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1
Kaszler, Nikolett, Péter Benkő, Árpád Molnár, Abigél Zámbori, Attila Fehér, and Katalin Gémes. "Absence of Arabidopsis Polyamine Oxidase 5 Influences the Cytokinin-Induced Shoot Meristem Formation from Lateral Root Primordia." Plants 12, no. 3 (January 18, 2023): 454. http://dx.doi.org/10.3390/plants12030454.
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Lateral root primordia (LRPs) of Arabidopsis can be directly converted to shoot meristems (SMs) by the application of exogenous cytokinin. Here, we report that Arabidopsis POLYAMINE OXIDASE 5 (AtPAO5) contributes to this process, since the rate of SM formation from LRPs was significantly lower in the pao5-2 knockout mutant. Furthermore, the presented experiments showed that AtPAO5 influences SM formation via controlling the thermospermine (T-Spm) level. Gene expression analyses supported the view that the pao5-2 mutation as well as exogenous T-Spm downregulate the expression of the class 3 haemoglobin coding genes AtGLB1 and AtGLB2. AtGLB1 and 2 have been reported to augment cytokinin sensitivity, indirectly inhibiting the expression of type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs). In agreement, the same ARR-coding genes were found to be upregulated in the pao5-2 mutant. Although GLB proteins might also control cytokinin-induced nitric oxide (NO) accumulation, we could not find experimental evidence for it. Rather, the negative effect of NO-donor treatment on AtPAO5 gene expression and SM formation was seen. Nevertheless, a hypothetical pathway is set up explaining how AtPAO5 may affect direct shoot meristem formation, controlling cytokinin sensitivity through T-Spm and GLBs.
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Israel, David, Shanjida Khan, Charles R. Warren, Janusz J. Zwiazek, and T. Matthew Robson. "The contribution of PIP2-type aquaporins to photosynthetic response to increased vapour pressure deficit." Journal of Experimental Botany 72, no. 13 (April 30, 2021): 5066–78. http://dx.doi.org/10.1093/jxb/erab187.
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Abstract The roles of different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using knockout mutants. Since multiple Arabidopsis PIPs are implicated in CO2 transport across cell membranes, we focused on identifying the effects of the knockout mutations on photosynthesis, and whether they are mediated through the control of stomatal conductance of water vapour (gs), mesophyll conductance of CO2 (gm), or both. We grew Arabidopsis plants in low and high humidity environments and found that the contribution of PIPs to gs was larger under low air humidity when the evaporative demand was high, whereas any effect of a lack of PIP function was minimal under higher humidity. The pip2;4 knockout mutant had 44% higher gs than wild-type plants under low humidity, which in turn resulted in an increased net photosynthetic rate (Anet). We also observed a 23% increase in whole-plant transpiration (E) for this knockout mutant. The lack of functional plasma membrane aquaporin AtPIP2;5 did not affect gs or E, but resulted in homeostasis of gm despite changes in humidity, indicating a possible role in regulating CO2 membrane permeability. CO2 transport measurements in yeast expressing AtPIP2;5 confirmed that this aquaporin is indeed permeable to CO2.
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Jellen-Ritter, A. S., and W. V. Kern. "Enhanced Expression of the Multidrug Efflux Pumps AcrAB and AcrEF Associated with Insertion Element Transposition in Escherichia coli Mutants Selected with a Fluoroquinolone." Antimicrobial Agents and Chemotherapy 45, no. 5 (May 1, 2001): 1467–72. http://dx.doi.org/10.1128/aac.45.5.1467-1472.2001.
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ABSTRACT The development of fluoroquinolone resistance in Escherichia coli may be associated with mutations in regulatory gene loci such as marRAB that lead to increased multidrug efflux, presumably through activation of expression of the AcrAB multidrug efflux pump. We found that multidrug-resistant (MDR) phenotypes with enhanced efflux can also be selected by fluoroquinolones frommarRAB- or acrAB-inactivated E. coli K-12 strains having a single mutation in the quinolone-resistance-determining region of gyrA. Mutant 3-AG100MKX, obtained from a mar knockout strain after two selection steps, showed enhanced expression of acrB in a reverse transcriptase PCR associated with insertion of IS186 into the AcrAB repressor gene acrR. In vitro selection experiments with acrAB knockout strains yielded MDR mutants after a single step. Enhanced efflux in these mutants was due to increased expression of acrEF and associated with insertion of IS2 into the upstream region ofacrEF, presumably creating a hybrid promoter. These observations confirm the importance of efflux-associated nontarget gene mutations and indicate that transposition of genetic elements may have a role in the development of fluoroquinolone resistance in E. coli.
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Ohtani, Kaori, Yonghui Yuan, Sufi Hassan, Ruoyu Wang, Yun Wang, and Tohru Shimizu. "Virulence Gene Regulation by the agr System in Clostridium perfringens." Journal of Bacteriology 191, no. 12 (April 10, 2009): 3919–27. http://dx.doi.org/10.1128/jb.01455-08.
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ABSTRACT A gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissue. The agr system is known to be important for the regulation of virulence genes in a quorum-sensing manner in Staphylococcus aureus. A homologue for S. aureus agrBD (agrBDSa ) was identified in the C. perfringens strain 13 genome, and the role of C. perfringens agrBD (agrBDCp ) was examined. The agrBDCp knockout mutant did not express the theta-toxin gene, and transcription of the alpha- and kappa-toxin genes was also significantly decreased in the mutant strain. The mutant strain showed a recovery of toxin production after the addition of the culture supernatant of the wild-type strain, indicating that the agrBDCp mutant lacks a signal molecule in the culture supernatant. An agr-virR double-knockout mutant was constructed to examine the role of the VirR/VirS two-component regulatory system, a key virulence regulator, in agrBDCp -mediated regulation of toxin production. The double-mutant strain could not be stimulated for toxin production with the wild-type culture supernatant. These results indicate that the agrBDCp system plays an important role in virulence regulation and also suggest that VirR/VirS is required for sensing of the extracellular signal and activation of toxin gene transcription in C. perfringens.
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Buckhaults, Phillip J., Sana Khalili, Carolyn Banister, Prashanth R. Gokare, Dave Pocalyko, and Kurtis Bachman. "Abstract 251: Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids." Cancer Research 83, no. 7_Supplement (April 4, 2023): 251. http://dx.doi.org/10.1158/1538-7445.am2023-251.
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Abstract The promise of precision medicine is based on the idea that genetic alterations present only in tumor cells create vulnerabilities that can be targeted for therapeutic intent. In some instances, somatically mutated driver genes such as KRAS, BRAF or PIK3CA can be targeted with small molecule inhibitors specific for mutant oncoproteins. In other cases therapeutic vulnerability is an indirect consequence, such as response to immune checkpoint inhibitors caused by somatic inactivation of mismatch repair genes and subsequent creation of neo-antigens in tumors. Synthetic lethal vulnerabilities dependent on common somatic alterations would be highly specific therapeutic targets applicable to a large number of cancer patients. We sought to identify potential therapeutic vulnerabilities for colon cancer by performing a genome wide CRISPR knockout screen in a colon cancer organoid in both TP53-Wild-Type and TP53-Knockout backgrounds. We identified 1784 gene knockouts with TP53-dependent effects on Darwinian fitness. Examples include MDM2 and PPM1D knockouts, both of which selectively harmed the TP53 WT organoids. 250 gene knockouts selectively harmed the TP53 KO organoids and represent novel avenues for development of TP53 synthetic lethal targeted therapeutics. We also identified ~1000 gene knockouts under significant negative Darwinian selection in both TP53 WT and TP53 KO organoids which are not known to be common essential genes. Several of these organoid model specific vulnerabilities are in pathways that are downstream of the somatic mutations present in the tumor and are therefore candidates for highly-specific targeted therapeutic intervention. Examples include Werner’s pathway dependencies (WRN, EME1, MUS81) which are known to result from mismatch repair deficiency, WNT pathway dependencies (CTNNB1, PORCN) which result from RNF43 inactivating somatic mutation, and BRAF dependency resulting from its own activating oncogenic somatic mutation. In summary, genome-wide CRISPR knockout library screening of human colon cancer organoids can provide a comprehensive overview of dependencies interacting with either natural or engineered mutations and are a promising novel platform for discovering personalized or pathway based therapeutic targets. Future work will focus on target validation and additional primary screens using our biobank of tumor-normal organoid pairs. Citation Format: Phillip J. Buckhaults, Sana Khalili, Carolyn Banister, Prashanth R. Gokare, Dave Pocalyko, Kurtis Bachman. Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids [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 251.
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Mayo-Muñoz, David, Fei He, Jacob Jørgensen, Poul Madsen, Yuvaraj Bhoobalan-Chitty, and Xu Peng. "Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2." Viruses 10, no. 12 (December 8, 2018): 695. http://dx.doi.org/10.3390/v10120695.
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Genetic engineering of viruses has generally been challenging. This is also true for archaeal rod-shaped viruses, which carry linear double-stranded DNA genomes with hairpin ends. In this paper, we describe two different genome editing approaches to mutate the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) using the archaeon Sulfolobus islandicus LAL14/1 and its derivatives as hosts. The anti-CRISPR (Acr) gene acrID1, which inhibits CRISPR-Cas subtype I-D immunity, was first used as a selection marker to knock out genes from SIRV2M, an acrID1-null mutant of SIRV2. Moreover, we harnessed the endogenous CRISPR-Cas systems of the host to knock out the accessory genes consecutively, which resulted in a genome comprised solely of core genes of the 11 SIRV members. Furthermore, infection of this series of knockout mutants in the CRISPR-null host of LAL14/1 (Δarrays) confirmed the non-essentiality of the deleted genes and all except the last deletion mutant propagated as efficiently as the WT SIRV2. This suggested that the last gene deleted, SIRV2 gp37, is important for the efficient viral propagation. The generated viral mutants will be useful for future functional studies including searching for new Acrs and the approaches described in this case are applicable to other viruses.
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Kaplan, Kara, Ashley E. Echert, Ben Massat, Madeleine M. Puissant, Oleg Palygin, Aron M. Geurts, and Matthew R. Hodges. "Chronic central serotonin depletion attenuates ventilation and body temperature in young but not adult Tph2 knockout rats." Journal of Applied Physiology 120, no. 9 (May 1, 2016): 1070–81. http://dx.doi.org/10.1152/japplphysiol.01015.2015.
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Genetic deletion of brain serotonin (5-HT) neurons in mice leads to ventilatory deficits and increased neonatal mortality during development. However, it is unclear if the loss of the 5-HT neurons or the loss of the neurochemical 5-HT led to the observed physiologic deficits. Herein, we generated a mutant rat model with constitutive central nervous system (CNS) 5-HT depletion by mutation of the tryptophan hydroxylase 2 ( Tph2) gene in dark agouti (DA Tph2−/−) rats. DA Tph2−/− rats lacked TPH immunoreactivity and brain 5-HT but retain dopa decarboxylase-expressing raphe neurons. Mutant rats were also smaller, had relatively high mortality (∼50%), and compared with controls had reduced room air ventilation and body temperatures at specific postnatal ages. In adult rats, breathing at rest and hypoxic and hypercapnic chemoreflexes were unaltered in adult male and female DA Tph2−/− rats. Body temperature was also maintained in adult DA Tph2−/− rats exposed to 4°C, indicating unaltered ventilatory and/or thermoregulatory control mechanisms. Finally, DA Tph2−/− rats treated with the 5-HT precursor 5-hydroxytryptophan (5-HTP) partially restored CNS 5-HT and showed increased ventilation ( P < 0.05) at a developmental age when it was otherwise attenuated in the mutants. We conclude that constitutive CNS production of 5-HT is critically important to fundamental homeostatic control systems for breathing and temperature during postnatal development in the rat.
