populations in the TP53mut tumors in six, a mixed pattern in four, exclusively increased immune cell populations in two, and no significant alterations in 20 cancer subtypes. The analysis of a large cohort of human tumors complements results from experimental studies and support the development of novel strategies for therapeutic targeting of TP53mut. Citation Format: Jan Budczies, Eva Romanovsky, Klaus Kluck, Iordanis Ourailidis, Michael Menzel, Susanne Beck, Markus Ball, Daniel Kazdal, Petros Christopoulos, Peter Schirmacher, Thorsten Stiewe, Albrecht Stenzinger. Homogenous TP53mut-associated tumor biology across mutation and cancer types revealed by comprehensive mRNA expression analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2607.

Abstract 4780: Comparison of safety and efficacy outcomes with nonthermal plasma and tirapazamine in porcine skin and mouse melanoma models

Abstract A novel preclinical experimental therapy for various cancer types including melanoma is the combination of nonthermal plasma (NTP) and tirapazamine (TPZ) delivered in situ. This patented combination therapy delivers reactive oxygen species (ROS) and reactive nitrogen species (RNS) from the atmosphere that are activated and ionized with helium gas to create the state of matter known as plasma. The combination with the prodrug, TPZ, that only activates to become a ROS in hypoxic environments, shows synergistic targeting of cancer cells. In this study, we assayed the effects of the dual therapy on porcine skin, which is the closest model to represent human skin and is commonly used for testing drug therapy. The results demonstrated that porcine skin showed no significant cell killing with the therapy. We also compared the combination therapy in normal skin keratinocytes and 1205Lu cells derived from a melanoma metastasis to the lung. The cells were treated with NTP and TPZ and maintained in a hypoxic chamber. Four days following therapy the keratinocytes continued to grow in culture, whereas the melanoma cells expressing endogenous gap junctions, or overexpressed Cx43 gap junctions showed decreased viability. However, the melanoma cells with a dominant negative Cx43 mutant did not show this loss in viability. This confirms our previous report that gap junctions mediate the passage of derivatives of ROS to increase the extent of cell death. To determine the effects of NTP on regulating the lethal dose of TPZ, we performed IC50 curves for TPZ in the presence or absence of NTP in B16-F10 murine metastatic melanoma cells. We demonstrated a decrease in the IC50 from 28 μM without NTP to 17.4 μM with NTP. In further analysis, the effects of treating with NTP in the presence or absence of tissue culture media were compared. The results showed a highly significant increased efficacy of NTP in the absence of media. The combination therapy was more effective than each independent treatment. To further confirm the experimental analysis, we induced tumor formation with the B16-F10 cells in a syngeneic C57BL/6 mouse model. The tumors from mice that were untreated or treated with NTP or TPZ alone, showed a significant increase in tumor volume as compared to the tumors from mice receiving the combination treatment in situ. In comparison of the tumor growth rates, the control, NTP-, or TPZ-treated tumors had increased by approximately 350% by day 10, whereas the tumors in the mice that received the dual therapy only increased by an average of 70%. The results from these studies collectively implicate the combination of NTP and TPZ as a potential synergistic treatment for in situ melanomas that offer selectivity as compared to normal tissue. Citation Format: Matthew Yehl, Dominik Kucharski, Michelle Eubank, Xavier Ramadan, Ahmad Dianat, Bianca Pham, Timothy C. Hutcherson, Sandra Sexton, Shoshanna N. Zucker. Comparison of safety and efficacy outcomes with nonthermal plasma and tirapazamine in porcine skin and mouse melanoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4780.

Abstract 1370: Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer

Abstract Rab27B is a member of the Rab small GTPases that belong to the RAS-like GTPase superfamily. Rab27B has been shown to regulate endosomal trafficking, exosome secretion, and delivery of secretory granules. However, the role of Rab27B in colorectal cancer (CRC) tumor biology is unknown. Using CRC tissue microarrays, a significant upregulation of Rab27B was detected in staged adenocarcinoma compared to normal colon tissue. Interestingly, microsatellite unstable (MSI) tumors showed a higher increase in Rab27B (2.4-fold) than microsatellite stable (MSS) tumors (1.6-fold), consistent with TCGA data. To study the role of Rab27B in CRC, we targeted Rab27B in MSI HCT116 cell line by CRISPR-Cas9 deletion and siRNA knockdown. In both cases, loss of Rab27B resulted in a dysregulation of cellular autophagy and reduction in extracellular vesicle secretion. Rab27B-depleted cells showed an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62, indicating a defect in autophagy flux. To characterize this autophagy defect, we used eGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining to identify that autophagy flux is blocked at the autophagosome/lysosome fusion step when Rab27B is lost. To test whether the GTPase activity of Rab27B is needed for the autophagy role, we added back constitutively active and dominant negative GTPase mutants of Rab27B in Rab27B KO cells and observed autophagy phenotype rescue with only wildtype and constitutively active GTPase forms. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that this defect in autophagy flux resulting from Rab27B deletion will impact cellular stress response and reduce tumor growth. While Rab27B knockout (KO) cells show similar in vitro 2D-growth characteristics as wildtype cells under normal conditions, loss of Rab27B resulted in a 60% reduction in cell viability in response to starvation conditions. Similarly, a 94% reduction in colony formation was observed when grown in soft agar, along with deficiencies in spheroid formation. In vivo, Rab27B KO cells showed a major reduction (p<0.0001) in tumor growth xenografts compared to wildtype HCT116 cells. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on further characterizing the relationship among Rab27B overexpression, microsatellite instability, and autophagy in vitro and in vivo using Rab27B knockout mouse model, as well as investigating whether Rab27B can be targeted as a potential new therapeutic strategy. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan A. Dixon. Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1370.