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Jones, Alexis M., Aphrothiti J. Hanrahan, Moriah H. Nissan, Sebastien Monette, Ziyu Chen, Wenhuo Hu, Sandra Misale, et al. "Abstract 2: Vertical MAPK pathway targeting in novel genetically engineered mouse and cell line models of NF1-altered melanoma: the mSK-Mel murine cohort." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2. http://dx.doi.org/10.1158/1538-7445.am2023-2.
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Abstract Large scale clinical genomic sequencing efforts have revealed inactivating mutations in the RAS-GTPase Neurofibromin 1 (NF1) in a significant subset of melanomas. To date, immunotherapy and MAPK pathway-directed targeted therapies have been largely inactive in this molecularly defined cohort and immunogenic models that reflect the distinct co-mutation patterns found in NF1-mutant melanoma patients are lacking. Leveraging a population-scale tumor genomic profiling initiative, we identified TP53 as a gene significantly co-altered with NF1 in melanoma. We thus generated and molecularly characterized a cohort of genetically engineered mice with targeted deletion of NF1 in melanocytes. Melanocyte-specific, homozygous knockout of NF1 induced hyperpigmentation yet was insufficient for tumorigenesis. Addition of TP53 knockout and/or conditional activating mutation in BRAF (BRAFVE), resulted in melanoma formation with variable and high penetrance, respectively, along with histologic features consistent with human melanomas. Tumor latency and overall survival in NF1/TP53 double knockout mice was similar to NF1/BRAFVE double mutants. NF1 knockout did not shorten the latency to tumor formation in the setting of BRAFVE/TP53 mutation but did intensify melanocytic hyperpigmentation in all genetic backgrounds tested. To facilitate preclinical and functional studies, we derived 22 congenic cell lines from harvested mouse tumors from NF1 knockout mice, with and without BRAFVE mutation, and tested their sensitivity to targeted agents. As expected, loss of NF1 conditioned the response to BRAF inhibition, while NF1-mutant cells retained sensitivity to MEK inhibition. To abrogate the effects of adaptive RAS reactivation after MEK inhibitor therapy, combined MEK/SHP inhibition in NF1/TP53 knockout cells and BRAF/SHP inhibition in NF1/TP53/BRAFVE mutant cells strongly blunted ERK phosphorylation and cell proliferation better than single agent therapy. However, this response to the addition of SHP inhibition was transient and ERK rebound was driven by continued MEK activation and dependance. In syngeneic xenograft models of NF1/TP53/BRAFVE mutation, MEK inhibition alone, or in combination with RAF and/or SHP inhibition, induced tumor regression and delayed the onset of resistance and progression as compared to doublet RAF/SHP inhibitor therapy. Overall, we demonstrated the efficacy and feasibility of vertical MAPK pathway targeting in a novel cohort of genetically relevant mouse and cell line models of NF1-mutant melanoma and provide justification for future studies of vertical MAPK pathway targeting to achieve maximal ERK pathway inhibition in this molecularly defined patient cohort. Citation Format: Alexis M. Jones, Aphrothiti J. Hanrahan, Moriah H. Nissan, Sebastien Monette, Ziyu Chen, Wenhuo Hu, Sandra Misale, Isabell Schulze, Naresh Vasani, Cailian Liu, Xia Yang, Mohsen Abu-Akeel, Elisa de Stanchina, Nikolaus Schultz, Michael F. Berger, Neal Rosen, Taha Merghoub, David B. Solit. Vertical MAPK pathway targeting in novel genetically engineered mouse and cell line models of NF1-altered melanoma: the mSK-Mel murine cohort [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 2.
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Grant, Kevron, Nicole M. Carey, Miguel Mendoza, John Schulze, Marinus Pilon, Elizabeth A. H. Pilon-Smits, and Doug van Hoewyk. "Adenosine 5′-phosphosulfate reductase (APR2) mutation in Arabidopsis implicates glutathione deficiency in selenate toxicity." Biochemical Journal 438, no. 2 (August 12, 2011): 325–35. http://dx.doi.org/10.1042/bj20110025.
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APR2 is the dominant APR (adenosine 5′-phosphosulfate reductase) in the model plant Arabidopsis thaliana, and converts activated sulfate to sulfite, a key reaction in the sulfate reduction pathway. To determine whether APR2 has a role in selenium tolerance and metabolism, a mutant Arabidopsis line (apr2-1) was studied. apr2-1 plants had decreased selenate tolerance and photosynthetic efficiency. Sulfur metabolism was perturbed in apr2-1 plants grown on selenate, as observed by an increase in total sulfur and sulfate, and a 2-fold decrease in glutathione concentration. The altered sulfur metabolism in apr2-1 grown on selenate did not reflect typical sulfate starvation, as cysteine and methionine levels were increased. Knockout of APR2 also increased the accumulation of total selenium and selenate. However, the accumulation of selenite and selenium incorporation in protein was lower in apr2-1 mutants. Decreased incorporation of selenium in protein is typically associated with increased selenium tolerance in plants. However, because the apr2-1 mutant exhibited decreased tolerance to selenate, we propose that selenium toxicity can also be caused by selenate's disruption of glutathione biosynthesis leading to enhanced levels of damaging ROS (reactive oxygen species).
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So, Jonathan, Nathaniel Mabe, Bernhard Englinger, Jason Kwon, Brian Shim, Mariella Filbin, Kimberly Stegmaier, and William Hahn. "Abstract 2147: Synthetic lethality of VRK1 in VRK2-methylated cancers." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2147. http://dx.doi.org/10.1158/1538-7445.am2022-2147.
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Abstract Paralogs are a source of synthetic lethal interactions which lend themselves to novel bio-marker linked targeted therapeutics. From genome-wide essentiality measurements in over 900 cancer cell lines, we find VRK1 dependency in VRK2-methylated adult and pediatric gliomas and neuroblastomas. VRK2 methylation was mainly seen in IDH-mutant gliomas and H3.3-G34R-mutant DIPG. Knockout of VRK2 was able to sensitize cells to VRK1 knockout. Overexpression of kinase-active but not kinase inactive VRK2 rescued VRK1 knockout. Global phosphoproteomics in VRK1 and VRK2 knockout cells demonstrate increased phosphorylation of targets involved with DNA damage and decreased phosphorylation of nuclear membrane targets. VRK1 and VRK2 were found to phosphorylate BANF1, which is involved in nuclear membrane re-formation during mitosis. Taken together, we show that VRK1 is a viable target in VRK2-methylated adult and pediatric gliomas, and neuroblastomas. Citation Format: Jonathan So, Nathaniel Mabe, Bernhard Englinger, Jason Kwon, Brian Shim, Mariella Filbin, Kimberly Stegmaier, William Hahn. Synthetic lethality of VRK1 in VRK2-methylated cancers [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 2147.
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Kozhin, PM, DD Romashin, AL Rusanov, and NG Luzgina. "Knockout of mutant TP53 in the HaCaT cells enhances their migratory activity." Bulletin of Russian State Medical University, no. 2022(6) (December 2022): 110–15. http://dx.doi.org/10.24075/brsmu.2022.070.
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The HaCaT cell line represents the spontaneously immortalized non-carcinogenic human keratinocytes that are used as a model for studying the function of normal human keratinocytes. There are two TP53 alleles in the HaCaT cell genome, which comprise two gain-of-function (GOF) mutations acquired through spontaneous immortalization (mutTP53). Mutations result in the increased proliferation rate and violation of the stratification program. The study was aimed to assess the effects of the mutTP53 gene knockout on the HaCaT keratinocytes capability of proliferation and migration in the in vitro model of epidermal injury and regeneration (scratch test), and on the ability to form stratified epithelium in the organotypic epidermal model. To perform the scratch-test, cells were cultured until monolayer was formed, then the standardized injury was created. The organotypic model was obtained by growing keratinocytes in the polycarbonate membrane inserts with the pore size of 0.4 μm at the interface between the phases (air-liquid). It has been shown that the mutant TP53 gene knockout results in the increased migration capability of the HaCaT keratinocytes: in the HaCaT with the mutTP53 knockout, the defect closure occurred faster than in the appropriate group of the WT HaCaT (p < 0.05), on day three the defect size was 12% ± 3% and 66% ± 5% of the initial size. There is evidence that mutant TP53 in the HaCaT cells is a negative regulator of the laminin 5 expression (LAMC2 expression was 9.96 ± 1.92 times higher in the cells with the mutTP53 knockout, p < 0.05), however, this does not promote normalization of the program of epithelial differentiation and stratification followed by formation of the stratum corneum in the organotypic model.
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Patel, Sonia, Monique Nilsson, Yan Yang, Xiaoxing Yu, Fahao Zhang, Alissa Poteete, Xiaoyang Ren, et al. "Abstract 3125: IL-6 contributes to the suppression of T and NK cell anti-tumor activity in EGFR-mutant NSCLC." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3125. http://dx.doi.org/10.1158/1538-7445.am2022-3125.
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Abstract Around 10-15% of non-small cell lung cancer (NSCLC) cases in the United States harbor an activating mutation in epidermal growth factor receptor (EGFR) which are initially highly sensitive to treatment EGFR tyrosine kinase inhibitors (TKI) before eventually developing resistance to these agents. Therefore, there is a current unmet clinical need to identify novel treatment options for this patent population. While anti-PD-1/PD-L1 immunotherapy has been effective for many NSCLC patients, EGFR-mutant tumors have response rates of less than 10%. The mechanisms driving resistance to immunotherapy in EGFR-mutant NSCLC are not well understood. We previously reported that IL-6 is highly upregulated in NSCLC cells with acquired resistance to EGFR-TKIs. Because IL-6 is a pleiotropic cytokine known to impact immune populations within the tumor microenvironment, we hypothesized that IL-6 may drive immunosuppression in EGFR mutant NSCLC. Using genetically engineered mouse models (GEMMs) of EGFR-mutant NSCLC with and without IL-6 knockout, we evaluated the effects of IL-6 on the tumor microenvironment. EGFR-mutant mice with knockout of IL-6 had a significantly increased overall survival compared to those with intact-IL-6. Knockout of IL-6 increased total immune infiltration in these tumors as assessed by flow cytometry. Infiltrating T cells from IL-6 knockout mice displayed a smaller population of T helper 17 cells compared to IL-6-expressing tumors. Knockout of IL-6 also increased the population of infiltrating activated CD8 T cells and a reduced T-regulatory cell population. Due to these changes in T cell activity, we wanted to determine any potential synergistic effects of IL-6 blockade used during anti-PD-1 therapy. We observed that combination treatment extended survival in EGFR-mutant NSCLC mice. Corresponding in vitro T cell cytotoxicity assays confirmed that EGFR-TKI resistant cells treated with IL-6 blocking antibody were more sensitive to T cell-mediated killing. Furthermore, infiltration of NK cells was increased in tumors with knockout of IL-6. Infiltrating NK cells found in these IL-6 knockout mice displayed a more activated phenotype as demonstrated by increased expression of NKG2D. Next, we evaluated the effects of IL-6 from conditioned media collected from human EGFR-mutant and TKI-resistant cells lines on human T and NK cells cultured ex vivo. e determined that IL-6 suppressed expression of activation markers including granzyme B and IFN-y on the surface of both T and NK cells. Blockade of IL-6 increased NK-mediated cytotoxic killing of EGFR-TKI resistant cells in vitro. In conclusion, our data indicates that in EGFR-TKI resistance, upregulated IL-6 suppresses T and NK cell cytotoxic potential and drives immunosuppression. Citation Format: Sonia Patel, Monique Nilsson, Yan Yang, Xiaoxing Yu, Fahao Zhang, Alissa Poteete, Xiaoyang Ren, Li Shen, Jing Wang, Xiuning Le, John Heymach. IL-6 contributes to the suppression of T and NK cell anti-tumor activity in EGFR-mutant NSCLC [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 3125.
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Kent, Matthew R., Delia Calderon, Katherine M. Silvius, Jack P. Kucinski, Collette A. LaVigne, Matthew V. Cannon, and Genevieve C. Kendall. "Abstract 3533: Zebrafish her3 knockout impacts developmental and rhabdomyosarcoma-related gene signatures." Cancer Research 83, no. 7_Supplement (April 4, 2023): 3533. http://dx.doi.org/10.1158/1538-7445.am2023-3533.
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Abstract HES3 is a basic helix-loop-helix transcription factor that regulates neural stem cell renewal during development. HES3 overexpression is predictive of reduced overall survival in patients with fusion-positive rhabdomyosarcoma, a pediatric cancer that resembles immature and undifferentiated skeletal muscle and is most commonly driven by the fusion-oncogene PAX3-FOXO1. However, the mechanisms of HES3 cooperation in PAX3-FOXO1 driven rhabdomyosarcoma are unclear and are likely related to her3/HES3’s role in neurogenesis. To investigate HES3’s function during development, we generated a zebrafish CRISPR/Cas9 null mutation of her3, the zebrafish ortholog of HES3. Phenotypic characterization revealed that her3 null mutation is not embryonic lethal as they are present at expected Mendelian ratios. We observed a temporary growth delay her3 zebrafish null mutants and, rarely, eye defects in adults. Transcriptomic analysis of her3 null mutant embryos showed early dysregulation of a known downstream target, neurog1 and downregulation of genes involved in organ development, such as pctp and grinab, while genes pointing toward a terminal differentiation state, such as tmod, are upregulated. Differentially expressed genes in her3 null mutant embryos are enriched for HOX and SOX10 motifs, suggesting these may be key genes involved during HES3 dysregulation. Several cancer-related gene pathways are impacted, including the inhibition of matrix metalloproteinases and the tumor microenvironment pathway. To complement our zebrafish model, we are developing a double-inducible cell line model consisting of a tetracycline-inducible HES3 and a cumate-inducible PAX3-FOXO1 to further investigate the hypothesized cooperation between her3/HES3 and PAX3-FOXO1 in fusion-positive rhabdomyosarcoma. These two systems will allow us to elucidate conserved mechanisms of cooperation between HES3 and PAX3-FOXO1, and identify new therapeutic opportunities for children with fusion-driven rhabdomyosarcoma. Citation Format: Matthew R. Kent, Delia Calderon, Katherine M. Silvius, Jack P. Kucinski, Collette A. LaVigne, Matthew V. Cannon, Genevieve C. Kendall. Zebrafish her3 knockout impacts developmental and rhabdomyosarcoma-related gene signatures. [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 3533.
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De, Archana, Vijayalaxmi Gupta, Amlan Das, Arnab Ghosh, Inamul Haque, Laster Lau, Sushanta K. Banerjee, and Snigdha Banerjee. "Abstract 536: The role of CCN1 in mutant K-RAS addiction in pancreatic ductal adenocarcinoma." Cancer Research 83, no. 7_Supplement (April 4, 2023): 536. http://dx.doi.org/10.1158/1538-7445.am2023-536.
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Abstract Mutant K-RAS addiction of pancreatic ductal adenocarcinoma cells (PDAC) coordinates their transformation, proliferation, and survival. Therefore, initially, it had an impression that targeting mutant K-RAS could be an ideal strategy for treating PDAC. However, after decades of studies, no effective RAS inhibitors reached the clinic, indicating that RAS-oncoprotein is an undruggable target in PDAC. Thus, identifying a new target linked with K-RAS signaling was most needed. Using genetically engineered cell lines and K-RAS-driven genetically engineered mouse models (KC or KPC) with or without pancreas-specific CCN1 conditional knockout background, we identified a positive feedback circuit between CCN1 and mutant K-RAS expression. Moreover, blocking this feedback via conditional knockout of CCN1 in the pancreas significantly reduced invasive phenotypes, PDAC growth, and metastasis in the lung in KC and KPC mice. Mechanistically we found that the K-RAS-CCN1 feedback circuit is regulated by microRNA miR-145 via a feedforward mechanism. Collectively, these studies highlight the clinical potential of targeting CCN1 in PDAC. (This work is supported by VA Merit grants). Citation Format: Archana De, Vijayalaxmi Gupta, Amlan Das, Arnab Ghosh, Inamul Haque, Laster Lau, Sushanta K. Banerjee, Snigdha Banerjee. The role of CCN1 in mutant K-RAS addiction in pancreatic ductal adenocarcinoma [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 536.
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Boes, Nelli, Kerstin Schreiber, Elisabeth Härtig, Lothar Jaensch, and Max Schobert. "The Pseudomonas aeruginosa Universal Stress Protein PA4352 Is Essential for Surviving Anaerobic Energy Stress." Journal of Bacteriology 188, no. 18 (September 15, 2006): 6529–38. http://dx.doi.org/10.1128/jb.00308-06.
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ABSTRACT During infection of the cystic fibrosis (CF) lung, Pseudomonas aeruginosa microcolonies are embedded in the anaerobic CF mucus. This anaerobic environment seems to contribute to the formation of more robust P. aeruginosa biofilms and to an increased antibiotic tolerance and therefore promotes persistent infection. This study characterizes the P. aeruginosa protein PA4352, which is important for survival under anaerobic energy stress conditions. PA4352 belongs to the universal stress protein (Usp) superfamily and harbors two Usp domains in tandem. In Escherichia coli, Usp-type stress proteins are involved in survival during aerobic growth arrest and under various other stresses. A P. aeruginosa PA4352 knockout mutant was tested for survival under several stress conditions. We found a decrease in viability of this mutant compared to the P. aeruginosa wild type during anaerobic energy starvation caused by the missing electron acceptors oxygen and nitrate. Consistent with this phenotype under anaerobic conditions, the PA4352 knockout mutant was also highly sensitive to carbonyl cyanide m-chlorophenylhydrazone, the chemical uncoupler of the electron transport chain. Primer extension experiments identified two promoters upstream of the PA4352 gene. One promoter is activated in response to oxygen limitation by the oxygen-sensing regulatory protein Anr. The center of a putative Anr binding site was identified 41.5 bp upstream of the transcriptional start site. The second promoter is active only in the stationary phase, however, independently of RpoS, RelA, or quorum sensing. This is the second P. aeruginosa Usp-type stress protein that we have identified as important for survival under anaerobic conditions, which resembles the environment during persistent infection.
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Shi, Wei, Nora Heisterkamp, John Groffen, Jingsong Zhao, David Warburton, and Vesa Kaartinen. "TGF-β3-null mutation does not abrogate fetal lung maturation in vivo by glucocorticoids." American Journal of Physiology-Lung Cellular and Molecular Physiology 277, no. 6 (December 1, 1999): L1205—L1213. http://dx.doi.org/10.1152/ajplung.1999.277.6.l1205.
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Newborn transforming growth factor (TGF)-β3-null mutant mice exhibit defects of palatogenesis and pulmonary development. Glucocorticoids, which play a central role in fetal lung maturation, have been postulated to mediate their stimulatory effects on tropoelastin mRNA expression through TGF-β3 in cultured lung fibroblasts. In the present study, we analyzed the abnormally developed lungs in TGF-β3-null mutant mice and compared the effects of glucocorticoids on gene expression and lung morphology between TGF-β3 knockout and wild-type mice. Lungs of TGF-β3-null mutant mice on embryonic day 18.5 did not form normal saccular structures and had a thick mesenchyme between terminal air spaces. Moreover, the number of surfactant protein C-positive cells was decreased in TGF-β3-null mutant lungs. Interestingly, glucocorticoids were able to promote lung maturation and increased expression of both tropoelastin and fibronectin but decreased the relative number of surfactant protein C-positive cells in fetal lungs of both genotypes. This finding provides direct evidence that glucocorticoid signaling in the lung can use alternative pathways and can exert its effect without the presence of TGF-β3.
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Wu, Wenbi, and A. Lorena Passarelli. "Autographa californica Multiple Nucleopolyhedrovirus Ac92 (ORF92, P33) Is Required for Budded Virus Production and Multiply Enveloped Occlusion-Derived Virus Formation." Journal of Virology 84, no. 23 (September 22, 2010): 12351–61. http://dx.doi.org/10.1128/jvi.01598-10.
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ABSTRACT The Autographa californica multiple nucleopolyhedrovirus orf92 (p33), ac92, is one of 31 genes carried in all sequenced baculovirus genomes, thus suggesting an essential function. Ac92 has homology to the family of flavin adenine dinucleotide-linked sulfhydryl oxidases and is related to the ERV/ALR family of sulfhydryl oxidases. The role of ac92 during virus replication is unknown. Ac92 was associated with the envelope of both budded and occlusion-derived virus (ODV). To investigate the role of Ac92 during virus replication, an ac92-knockout bacmid was generated through homologous recombination in Escherichia coli. Titration and plaque assays showed no virus spread in ac92-knockout bacmid DNA-transfected insect cells. Deletion of ac92 did not affect viral DNA replication. However, ac92-knockout bacmid DNA-transfected cells lacked multiply enveloped occlusion-derived nucleocapsids; instead, singly enveloped nucleocapsids were detected. To gain insight into the requirement for sulfhydryl oxidation during virus replication, a virus was constructed in which the Ac92 C155XXC158 amino acids, important for sulfhydryl oxidase activity, were mutated to A155XXA158. The mutant virus exhibited a phenotype similar to that of the knockout virus, suggesting that the C-X-X-C motif was essential for sulfhydryl oxidase activity and responsible for the altered ODV phenotype.
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Metz, Alexander, Marya Kozinova, Robert Uzzo, Jessica Peskin, Michael Slifker, Janusz Franco-Barraza, Edna Cukierman, and Philip Abbosh. "Abstract 3725: SETD2 loss in renal carcinoma cells induces the unfolded protein response." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3725. http://dx.doi.org/10.1158/1538-7445.am2022-3725.