Abstract 5273: The E3 ubiquitin ligase SIAH2 promotes clear cell renal cell carcinoma epithelial-mesenchymal transition via regulation of SETD2 stability

Abstract Introduction & Objective: SETD2, a major histone H3K36 trimethyltransferase, has been shown to play an important role in multiple biological processes. Mutations in SETD2 have been evidenced in several types of cancer, including clear cell renal cell carcinoma (ccRCC). Previous studies reported the potential involvement of SETD2 in driving ccRCC tumorigenesis. However, the mechanism by which SETD2 causes cell metastasis remains poorly understood. Methods: Genome-wide CRISPR-Cas9-based screens and immunoprecipitation-mass spectrometry were used to identify candidates for regulating SETD2 stability. Immunohistochemistry staining of the ccRCC tissue array and bioinformatics analysis of TCGA database were used to investigate the clinical relevance of SETD2 in ccRCC. The gene expression was detected by qRT-PCR, and the protein level was detected by western blotting. Wound healing and transwell migration assay were used to explore the cell migration ability. SIAH2 was deleted from ccRCC cell lines using CRISPR/Cas9-mediated gene editing. Furthermore, we used BALB/c-nude mice to construct xenograft mouse models and pulmonary metastasis models. Moreover, RNA-seq, H3K36me3 CUT&Tag and RNA N6-methyladenosine-IP were performed to uncover the mechanism. Results: SETD2 mutant or low expression was correlated with poor prognosis in patients with ccRCC based on TCGA data and tissue array. Through the CRISPR/Cas9 screening system and immunoprecipitation-mass spectrometry, we identified the E3 ubiquitin ligase SIAH2 that regulates the stability of SETD2. Then, we verified that the protein stability of SETD2 was downregulated by SIAH2 in a dose-dependent manner and further confirmed that the SIAH2 promotes SETD2 degradation in a proteasome-dependent pathway. In addition, we constructed a dominant negative mutant of SIAH2 and proved the degradation dependent on its enzymatic activity. Moreover, we showed that SIAH2 knockout increased the protein level of SETD2. In SETD2 stable knockdown ccRCC cell lines, loss of SETD2 enhanced the capability of cell migration, while knockdown of SIAH2 resulted in the rescue of SETD2 deficient effect. Moreover, SETD2 ablation significantly decreases H3K36me3 peaks of E-cadherin, further reducing the m6A signal and stability of E-cadherin mRNA. Conclusion: In this study, we firstly revealed the mechanism that E3 ubiquitin ligase SIAH2 interacted with SETD2 and promoted the ubiquitination degradation of SETD2. Moreover, SETD2 ablation in ccRCC cells enhanced EMT through impaired epigenetic regulation of E-cadherin, suggesting SIAH2/SETD2/E-cadherin axis as a potential therapeutic target for ccRCC. Citation Format: Mengxue Yu, Lingao Ju, Gang Wang. The E3 ubiquitin ligase SIAH2 promotes clear cell renal cell carcinoma epithelial-mesenchymal transition via regulation of SETD2 stability. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5273.