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Abstract Introduction: SETD2 encodes a histone H3-K36 methyltransferase which is frequently inactivated in clear cell renal carcinomas (ccRCCs) and papillary RCCs via 3p deletion/LOH and deleterious mutations. Histone H3-K36 trimethylation is facilitated by SETD2, which is necessary for proper pre-mRNA intron splicing. Improperly spliced mature mRNA may lead to aberrant translation of retained introns (ATaRI), which represents potential therapeutic vulnerabilities. We explored this hypothesis using real world data and RCC models. Methods and Results: Gene set enrichment analysis comparing SETD2-mutant to WT tumors using samples from the TCGA KIRC data set revealed that the unfolded protein response (UPR) was strongly enriched, as well as several immunotherapy-relevant pathways. This suggested that peptides arising from ATaRI may be present, since they would not be expected to fold properly and thus need to be addressed by the UPR pathway to maintain homeostasis. To investigate this further, we generated Setd2-isogenic RENCA cells using CRISPR. Knockout was confirmed by sequencing and immunoblot. H3K36 trimethylation was decreased or eliminated in monoclonal knockout cell lines, confirming a functional effect. Markers of UPR activation, including Atf4 and cleaved Atf6, were found to be upregulated in Setd2-mutant RENCA cells compared to controls as measured by immunoblot. Cleaved ATF6 translocates to the nucleus to induce the UPR transcriptional program. Consistent with this, Atf6 was found to localize to the nucleus in Setd2-knockout cells using immunofluorescence (IF). Analysis of tissue microarrays of human SETD2-mutant vs -WT ccRCC revealed increased ATF6 signal in areas with low H3K36 tri-methylation, indicating UPR activation in vivo. Interestingly, CHOP, another downstream effector of the UPR pathway which predominantly regulates cell death, did not become upregulated in Setd2-knockout cells, suggesting activation of a compensatory cell survival pathway. Geldanamycin was shown to destabilize the Perk and Ire1a arms of the UPR, which resulted in increased cell death in Setd2 mutant cells using Cell Titre glo. Additionally, H3K36 trimethylation has been implicated in directing homologous repair of DNA, and blunted responses to agents that induce DNA double-strand breaks of p-Atm, p53, and Rad51 were observed by immunoblot and IF. The inability to detect DNA damage in Setd2 mutant cells however did not confer sensitivity to PARP inhibitors. Conclusions: We identify activation of the UPR upon Setd2 loss and suggest that activation of part of the UPR pathway may represent a new therapeutic vulnerability for exploitation as a rationale for personalized medicine. We further characterize the altered DNA damage response in the setting of Setd2 loss. We continue to evaluate the generation of peptides arising from ATaRI in Setd2-mutant contexts. Citation Format: Alexander Metz, Marya Kozinova, Robert Uzzo, Jessica Peskin, Michael Slifker, Janusz Franco-Barraza, Edna Cukierman, Philip Abbosh. SETD2 loss in renal carcinoma cells induces the unfolded protein response [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 3725.
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More, Aditya, Valsala Haridas, Ichiaki Ito, Saikat Chowdhury, Yue Gu, Natalie W. Fowlkes, and John P. Shen. "Abstract 822: Oncogene addiction to GNAS in GNASR201 mutant tumors." Cancer Research 82, no. 12_Supplement (June 15, 2022): 822. http://dx.doi.org/10.1158/1538-7445.am2022-822.
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Abstract Background: The GNASR201 mutation is the single most frequent cancer-causing mutation across all heterotrimeric G proteins. This gain of function mutation in GNAS drives oncogenesis in appendiceal, colorectal, and gastric adenocarcinoma, as well as Intraductal Papillary Mucinous Neoplasms (IPMN) and Small Cell Lung Cancer (SCLC). In this study, we investigated the role of GNAS in tumor growth using peritoneal models of colorectal cancer (CRC). Methods: GNAS was knocked out in multiple GNASR201C/H mutant colon cell lines (KM12, SNU175, and SKCO1) and overexpressed in GNASWT LS174T cells. Isogenic pairs of KM12 and LS174T cells were injected into the peritoneum of NSG mice to study the role of GNAS in cell line derived xenograft (CDX) models of peritoneal metastasis. Cell lines and CDX were profiled with RNAseq, reverse phase protein assay (RPPA), and immunohistochemistry (IHC) to identify potential pathways regulated by mutant GNAS. Identified mediators of GNAS signaling were then validated using chemical inhibitors of PKA (H-89) and β-catenin (LF3). Results: GNAS knockout significantly decreased 2D colony formation by KM12 (68%), SNU175 (76%), and SKCO1 (85%) cells (all p < 0.0001), and decreased 3D organoid area of KM12 cells by 62% (p = 0.043). There was significant increase in colony formation by LS174T cells overexpressing GNASR201C (193%, p = 0.016) and GNASR201H (170%, p =0.0037). Mice injected with KM12 GNAS-knockout tumors exhibited a significant 68% reduction in tumor growth (n = 6, p = 0.016) and were more likely to survive at 7 weeks (0% vs. 100%, p = 0.0007) relative to those with parent KM12-GNASR201H tumors. Likewise, mice injected with GNASR201H LS174T cells showed significant increase in tumor growth (934% vs. 100%, n = 3, p = 0.042). Histology of GNAS-knockout tumors showed a marked decrease in mucinous stroma and increased lytic necrosis, suggesting an interaction between oncogenic GNAS signaling and the peritoneal environment. GNAS is known to stimulate adenylate cyclase and GNAS knockout decreased levels of cyclic AMP in KM12 cells, confirming an on-target effect. RPPA and RNAseq profiling of KM12 and LS174T PDX tumors identified phosphorylation of β-catenin and activation of Wnt/β-catenin targets (NES = 1.25, 1.24) as critical downstream effects of mutant GNAS signaling, confirmed by a 4.9-fold (p = 0.001) increase in nuclear β-catenin intensity in LS174T GNASR201H tumors (19% vs. 99% nuclei stained positive, p = 0.0002). Chemical inhibition of both PKA and β-catenin reduced growth of GNAS mutant organoids by 79% (p = 0.004) and 67% (p = 0.007) respectively, supporting the involvement of the cAMP/PKA and β-catenin pathways in mutant GNAS signaling. Conclusions: Our findings demonstrate oncogene addiction to GNAS in peritoneal models of GNASR201C/H tumors, which signal through the cAMP/PKA and Wnt/β-catenin pathways. Thus, GNAS and its downstream mediators are promising therapeutic targets for GNAS mutant tumors. Citation Format: Aditya More, Valsala Haridas, Ichiaki Ito, Saikat Chowdhury, Yue Gu, Natalie W. Fowlkes, John P. Shen. Oncogene addiction to GNAS in GNASR201 mutant tumors [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 822.
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Haase, Jacob, Talia A. Gebhard, Qi Liu, Sara G. Bernabé, Jingzhu Hao, Chisom Unegbu, Athanasios Bikas, Jun Qi, and Iñigo Landa. "Abstract 3068: CREBBP/EP300 disruption promotes tumor progression and confers synthetic lethality in anaplastic thyroid cancers." Cancer Research 83, no. 7_Supplement (April 4, 2023): 3068. http://dx.doi.org/10.1158/1538-7445.am2023-3068.
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Abstract Anaplastic thyroid cancers (ATC) are fast-growing, undifferentiated tumors and almost invariably fatal, primarily due to the lack of effective therapeutic options. The recent approval of dabrafenib plus trametinib for the treatment of BRAFV600E-mutant ATCs improved the prognosis of a subset of patients, but ineligibility and acquired resistance still limit their use. Overall, ATC patients remain in great need of tailored therapeutic options. We previously showed that loss-of-function alterations targeting histone acetyltransferase (HAT) genes, namely CREBBP and EP300, occur in 15-20% of ATCs, but only in <1% of their well-differentiated counterparts. What remains unknown are the specific mechanisms by which HAT disruptions perturb chromatin architecture, impact gene homeostasis, and unleash cellular processes in these aggressive tumors. We are assessing the HAT-mediated thyroid cancer progression. CREBBP/EP300 knockouts enhanced thyroid cancer cell proliferation in vitro and induced thyroid gland growth in a thyroid-specific mouse model of HAT loss. We are leveraging these animals to characterize the in vivo effects of Crebbp/Ep300 knockout, alone or in combination with BrafV600E, in thyroid cancer phenotypes and epigenetic reconfiguration. In addition, we are exploiting the molecular consequences of HAT loss to explore tailored treatments. CRISPR/Cas9 screens identified a mutual CREBBP/EP300-dependency of HAT-mutant human cancer cell models. Our experiments in thyroid cancer cells employing CREBBP/EP300-targeting proteolysis-targeting chimera (PROTAC) compounds specifically degraded these proteins and decreased histone acetylation. Our findings prove the oncogenicity of HAT loss in thyroid cancer progression and support exploring synthetic lethality dependencies in CREBBP/EP300-mutant ATCs. In summary, we provide pre-clinical basis to inform genomics-driven and mechanism-oriented decisions for the clinical management of patients with HAT-altered ATC. Citation Format: Jacob Haase, Talia A. Gebhard, Qi Liu, Sara G. Bernabé, Jingzhu Hao, Chisom Unegbu, Athanasios Bikas, Jun Qi, Iñigo Landa. CREBBP/EP300 disruption promotes tumor progression and confers synthetic lethality in anaplastic thyroid cancers [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 3068.
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Hao, Mingyue, Minghui Wang, Danyu Zhao, Yong Shi, Ye Yuan, Junmei Li, Yunyi Zhai, et al. "Alr Gene in Brucella suis S2: Its Role in Lipopolysaccharide Biosynthesis and Bacterial Virulence in RAW264.7." International Journal of Molecular Sciences 24, no. 13 (June 28, 2023): 10744. http://dx.doi.org/10.3390/ijms241310744.
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Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain’s rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella’s survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.
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Hong, Jiaqian, Yiming Song, Jiayan Xie, Jianhua Xie, Yi Chen, Ping Li, Danyang Liu, Xiaobo Hu, and Qiang Yu. "Acrolein Promotes Aging and Oxidative Stress via the Stress Response Factor DAF-16/FOXO in Caenorhabditis elegans." Foods 11, no. 11 (May 28, 2022): 1590. http://dx.doi.org/10.3390/foods11111590.
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For this investigation, Caenorhabditis elegans (C. elegans) served, for the first time, as a model organism to evaluate the toxic effect and possible underlying mechanisms under acrolein (ACR) exposure. The results showed that ACR exposure (12.5–100 μM) shortened the lifespan of C. elegans. The reproductive capacity, body length, body width, and locomotive behavior (head thrash) of C. elegans were diminished by ACR, especially the doses of 50 and 100 μM. Furthermore, ACR significantly enhanced the endogenous ROS levels of C. elegans, inhibited the antioxidant-related enzyme activities, and affected the expression of antioxidant related genes. The increasing oxidative stress level promoted the migration of DAF-16 into the nucleus that was related to the DAF-16/FOXO pathway. It was also confirmed by the significant decrease of the lifespan-shortening trend in the daf-16 knockout mutant. In conclusion, ACR exposure induced aging and oxidative stress in C.elegans, resulting in aging-related decline and defense-related DAF-16/FOXO pathways’ activation.
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Kim, Sunny, Jonathan P. Kastan, Hsiu-Chi Ting, Youngkyu Park, and Dave Tuveson. "Abstract 2974: A complex of tumor-suppressor NF1 and SPRED2 promotes pancreatic cancer tumorigenesis." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2974. http://dx.doi.org/10.1158/1538-7445.am2022-2974.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with poor prognosis due to late detection and lack of effective therapeutic options. >90% of PDAC tumors are driven by activating mutations in the KRAS oncogene, making it an appealing subject for further investigation into understanding PDAC biology and offering a potential genetic vulnerability to target in patients. Our laboratory has previously reported a mutant-KRAS-proximal complex at the plasma membrane that consists of tumor-suppressor Neurofibromatosis type 1 (NF1), Sprouty Related EVH1 Domain Containing 2 (SPRED2), and Ribosomal S6 Kinase 1 (RSK1). This complex mechanistically suppresses WT KRAS signaling via RSK1, a suppression which is then relieved upon mutant KRAS inhibition, ultimately promoting adaptive resistance for PDAC cells under these conditions. These data highlighted a potential paradoxical role for two canonical tumor-suppressors, NF1 and SPRED2, as promoters of PDAC tumorigenesis when mutant KRAS is inhibited. We find that knockout of NF1 in PDAC organoids induces a substantial growth defect in vivo, and our preliminary data indicates that SPRED2 knockout PDAC cells have reduced proliferation in vitro. Moreover, both SPRED2 and NF1 KO promotes a diminished migratory phenotype in vitro. Given NF1's well-established role as a tumor-suppressor, our observations of decreased tumorigenic properties upon NF1 deletion are unexpected. We hypothesize that NF1 and Spred2 promote tumorigenesis via recruitment of RSK1 to the plasma membrane, inducing the phosphorylation of putative substrates to stimulate a pro-migratory cellular behavior. Our work suggests the existence of a unique RSK1 program localized at the plasma membrane by mutant KRAS, providing a new facet of oncogenesis to investigate for fundamental understanding and potential therapeutic opportunities. Citation Format: Sunny Kim, Jonathan P. Kastan, Hsiu-Chi Ting, Youngkyu Park, Dave Tuveson. A complex of tumor-suppressor NF1 and SPRED2 promotes pancreatic cancer tumorigenesis [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 2974.
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Lee, Su-Chan, Lujain Alaali, and Charles G. Eberhart. "Abstract 2557: Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2557. http://dx.doi.org/10.1158/1538-7445.am2023-2557.
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Abstract Retinoblastoma is the most common intraocular cancer of childhood, with approximately 8,000 new cases each year worldwide. While survival rates are relatively high in developed countries, death can occur in up to half of children in some regions. In addition, blindness is common in patients who survive, and improved therapies are clearly needed. While loss of RB gene function is essential for the initiation of almost all retinoblastoma, additional molecular changes driving their growth and dissemination are poorly understood. Improved understanding of the molecular pathways driving retinoblastoma growth and dissemination may allow us to develop more effective and less toxic therapies. Alterations leading to BCL-6 corepressor (BCOR) loss of function are the second most common mutations in retinoblastoma after changes affecting the RB locus, and are found in more aggressive tumors. BCOR mutations are also associated with metastasis and poor prognosis in other types of pediatric cancer, suggesting that targeting it will represent a new therapeutic approach for aggressive retinoblastoma. Here, we found that BCOR knockdown or knockout in two retinoblastoma cell lines with high baseline levels of protein caused significantly increased invasion/migration, proliferation and clonogenicity. In contrast, overexpression of BCOR in retinoblastoma cells with low/no baseline expression showed opposite effects. In orthotopic xenograft models, BCOR knockout dramatically increased the size and spread of retinoblastoma xenografts as compared to parental controls. In order to determine the molecular pathways modulated by BCOR, we used RNA sequencing and found that loss of function induces IGF1 transcription, resulting in activation of IGF1R and the ERK signaling pathway. Pharmacological inhibition of IGF1/IGF1R signaling using Lincitinib or AEW541 inhibited proliferation and migration in BCOR knockout retinoblastoma cells in vitro, and Lincitinib dramatically slowed xenograft growth in vivo, suggesting that induction of IGF1 represents an important downstream effector following BCOR loss. Our studies support a functional role for BCOR mutations in aggressive retinoblastoma, and suggest that this is mediated at least in part by increased IGF1R signaling, which represents a new therapeutic target for these malignant childhood tumors of the eye. Citation Format: Su-Chan Lee, Lujain Alaali, Charles G. Eberhart. Targeting the IGF1R signaling pathway inhibits cell growth and dissemination in BCOR mutant retinoblastoma [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 2557.
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Kolandaivelu, Kumaran, and Chi-Sang Poon. "A miniature mechanical ventilator for newborn mice." Journal of Applied Physiology 84, no. 2 (February 1, 1998): 733–39. http://dx.doi.org/10.1152/jappl.1998.84.2.733.
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Kolandaivelu, Kumaran, and Chi-Sang Poon.A miniature mechanical ventilator for newborn mice. J. Appl. Physiol. 84(2): 733–739, 1998.—Transgenic/knockout mice with predefined mutations have become increasingly popular in biomedical research as models of human diseases. In some instances, the resulting mutation may cause cardiorespiratory distress in the neonatal or adult animals and may necessitate resuscitation. Here we describe the design and testing of a miniature and versatile ventilator that can deliver varying ventilatory support modes, including conventional mechanical ventilation and high-frequency ventilation, to animals as small as the newborn mouse. With a double-piston body chamber design, the device circumvents the problem of air leakage and obviates the need for invasive procedures such as endotracheal intubation, which are particularly important in ventilating small animals. Preliminary tests on newborn mice as early as postnatal day 0 demonstrated satisfactory restoration of pulmonary ventilation and the prevention of respiratory failure in mutant mice that are prone to respiratory depression. This device may prove useful in the postnatal management of transgenic/knockout mice with genetically inflicted respiratory disorders.
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Xu, Mingjing, Yin Kau Lam, Jianqing Yu, Kelvin Kwok Chai Ng, and Nathalie Wong. "Abstract 2590: TP53 R249S mutation confers hepatic organoids with gain-of-function (GOF) tumorigenic features through transcriptional activation of ZMIZ2." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2590. http://dx.doi.org/10.1158/1538-7445.am2023-2590.
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Abstract Hepatocellular Carcinoma (HCC) represents the third leading cause of cancer-related mortality worldwide. TP53 mutations are pivotal genomic drivers for HCC development through frequent concurrent loss-of-function (LOF) aberrations and protein-altering missense mutations. Over 90% of the TP53 missense mutations are distributed within the core DNA binding domain (DBD), where R249S is the most common. Cumulative studies have demonstrated the cancer-promoting properties of TP53R249S mutation in HCC, but its biological impact on tumor initiation remains to be defined. To mimic the early stages of liver carcinogenesis, we generated normal hepatic organoids from human liver tissues of 3 individuals. CRISPR-Cas9 mediated knockout of TP53 in liver organoids resulted in pleomorphic malignant features, including dysplasia, hyperchromasia, atypical and frequent mitosis, loss of polarity, and increased nuclear to cytoplasm ratio. To recapitulate TP53R249S genotype in HCC, we ectopically expressed R249S mutant in liver organoids through lentiviral infection. TP53R249S liver organoids displayed tumorigenic properties as evidenced by increased lesion forming incidence (37.5%) in subcutaneous xenografts when compared with TP53KO (17%) and TP53 wild-type (WT) organoids (0%). Chromatin immunoprecipitation sequencing (ChIP-seq) analysis with HCC cell lines substantiated the specific gain-of-function (GOF) transcriptional activities of R249S mutant. A unique subset of transcription start site-proximal peaks was exclusively found in R249S mutant cells when compared with other TP53 missense mutants and WT. Integration of ChIP-seq and RNA-seq analysis identified transcription factor ZMIZ2 as a direct transcription target of R249S mutant. Our results showed that ZMIZ2 is preferentially overexpressed in HCC patients carrying TP53R249S mutation and exerts a vital role for proliferation of R249S mutant HCC cells. Knockdown of ZMIZ2 profoundly suppressed global H3K27 acetylation (H3K27ac) and H3K4 trimethylation (H3K4me3) in HCC cells. Transcriptome profiling of ZMIZ2 knockdown cells identified multiple downstream targets enriched in chromatin binding and interaction with histone deacetylase, further reinforcing its involvement in epigenetic regulation. In summary, our study revealed that TP53R249S mutation confers distinct advantages in increased tumorigenicity to human liver organoids through GOF transcriptional activities. ZMIZ2 serves as a direct downstream effector contributing to the oncogenic growth arising from R249S mutation plausibly through altering chromatin remodelling. Citation Format: Mingjing Xu, Yin Kau Lam, Jianqing Yu, Kelvin Kwok Chai Ng, Nathalie Wong. TP53 R249S mutation confers hepatic organoids with gain-of-function (GOF) tumorigenic features through transcriptional activation of ZMIZ2 [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 2590.
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Alobaidi, Ryyan, Nusrat Islam, Yanjun R. Zhang, Mathew M. Shamo, Samuel Allsup, Cynthia M. Simbulan-Rosenthal, and Dean S. Rosenthal. "Abstract 6142: Inhibition of both MAPK and AKT pathways overcomes resistance of NRAS-mutant melanoma stem cells to apoptosis." Cancer Research 83, no. 7_Supplement (April 4, 2023): 6142. http://dx.doi.org/10.1158/1538-7445.am2023-6142.
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Abstract Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and characterized by elevated expression of stem cell markers, such as CD133. We have shown that siRNA knockdown of CD133 enhanced apoptosis induced by the MEK inhibitor trametinib in melanoma cells. The current study investigates the underlying mechanisms of CD133's anti-apoptotic activity in patient-derived BAKP melanoma, harboring the difficult-to-treat NRASQ61K driver mutation, after either CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. CD133 knockout in BAKP cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in trametinib-treated cells, coincident with elevated pro-survival p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. Inhibition of MEK with trametinib, in combination with pan-AKT inhibitor capivasertib (AZD5363) in BAKP cells with either CD133 overexpression or knockout in vitro reduced cell survival as measured by XTT, FACS analysis and colony formation assays. Further, in vivo studies with nude mice xenografted with Dox-inducible BAKP melanoma cells, showed significantly decreased tumor growth after xenografted mice were treated with trametinib alone or in combination with AZD5363. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the AKT and MAPK survival pathways with both trametinib and AZD5363 highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma. Citation Format: Ryyan Alobaidi, Nusrat Islam, Yanjun R. Zhang, Mathew M. Shamo, Samuel Allsup, Cynthia M. Simbulan-Rosenthal, Dean S. Rosenthal. Inhibition of both MAPK and AKT pathways overcomes resistance of NRAS-mutant melanoma stem cells to apoptosis [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 6142.
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Ramanathan, Gajalakshmi, Rebecca Johnson, Jane Huishien Chen, Aaron Jia-En Huang, Bishop Bliss, Michael T. Kleinman, and Angela Fleischman. "Cigarette Smoke and E-Cigarette Aerosols Lead to Clonal Expansion of Tet2 -/- and Dnmt3a R878H Cells In Vivo." Blood 138, Supplement 1 (November 5, 2021): 2167. http://dx.doi.org/10.1182/blood-2021-151322.