158 Controlling Glioblastoma Invasion In Vivo With CD97

INTRODUCTION: Increased levels of adhesion G Protein-Coupled Receptor (GPCR) CD97 have been detected in multiple metastatic cancers. Glioblastoma (GBM), a Grade-IV Glioma, also overexpress this receptor, and its levels positively correlate with more aggressive GBM invasion. METHODS: CD97 consists of two distinct domains: the large extracellular fragment interacting with ECM and other cell surface receptors and the GPCR core that activates heterotrimeric G Proteins G12/13. To investigate the mechanism of action of CD97, we generated a CD97 knockdown (shCD97) and overexpression (CD97-WT) constructs. Additionally, we generated two functional mutants: a constitutively active mutant lacking the N-Terminus (dNTF) and dominant-negative mutant (H436A) that cannot activate G-Proteins downstream. Glioma Stem Cells (GSC) were transduced by control and CD97 lentiviral constructs and implanted into mouse brains. Once tumors formed the brains were harvested, sliced, and immunostained to evaluate invasion and proliferation. Comparing mutants to control, dunnett’s multiple comparisons test was used for statistical analysis. RESULTS: Similar to CD97-WT, we found that both dNTF and H436A, increase tumor volume (p < 0.05). In contrast, the shCD97 mutant decreased tumor size (p < 0.05). Interestingly, only dNTF significantly increased tumor cell proliferation (p < 0.005), yet CD97-WT and H436A affected proliferation of non-tumor cells in the tumor bulk, indicating that this process requires the N-Terminus Fragment. In addition, over-expression of CD97-WT dramatically increases tumor invasion along myelinated fibers of corpus collosum, and tumor spread in the brain tissue (p < 0.05). Yet, the H436A mutant produced large, compact, and non-invasive tumors with sharp borders (p < 0.05) indicating that cell migration requires activation of G12/13. Surprisingly, immunostaining revealed that CD97 knockdown cells displayed invasive phenotype. CONCLUSIONS: Our results demonstrate the dual role CD97 plays in GBM invasion and proliferation, alluding to the molecular mechanisms that can potentially be targeted in GBM treatment.

Ubiquitin‐dependent regulation of MYC: A promising therapeutic target

Ubiquitin‐dependent regulation of MYC: A promising therapeutic target

Phosphorylated S6K1 and 4E-BP1 play different roles in constitutively active Rheb-mediated retinal ganglion cell survival and axon regeneration after optic nerve injury.

Ras homolog enriched in brain (Rheb) is a small GTPase that activates mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that constitutively active Rheb can enhance the regeneration of sensory axons after spinal cord injury by activating downstream effectors of mTOR. S6K1 and 4E-BP1 are important downstream effectors of mTORC1. In this study, we investigated the role of Rheb/mTOR and its downstream effectors S6K1 and 4E-BP1 in the protection of retinal ganglion cells. We transfected an optic nerve crush mouse model with adeno-associated viral 2-mediated constitutively active Rheb and observed the effects on retinal ganglion cell survival and axon regeneration. We found that overexpression of constitutively active Rheb promoted survival of retinal ganglion cells in the acute (14 days) and chronic (21 and 42 days) stages of injury. We also found that either co-expression of the dominant-negative S6K1 mutant or the constitutively active 4E-BP1 mutant together with constitutively active Rheb markedly inhibited axon regeneration of retinal ganglion cells. This suggests that mTORC1-mediated S6K1 activation and 4E-BP1 inhibition were necessary components for constitutively active Rheb-induced axon regeneration. However, only S6K1 activation, but not 4E-BP1 knockdown, induced axon regeneration when applied alone. Furthermore, S6K1 activation promoted the survival of retinal ganglion cells at 14 days post-injury, whereas 4E-BP1 knockdown unexpectedly slightly decreased the survival of retinal ganglion cells at 14 days post-injury. Overexpression of constitutively active 4E-BP1 increased the survival of retinal ganglion cells at 14 days post-injury. Likewise, co-expressing constitutively active Rheb and constitutively active 4E-BP1 markedly increased the survival of retinal ganglion cells compared with overexpression of constitutively active Rheb alone at 14 days post-injury. These findings indicate that functional 4E-BP1 and S6K1 are neuroprotective and that 4E-BP1 may exert protective effects through a pathway at least partially independent of Rheb/mTOR. Together, our results show that constitutively active Rheb promotes the survival of retinal ganglion cells and axon regeneration through modulating S6K1 and 4E-BP1 activity. Phosphorylated S6K1 and 4E-BP1 promote axon regeneration but play an antagonistic role in the survival of retinal ganglion cells.

A Mouse Model with a Frameshift Mutation in the Nuclear Factor I/X (NFIX) Gene Has Phenotypic Features of Marshall-Smith Syndrome.