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Abstract Background: Environmental factors must play a significant role in the emergence of clonal hematopoiesis since only a fraction of individuals harboring clonal hematopoiesis of indeterminate potential (CHIP) mutations develop hematologic malignancy. Mouse models of chronic inflammation have demonstrated clonal expansion of Tet2 and Dnmt3a knockout hematopoietic cells while Ppm1d mutated clones exhibit clonal dominance in response to cytotoxic DNA damaging chemotherapy stress. Epidemiologic studies have associated smoking behavior with clonal hematopoiesis but the leukemogenic effects of cigarette smoke on hematopoietic stem cells (HSCs) are poorly defined. In addition, the exploding use of electronic (e)-cigarettes has led to significant concern on their detrimental health effects plus studies of E-cigarettes on the hematopoietic system are non-existent. Here, we investigated the role of cigarette smoke and E-cigarette aerosols in promoting clonal expansion of common CHIP mutations. We hypothesize that one or more specific somatic CHIP mutation displays a fitness advantage in the presence of cigarette smoke and/or e-cigarette aerosols. Methods: Competitive bone marrow transplant assays were used to determine the development of clonal expansion in response to cigarette smoke and E-cigarette aerosols using Tet2 knockout (Tet2 -/-), Dnmt3a R878H and Jak2 V617F genetically modified mice. Lethally irradiated recipient mice in the CD45.1/2 background were transplanted with whole bone marrow cells from wild type (WT) (CD45.1) and mutant (CD45.2) mice. Ratio of cells transplanted were 1:10 for Tet2 -/- and WT; 1:5 for Dnmt3a R878H and WT and 1:1 for Jak2 V617F and WT. Transplanted mice were exposed to cigarette smoke or E-cigarette aerosols using a nose-only inhalation exposures system for 2 hours/day, 4 days/week for 2 or 3 months. Control mice were exposed to room air using the nose-only inhalation approach. Results: After 2 months of exposure, we observed that Tet2 -/- cells had significantly expanded in the smoke group (paired t-test, p<0.05) while there was no significant difference in the air group (Fig. 1A). Furthermore, this increase in Tet2 -/- cells was more pronounced in the myeloid cell subset (Fig. 1B). While the knock-in mouse model of Jak2 V617F does not display a competitive advantage in a lethally irradiated bone marrow transplant setting, we observed persisting levels of Jak2 V617F mutant cells following smoke exposure but significantly reduced levels in the air group, illustrating that the mutant cells prevail in a smoke environment (Fig. 1C). During sacrifice of the Jak2 V617F transplanted and exposed mice, long-term HSCs in the bone marrow exhibited a trend towards increased bromodeoxyuridine (BrdU) incorporation and increased DNA damage as determined by H2AX staining (Fig 1D). Meanwhile, E-cigarette aerosol exposure of mice transplanted with Dnmt3a R878H cells, displayed increased levels of circulating mutant cells compared to the air group (Fig. 1E) (repeated measures, 2-way ANOVA). Conclusion: In vivo exposure of mouse models of CHIP to cigarette smoke and E-cigarette aerosols promotes mutant cell expansion over time. Our data indicate that more than one mutation is selected by environmental factors in the form of tobacco products. This data is important to guide us in preventive medicine and early detection of clonal hematopoiesis. Future research is aimed at deciphering the molecular responses of WT cells to cigarette smoke and E-cigarette aerosols and strategies to preserve WT stem cell fitness in the context of smoking and E-cigarette usage. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Shimada, Tsuyoshi, Nobuhiko Hiramatsu, Kunihiro Hayakawa, Shuhei Takahashi, Ayumi Kasai, Yasuhiro Tagawa, Mai Mukai, Jian Yao, Yoshiaki Fujii-Kuriyama, and Masanori Kitamura. "Dual suppression of adipogenesis by cigarette smoke through activation of the aryl hydrocarbon receptor and induction of endoplasmic reticulum stress." American Journal of Physiology-Endocrinology and Metabolism 296, no. 4 (April 2009): E721—E730. http://dx.doi.org/10.1152/ajpendo.90829.2008.
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Cigarette smoking decreases body weight, whereas molecular mechanisms underlying this phenomenon have not been elucidated. In this report, we investigated regulation of adipogenesis by cigarette smoke and involvement of aryl hydrocarbon receptor (AhR) and endoplasmic reticulum (ER) stress. We found that cigarette smoke extract (CSE) inhibited differentiation of preadipocytes into adipocytes dose dependently. It was associated with a decrease in lipid accumulation, blunted expression of adipocyte markers (adiponectin, PPAR-γ, and C/EBPα), and sustained expression of a preadipocyte marker MCP-1. CSE markedly induced activation of AhR, and AhR agonists (2,3,7,8-tetrachlorodibenzo- p-dioxin, benzo[ a]pyrene and 3-methylcholanthrene) reproduced the inhibitory effect of CSE on adipocyte differentiation. Furthermore, knockout of the AhR gene or blockade of AhR by a dominant-negative mutant attenuated the suppressive effects of CSE on adipocyte differentiation. We also found that CSE induced ER stress in preadipocytes, and ER stress inducers (thapsigargin, tunicamycin, and A23187) reproduced the suppressive effect of CSE on the differentiation of preadipocytes. Interestingly, AhR agonists did not cause ER stress, and ER stress inducers did not activate AhR. These results suggested that cigarette smoke has the potential to inhibit adipocyte differentiation via dual, independent mechanisms, i.e., through activation of the AhR pathway and induction of the unfolded protein response.
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Sedensky, Margaret M., Melissa A. Pujazon, and Phil G. Morgan. "Tail Clamp Responses in Stomatin Knockout Mice Compared with Mobility Assays in Caenorhabditis elegans during Exposure to Diethyl Ether, Halothane, and Isoflurane." Anesthesiology 105, no. 3 (September 1, 2006): 498–502. http://dx.doi.org/10.1097/00000542-200609000-00013.
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Background The gene unc-1 plays a central role in determining volatile anesthetic sensitivity in Caenorhabditis elegans. Because different unc-1 alleles cause strikingly different phenotypes in different volatile anesthetics, the UNC-1 protein is a candidate to directly interact with volatile anesthetics. UNC-1 is a close homologue of the mammalian protein stomatin, for which a mouse knockout was recently constructed. Because the stomatin gene is expressed in dorsal root ganglion cells, the authors hypothesized that the knockout would have an effect on anesthetic sensitivity in mice similar to that seen in nematodes. Methods Mice were placed in semiclosed chambers and exposed to continuous flows of diethyl ether, halothane, or isoflurane in air. Using lack of response to tail clamp as an endpoint, the authors determined the EC50s for the knockout strain compared with the nonmutated parental strain. They compared the differences seen in the mouse strains with the differences seen in the nematode strains. Results Stomatin-deficient mice had a 12% increase in sensitivity to diethyl ether but no significant change in sensitivity to halothane or isoflurane compared with wild type. No defect in locomotion was noted in the mutant mouse. Conclusions Nematodes and mice with deletions of the stomatin gene both have increased sensitivity to diethyl ether. Neither nematodes nor mice with stomatin deficiencies have significantly altered sensitivity to isoflurane or halothane. The effects of stomatin deficiency cross phylogenetic boundaries and support the importance of this protein in anesthetic response and the use of C. elegans as a model for anesthetic action in mammals.
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Baumann, C., B. Davies, M. Peters, U. Kaufmann-Reiche, M. Lessl, and F. Theuring. "AKR1B7 (mouse vas deferens protein) is dispensable for mouse development and reproductive success." Reproduction 134, no. 1 (July 2007): 97–109. http://dx.doi.org/10.1530/rep-07-0022.
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AKR1B7 (aldo–keto reductase family 1, member 7; also known as mouse vas deferens protein) is a member of the AKR superfamily, and has been suggested to play a role in detoxifying processes on account of its preferred substrates, 4-hydroxynonenal and isocaproaldehyde. High levels of protein expression were found in the vas deferens and the adrenal gland, where sustained expression is dependent on androgen or ACTH respectively. Recently, a remarkable induction of AKR1B7 expression has been reported in the ovary following exogenous injections of LH. In the present study, we confirm this regulation physiologically during the estrous cycle, observingAkr1b7expression to be restricted to the theca and stromal cells of the proestrus ovary. To further investigate the role of this detoxifying enzyme in both male and female reproduction, we generated knockout mice deficient in AKR1B7. Although AKR1B7 expression in the vas deferens is considerable and tightly regulated in the ovary of wild-type animals, homozygous mutant animals were found to be viable and no reproductive phenotype was observed. Ovarian follicle maturation and spermatozoa parameters remained normal in the absence of this protein. The determination of serum progesterone revealed an increase in hormone concentration in metestrus, while progesterone was found to be decreased in the estrus phase of the cycle in knockout females.
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Ahn, Kyung, Yuji Mishina, Mark C. Hanks, Richard R. Behringer, and E. Bryan Crenshaw. "BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb." Development 128, no. 22 (November 15, 2001): 4449–61. http://dx.doi.org/10.1242/dev.128.22.4449.
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We demonstrate that signaling via the bone morphogenetic protein receptor IA (BMPR-IA) is required to establish two of the three cardinal axes of the limb: the proximal-distal axis and the dorsal-ventral axis. We generated a conditional knockout of the gene encoding BMPR-IA (Bmpr) that disrupted BMP signaling in the limb ectoderm. In the most severely affected embryos, this conditional mutation resulted in gross malformations of the limbs with complete agenesis of the hindlimbs. The proximal-distal axis is specified by the apical ectodermal ridge (AER), which forms from limb ectoderm at the distal tip of the embryonic limb bud. Analyses of the expression of molecular markers, such as Fgf8, demonstrate that formation of the AER was disrupted in the Bmpr mutants. Along the dorsal/ventral axis, loss of engrailed 1 (En1) expression in the non-ridge ectoderm of the mutants resulted in a dorsal transformation of the ventral limb structures. The expression pattern of Bmp4 and Bmp7 suggest that these growth factors play an instructive role in specifying dorsoventral pattern in the limb. This study demonstrates that BMPR-IA signaling plays a crucial role in AER formation and in the establishment of the dorsal/ventral patterning during limb development.
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Doherty, Laura, Tenzin Sangpo, Peter Tsvetkov, John Davis, Navid Dianati, Wolfgang Schwede, Katja Zimmermann, et al. "Abstract 2682: Small molecule targeting the lipoic acid post-translational modification impacts proliferation of colorectal and PIK3CA-mutant cell lines." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2682. http://dx.doi.org/10.1158/1538-7445.am2022-2682.
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Abstract To identify novel therapeutic targets, we utilize the PRISM platform, a multiplexed cell line viability technology of 500 solid tumor cell lines and correlate responses to functional genomic and baseline genetic data. We describe ESD0140656, a small molecule with selective anti-proliferative effect on colorectal and PIK3CA-mutant cell lines. Response to ESD0140656 is correlated to sensitivity to CRISPR/Cas9 KO of components of the protein lipoylation pathway and OGDH complex members, which catalyze a step of the TCA cycle. Lipoylation is a rare post-translational modification attached to just four enzymes in humans, including the OGDH complex. Knockout of the protein that transfers lipoic acid to these four enzymes (LIPT1) sensitizes cells to ESD0140656, and ESD0140656 treatment leads to reduction of lipoic acid in cells. These results suggest ESD0140656 targets the lipoylation pathway and may represent a novel therapeutic angle for colorectal and PIK3CA-mutant tumors. Citation Format: Laura Doherty, Tenzin Sangpo, Peter Tsvetkov, John Davis, Navid Dianati, Wolfgang Schwede, Katja Zimmermann, Laura Evans, Aldo Amatucci, Henrik Seidel, Atanas Kamburov, Gizem Akcay, Todd Golub, Ashley Eheim, Nils Burkhardt, Knut Eis, Sven Christian, Matt Rees, Jennifer Roth. Small molecule targeting the lipoic acid post-translational modification impacts proliferation of colorectal and PIK3CA-mutant cell lines [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 2682.
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Zhou, Feng, Guimei Yang, Feng Tang Tang, Yan Zhang, Shengwei Yang, Wenqing Yang, Liting Xue, Ping Chen, and Renhong Tang. "Abstract 1724: SCR-8388, a potent and selective SOS1::KRAS inhibitor, is effective in KRAS-addicted cancers." Cancer Research 83, no. 7_Supplement (April 4, 2023): 1724. http://dx.doi.org/10.1158/1538-7445.am2023-1724.
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Abstract KRAS occurs Mutation in approximately one in seven of all human cancers, making it the most frequently mutated oncogene. The activation of KRAS protein is regulated via switching the cycle between the active form, guanosine- 5′-triphosphate (GTP)-bound state and the inactive form, guanosine-5′-diphosphate (GDP)-bound form. Guanine nucleotide-exchange factors (GEF), including SOS1/2 play a key role in catalyzing the transformation of KRAS from GDP to GTP form. In tumors with dysregulation of the MAPK signal axis, inhibition of SOS1/2 leads to abrogation of the Kras-mediated activation of downstream signals. However, double depletion of SOS1/2 is embryo lethal, and selective inhibition of SOS1 provides a more balanced therapy for Kras mutant tumors. Here we reported SCR-8388 as a highly potent and SOS1- specific inhibitor that effectively inhibited multiple Kras-mutant oncoproteins. SCR-8388 was potently bound to SOS1 and blocked the interaction of KRASG12D-GDP or KRASG12C-GDP with IC50 at single-digit nanomolar level. In contrast, SCR-8388 had no effect on the interaction of KRASG12C-GDP and SOS2. In H358 (KRASG12C) cells, SCR-8388 strongly inhibited the phosphorylation of ERK1/2 with an IC50 of 20 nM. The anti-proliferation activities of SCR-8388 on Kras mutant tumor cell lines were determined using the anchorage-independent three-dimensional (3-D) growth assay. The results showed that SCR-8388 strongly inhibited cell growth in five Kras-mutant cell lines which represent the different Kras mutation genotypes, including G12D, G12C, G12S and G13D mutants, the IC50s ranged from 17 to 120 nM, suggesting the broad activities of SOS1 inhibitor against pan-Kras mutation. In contrast, SCR-8388 (at 1 µM) showed no growth inhibition on two Kras non-addicted cancer cell lines, NCI-H520 and A375. Additionally, SCR-8388 demonstrated enhanced antiproliferation activity in a SOS2 knockout cell line (H358SOS2-/-) in comparison with the parental cells, indicating the high selectivity of SCR-8388 over SOS2. SCR-8388 displayed favorable PK properties, and higher tissue exposure, making SCR-8388 achieve effective tumor growth inhibition. In a subcutaneous MIA PaCa-2 xenograft model, oral administration of SCR-8388 resulted in superior tumor growth control in comparison with the clinical reference. Moreover, the robust anti-tumor efficacy was further observed in another KRASG13D-mutated DLD-1 xenograft model. Furthermore, SCR-8388 exhibited synergistic anti-tumor activity in combination with several MAPK pathway inhibitors, including KRASG12C, MEK, and ERK inhibitors as excepted. Notably, SCR-8388 demonstrated synergistic effects with the anti-EGFR antibody (cetuximab) both in Kras-mutant and WT cell lines in vitro and in vivo. In summary, SCR-8388 is a highly potent and selective SOS1 inhibitor, and effectively inhibits Kras-mutant tumors as mono- or combo therapeutics. Citation Format: Feng Zhou, Guimei Yang, Feng Tang Tang, Yan Zhang, Shengwei Yang, Wenqing Yang, Liting Xue, Ping Chen, Renhong Tang. SCR-8388, a potent and selective SOS1::KRAS inhibitor, is effective in KRAS-addicted cancers [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 1724.
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Krishna, Avantika, Michael J. Clowers, Bo Yuan, Milind Mutala, Maria Jose Arredondo Sancristobal, Jocelynn Colunga, Ryan De Maleki, Linda Phan, Humam Kadara, and Seyed Javad Moghaddam. "Abstract 652: Tumor cell-specific IL-1/IL-1R signaling promotes KRAS mutant lung tumorigenesis." Cancer Research 83, no. 7_Supplement (April 4, 2023): 652. http://dx.doi.org/10.1158/1538-7445.am2023-652.
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Abstract Despite improved diagnosis and treatment strategies such as immunotherapy, lung cancer is still the leading cause of cancer-related deaths worldwide in both men and women. Lung adenocarcinoma (LUAD) with driver mutations in the KRAS oncogene is the most prevalent molecular subtype of lung cancer. KRAS mutant LUAD exhibits aggressive biology in part due to enhanced pro-tumor inflammation mediated by activation of the nuclear factor-κB (NF-κB) and, consequently, elevated expression of various cytokines. While the pro-inflammatory cytokine IL-1β is a product of the NF-κB pathway it also acts as a potent activator, further amplifying the production of protumor cytokines via a positive feedback loop. We have shown that IL-1β blockade enhances anti-tumor immune responses while inhibiting immunosuppression in a mouse model of KRAS mutant LUAD driven by lung epithelial cell-specific expression of KRASG12D (CCSPCre/LSL-KRASG12D, CC-LR mouse). Cell lineage-specific mechanisms that underlie these anti-tumor effects following inhibition of IL-1β are still poorly understood. To fill this void, we here explored the effects of targeting the IL-1R receptor in KRAS mutant lung epithelial cells in the CC-LR mouse model. We studied tumor development and host immune response in 14-week-old CC-LR mice with conditional knockout of IL-1R in KRAS mutant lung epithelial cells (LR/IL-1RΔ/Δ) in comparison to age- and sex-matched control CC-LR littermates. LR/IL-1RΔ/Δ mice displayed markedly reduced tumor multiplicity (~50%) when compared to control CC-LR mice concomitant with decreased cell proliferation and angiogenesis evidenced by attenuated immunohistochemical expression of Ki-67 and ERG. Flow cytometry analysis showed an elevated inflammatory response, most evidently seen through the significant increase in infiltrating monocytes. Interestingly, a shift in type-1 conventional dendritic cells, commonly involved in antigen-cross presentation in tumorigenesis, to type-2 conventional dendritic cells was seen in LR/IL-1RΔ/Δ mice, suggesting a different method of cross-presentation resulting from the conditional knockout of IL-1R that could potentially lead to a protective T-cell response. These results were further confirmed via gene expression analysis of respective markers. Our findings provide further support for the role of the IL-1 cytokine family in the development and progression of KRAS mutant LUAD as well as warrant further studies targeted towards understanding the mechanistic effects IL-1β has on the tumor microenvironment. Citation Format: Avantika Krishna, Michael J. Clowers, Bo Yuan, Milind Mutala, Maria Jose Arredondo Sancristobal, Jocelynn Colunga, Ryan De Maleki, Linda Phan, Humam Kadara, Seyed Javad Moghaddam. Tumor cell-specific IL-1/IL-1R signaling promotes KRAS mutant lung tumorigenesis [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 652.
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Yau, Edwin H., Bojidar Kandar, Lei Deng, Te-An Chen, Wiam Bshara, Sean Glenn, Sarabjot Pabla, et al. "Abstract 1320: Immunogenomic analysis identifies the complement pathway as a therapeutic target in STK11 mutant non-small cell lung cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1320. http://dx.doi.org/10.1158/1538-7445.am2022-1320.
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Abstract Background: The use of immune PD-1/-L1 checkpoint inhibitors (ICI) has dramatically altered the treatment of advanced non-small cell lung cancer (NSCLC). However, a large proportion of patients with NSCLC do not derive clinical benefit from ICI treatment. Recent studies have identified certain genomic subsets of NSCLC as drivers of primary resistance to ICI treatment. Methods: We evaluated NSCLC patients treated at Roswell Park Comprehensive Cancer Center from 2017-2020 with successful genomic and immune profiling using a CLIA-certified laboratory developed test that included targeted next-generation genomic sequencing, PD-L1 assessment by immunohistochemistry (IHC), and targeted RNA-seq of 394 immune transcripts. Results: A total of 379 treatment-naive non-squamous NSCLC subjects were identified with 113 subjects treated with an FDA approved ICI regimen and evaluable for response. Using a gene expression signature based on differential gene expression analysis in the 113 subjects with and without disease control with ICI treatment, we performed unsupervised clustering of the larger 379 subject cohort and identified a responder (R) cluster and non-responder (NR) cluster. The R cluster was associated with high PD-L1 expression, T-cell infiltration, and KRAS mutations. The NR cluster was associated with negative PD-L1 expression, lack of T-cell infiltration, and mutations in STK11, APC/CTNNB1, and NFE2L2. Of the mutation subsets, STK11 was most associated with NR with a trend towards lower PD-L1 expression by clinical IHC, but much more significantly associated by gene expression signatures with low PD-L1 and T-cell inflammation. In our gene panel, we identified elevated TRIM29 expression in STK11 mutant NSCLC tumors and confirmed TRIM29 expression by IHC on a separate 64 subject cohort with known STK11 mutation status and archival formalin fixed paraffin embedded (FFPE) tissue. A subset of STK11 mutant NSCLC cell lines also demonstrated elevated TRIM29 expression and we generated knockouts of TRIM29 with CRISPR-Cas9 in these cell lines which resulted in decreased tumor growth in xenograft models. Using Nanostring nCounter multiplex inflammation gene expression panel on both the 64 patient FFPE cohort and TRIM29 knockout cell lines, we identified the innate immune complement pathway as a target of therapeutic interest and specifically the high expression of complement component C3 by TRIM29 high tumor cells. We then generated a syngeneic mouse model where loss of Stk11 resulted in elevated expression of Trim29 and C3 for preclinical studies to evaluate the therapeutic modulation of the complement pathway on response to ICI. Conclusion: Using a large clinical cohort of non-squamous NSCLC patients we characterized the immune environment of mutational subsets of NSCLC and identified the complement pathway as a potential therapeutic target in STK11 mutant NSCLC. Citation Format: Edwin H. Yau, Bojidar Kandar, Lei Deng, Te-An Chen, Wiam Bshara, Sean Glenn, Sarabjot Pabla, Antonios Papanicolau-Sengos, Mary Nesline, Hongbin Chen, Amy Early, Carl Morrison, Grace Dy. Immunogenomic analysis identifies the complement pathway as a therapeutic target in STK11 mutant non-small cell lung cancer [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 1320.
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Wei, Wei, Mitchell Geer, Xinyi Guo, Neville Sanjana, and Benjamin G. Neel. "Abstract 659: Mechanisms of resistance to SHP2 inhibition." Cancer Research 82, no. 12_Supplement (June 15, 2022): 659. http://dx.doi.org/10.1158/1538-7445.am2022-659.