The nuclear factor I/X (NFIX) gene encodes a ubiquitously expressed transcription factor whose mutations lead to two allelic disorders characterized by developmental, skeletal, and neural abnormalities, namely, Malan syndrome (MAL) and Marshall-Smith syndrome (MSS). NFIX mutations associated with MAL mainly cluster in exon 2 and are cleared by nonsense-mediated decay (NMD) leading to NFIX haploinsufficiency, whereas NFIX mutations associated with MSS are clustered in exons 6-10 and escape NMD and result in the production of dominant-negative mutant NFIX proteins. Thus, different NFIX mutations have distinct consequences on NFIX expression. To elucidate the in vivo effects of MSS-associated NFIX exon 7 mutations, we used CRISPR-Cas9 to generate mouse models with exon 7 deletions that comprised: a frameshift deletion of two nucleotides (Nfix Del2); in-frame deletion of 24 nucleotides (Nfix Del24); and deletion of 140 nucleotides (Nfix Del140). Nfix +/Del2, Nfix +/Del24, Nfix +/Del140, Nfix Del24/Del24, and Nfix Del140/Del140 mice were viable, normal, and fertile, with no skeletal abnormalities, but Nfix Del2/Del2 mice had significantly reduced viability (p < 0.002) and died at 2-3 weeks of age. Nfix Del2 was not cleared by NMD, and NfixDel2/Del2 mice, when compared to Nfix +/+ and Nfix +/Del2 mice, had: growth retardation; short stature with kyphosis; reduced skull length; marked porosity of the vertebrae with decreased vertebral and femoral bone mineral content; and reduced caudal vertebrae height and femur length. Plasma biochemistry analysis revealed Nfix Del2/Del2 mice to have increased total alkaline phosphatase activity but decreased C-terminal telopeptide and procollagen-type-1-N-terminal propeptide concentrations compared to Nfix +/+ and Nfix +/Del2 mice. Nfix Del2/Del2 mice were also found to have enlarged cerebral cortices and ventricular areas but smaller dentate gyrus compared to Nfix +/+ mice. Thus, Nfix Del2/Del2 mice provide a model for studying the in vivo effects of NFIX mutants that escape NMD and result in developmental abnormalities of the skeletal and neural tissues that are associated with MSS. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Porcine Enteric Alphacoronavirus Entry through Multiple Pathways (Caveolae, Clathrin, and Macropinocytosis) Requires Rab GTPases for Endosomal Transport.

Porcine enteric alphacoronavirus (PEAV) is a new bat HKU2-like porcine coronavirus, and its endemic outbreak has caused severe economic losses to the pig industry. Its broad cellular tropism suggests a potential risk of cross-species transmission. A limited understanding of PEAV entry mechanisms may hinder a rapid response to potential outbreaks. This study analyzed PEAV entry events using chemical inhibitors, RNA interference, and dominant-negative mutants. PEAV entry into Vero cells depended on three endocytic pathways: caveolae, clathrin, and macropinocytosis. Endocytosis requires dynamin, cholesterol, and a low pH. Rab5, Rab7, and Rab9 GTPases (but not Rab11) regulate PEAV endocytosis. PEAV particles colocalize with EEA1, Rab5, Rab7, Rab9, and Lamp-1, suggesting that PEAV translocates into early endosomes after internalization, and Rab5, Rab7, and Rab9 regulate trafficking to lysosomes before viral genome release. PEAV enters porcine intestinal cells (IPI-2I) through the same endocytic pathway, suggesting that PEAV may enter various cells through multiple endocytic pathways. This study provides new insights into the PEAV life cycle. IMPORTANCE Emerging and reemerging coronaviruses cause severe human and animal epidemics worldwide. PEAV is the first bat-like coronavirus to cause infection in domestic animals. However, the PEAV entry mechanism into host cells remains unknown. This study demonstrates that PEAV enters into Vero or IPI-2I cells through caveola/clathrin-mediated endocytosis and macropinocytosis, which does not require a specific receptor. Subsequently, Rab5, Rab7, and Rab9 regulate PEAV trafficking from early endosomes to lysosomes, which is pH dependent. The results advance our understanding of the disease and help to develop potential new drug targets against PEAV.

Systematic genetic identification of functional domains on collided di-ribosomes responsible for rescue pathways upon translation arrest in Saccharomyces cerevisiae.

Translation elongation becomes arrested when various obstacles arise, such as a series of inefficient rare codons or stable RNA secondary structures, thus causing ribosomal stalling along the mRNA. Certain wasteful and persistent stalling states are resolved by ribosome rescue pathways. For instance, collisions between stalled and subsequent ribosomes are thought to induce ubiquitination of ribosomal S20 protein by the E3 ubiquitin ligase Hel2, which triggers subsequent rescue reactions. Although structural studies have revealed specific contact sites between collided ribosomes, theribosomal regions crucial for the rescue reaction remain uncharacterized. In this study, we performed a systematic genetic analysis to identifythe molecular regions required for ribosome rescue in Saccharomyces cerevisiae. A series of dominant negative mutations capable of abolishing the rescue reaction were isolated in ribosomal proteins S20 and Asc1. Moreover, mutations in both proteins clustered on the surface of ribosomes between the collided ribosome interfaces, aligned in such a way that they seemingly faced each other. Further analysis via the application of the split-TRP1 protein assay revealed that the mutation of either protein distinctively affected the functional interaction between Hel2 and Asc1, suggesting the development of differential functionality at the interface between collided ribosomes. Our results provide novel and complementary insights into the detailed molecular mechanisms of ribosomal rescue pathways.