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Abstract SHP2 (PTPN11) is required for RAS activation, acting upstream of SOS1/2. Allosteric SHP2 inhibitors (SHP2is) stabilize auto-inhibition mediated by N-SH2/PTP interactions and prevent its activation by upstream stimuli. SHP2is impair the proliferation of oncogenic RTK- or cycling RAS mutant-expressing tumor cells and can overcome adaptive resistance to single agents targeting the RAS-MAPK-ERK pathway (e.g., EGFR, KRASG12C, BRAFV600E, MEK inhibitors). Multiple SHP2is are in clinical trials as single agents or in various combinations. As resistance to targeted therapy is universal, we sought to prospectively identify potential SHP2-extrinsic resistance mechanisms by performing genome-wide CRISPR/Cas9 knockout screens on two SHP2i-sensitive human FLT3-ITD driven AML cell lines, MOLM13 and MV4:11. We identified several expected “hits” based on known signaling pathways, including tumor suppressor (NF1, PTEN,CDKN1B) and “RASopathy” (LZTR1, RASA2) genes, as well as novel targets including INPPL1,MAP4K5, and some epigenetic modifiers. To test the generality of these findings, we built a“mini-CRISPR library of ~30 hits common to MOLM13 and MV4:11 cells and screened 15 SHP2i-sensitive lines. LZTR1 deletion conferred resistance in 14/15, followed by MAP4K5 (9/15),SPRED2 (6/15), STK40 (6/15), INPPL1 (5/15), NCOA6 (4/15), NCOR1 (4/15), and ELAVL1 (4/15). LZTR1 has been reported to regulate RIT or RAS stability. Notably, LZTR1 knockout universally increased RIT1, but in some lines, also increased RAS. INPPL1 encodes SHIP2, a 5’-inositide phosphatase that negatively regulates AKT activation in insulin signaling. However, INPPL1 deletion also increased RAS and ERK activity and activated ERK-dependent genes in FLT3-ITD AML lines. Experiments with INPPL1 mutant showed that SHC binding and an N-terminal region, but not the SH2 domain or phosphatase activity, is required for negative regulation of RAS. Interestingly, INPPL1 deletion also promoted resistance to SHP2 inhibition in several EGFR mutant cell lines. MAP4K5 deletion also increased ERK-dependent gene expression. Kinase activity, but not JNK activation, was required for MAP4K5 action. Our results predict multiple mechanisms of SHP2i resistance, emphasizing the need for detailed understanding of the resistance landscape to arrive a combinations that provide long term disease control. Citation Format: Wei Wei, Mitchell Geer, Xinyi Guo, Neville Sanjana, Benjamin G. Neel. Mechanisms of resistance to SHP2 inhibition [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 659.
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Valentine, Henkel L., Uttam Satyal, and Philip H. Abbosh. "Abstract 942: Development of customizable bladder cancer mouse model using CRISPR." Cancer Research 82, no. 12_Supplement (June 15, 2022): 942. http://dx.doi.org/10.1158/1538-7445.am2022-942.
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Abstract Introduction: In forward genetics, a phenotype (sensitivity to therapy) is attributed to a genotype (mutation in relevant gene) based on findings in anecdotal cases (e.g., extreme responders) with little systematic/experimental study. Although powerful, this approach is not widely applicable due to the limited availability of clinical materials and throughput thus limiting the number and type of hypotheses that can be rigorously tested. However, a genotype-therapy sensitivity relationship could be measured in isogenic systems using reverse genetics, whereby mutations in relevant genes are introduced followed by evaluation of a phenotype in wild-type and mutant contexts. Since no such pre-clinical system exists for bladder cancer, we developed an immunocompetent mouse model to induce customizable isogenic tumor formation in bladder urothelium using CRISPR. Methods: Bladder urothelium was harvested from transgenic mice harboring LSL-Cas9/GFP in Rosa26 locus (as described by Platt et al) and grown as organoids. These organoids had a basal phenotype. AAV harboring Cre and sgRNA(s) were used to induce Cas9 and engineer mutations. Based on their frequent somatic alteration pathway in TCGA-BLCA, Tp53 and Rb1 were chosen for knockout first to establish ‘base organoids’. Additional genes related to PI3K/AKT/mTOR pathway were edited in base organoids to develop a series of organoids with different genotypes predicted to activate this signaling pathway. Sanger sequencing and western blot were used to confirm knockout. Kinase inhibitors targeting the mTOR pathway at different levels were assessed in organoids with different genotypes. The edited urothelial cells were grafted in mouse bladder to develop isogenic tumor to assess in vivo therapeutic vulnerability of in-vitro-prioritized kinase inhibitors matching the genotype of the allografted tumor. Results: Rb1, Tp53, and Pten were successfully knocked out in urothelial organoids. When treated with Ipataseritib, an AKT inhibitor, the triple knockout organoid was found to be 9X more sensitive than Trp53/Rb1 knockout organoids. In contrast, triple knockout organoids were 15X less sensitive to Gefitinib, an EGFR inhibitor, than Trp53/Rb1 knockout organoids, in line with published data. Edited organoids generate a neo-urothelium in murine bladder in short term assays. Conclusions: We developed a flexible bladder cancer mouse model to study mutation-therapy relationship using reverse genetics. This system is a plausible way to uncover unappreciated therapeutic vulnerabilities that probably exist in bladder cancer. Citation Format: Henkel L. Valentine, Uttam Satyal, Philip H. Abbosh. Development of customizable bladder cancer mouse model using CRISPR [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 942.
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Chan, Pan F., and Simon J. Foster. "Role of SarA in Virulence Determinant Production and Environmental Signal Transduction in Staphylococcus aureus." Journal of Bacteriology 180, no. 23 (December 1, 1998): 6232–41. http://dx.doi.org/10.1128/jb.180.23.6232-6241.1998.
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ABSTRACT The staphylococcal accessory regulator (encoded bysarA) is an important global regulator of virulence factor biosynthesis in Staphylococcus aureus. To further characterize its role in virulence determinant production, ansarA knockout mutant was created by insertion of a kanamycin antibiotic resistance cassette into the sarAgene. N-terminal sequencing of exoproteins down-regulated bysarA identified several putative proteases, including a V8 serine protease and a novel metalloprotease, as the major extracellular proteins repressed by sarA. In kinetic studies, thesarA mutation delays the onset of α-hemolysin (encoded byhla) expression and reduces levels of hla to approximately 40% of the parent strain level. Furthermore, SarA plays a role in signal transduction in response to microaerobic growth since levels of hla were much lower in a microaerobic environment than after aerobic growth in the sarA mutant. An exoprotein exhibiting hemolysin activity on sheep blood, and up-regulated bysarA independently of the accessory gene regulator (encoded by agr), was specifically induced microaerobically. Transcriptional gene fusion and Western analysis revealed thatsarA up-regulates both toxic shock syndrome toxin 1 gene (tst) expression and staphylococcal enterotoxin B production, respectively. This study demonstrates the role ofsarA as a signal transduction regulatory component in response to aeration stimuli and suggests that sarAfunctions as a major repressor of protease activity. The possible role of proteases as regulators of virulence determinant stability is discussed.
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Ito, Chizuru, Kenji Yamatoya, Keiichi Yoshida, Lisa Fujimura, Hajime Sugiyama, Akiko Suganami, Yutaka Tamura, Masahiko Hatano, Kenji Miyado, and Kiyotaka Toshimori. "Deletion of Eqtn in mice reduces male fertility and sperm–egg adhesion." Reproduction 156, no. 6 (December 2018): 579–90. http://dx.doi.org/10.1530/rep-18-0394.
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A number of sperm proteins are involved in the processes from gamete adhesion to fusion, but the underlying mechanism is still unclear. Here, we established a mouse mutant, the EQUATORIN-knockout (EQTN-KO, Eqtn − / − ) mouse model and found that the EQTN-KO males have reduced fertility and sperm–egg adhesion, while the EQTN-KO females are fertile. Eqtn − / − sperm were normal in morphology and motility. Eqtn − / − -Tg (Acr-Egfp) sperm, which were produced as the acrosome reporter by crossing Eqtn − / − with Eqtn +/+ -Tg(Acr-Egfp) mice, traveled to the oviduct ampulla and penetrated the egg zona pellucida of WT females. However, Eqtn − / − males mated with WT females showed significant reduction in both fertility and the number of sperm attached to the zona-free oocyte. Sperm IZUMO1 and egg CD9 behaved normally in Eqtn − / − sperm when they were fertilized with WT egg. Another acrosomal protein, SPESP1, behaved aberrantly in Eqtn − / − sperm during the acrosome reaction. The fertility impairment of EQTN/SPESP1-double KO males lacking Eqtn and Spesp1 (Eqtn/Spesp1 − / − ) was more severe compared with that of Eqtn − / − males. Eqtn − / − -Tg (Eqtn) males, which were generated to rescue Eqtn − / − males, restored the reduced fertility.
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Riley, Amanda K., Athea Vichas, Naomi T. Nkinsi, Phoebe C. Parrish, Shriya Kamlapurkar, and Alice H. Berger. "Abstract 2972: Identification of USP9X as a novel regulator of RIT1 protein abundance and as a potential therapeutic target in RIT1-driven lung cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2972. http://dx.doi.org/10.1158/1538-7445.am2022-2972.
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Abstract Standard care of lung cancer treatment has shifted away from non-specific, cytotoxic chemotherapy in favor of targeted therapies based on genetic mutations within tumors. In 2014, somatic mutations in the small GTPase RIT1 (Ras-like in all tissues) were discovered as oncogenic drivers of lung adenocarcinoma. Thousands of patients per year are diagnosed with RIT1-driven cancer, but treatment options are limited. A targeted therapy for RIT1-driven tumors would address a major unmet clinical need. Little is known about how RIT1 drives cellular transformation. To genetically dissect RIT1 function, we performed a genome-wide CRISPR/Cas9 screen in isogenic PC9 lung adenocarcinoma cells. This screen took advantage of the observation that RIT1-mutant cells are resistant to EGFR inhibition. We leveraged this drug resistance phenotype to identify genetic dependencies (gene knockouts that are detrimental to cell growth) and cooperating factors (gene knockouts that are beneficial to cell growth) in RIT1-mutant cells. From this screen, we found that one of the top essential genes was the deubiquitinase USP9X. This is intriguing given that the protein abundance of RIT1 is known to be important for its function. Therefore, we sought out to test the hypothesis that USP9X regulates RIT1 abundance and that inhibition of USP9X could be an effective therapeutic strategy for abrogating RIT1-driven tumor growth. Our model suggests that USP9X promotes proteasome-mediated degradation of RIT1. To test this, we assessed RIT1 abundance in the context of USP9X knockout (KO). We found that RIT1 protein abundance was decreased in USP9X KO PC9 cells compared to parental cells. Furthermore, cycloheximide (CHX)-chase experiments revealed that RIT1 stability was decreased in USP9X KO cells, and RIT1 degraded faster than in parental cells. The average half-life of RIT1 in USP9X KO cells was 3.4 hrs while the average half-life in parental PC9 cells was 12.3 hrs (95% CI = -12.5 to -5.4 hrs). Treatment with the proteasome inhibitor bortezomib (BTZ) rescued RIT1 degradation by 99% in parental cells and 190% in USP9X KO cells (95% CI = 57 to 126%). In addition to assessing protein abundance and stability, we performed co-immunoprecipitation experiments in RIT1-expressing HEK293T cells and found that RIT1 and USP9X physically interact. Taken together, these data support the hypothesis that RIT1 is a substrate of USP9X. In addition to providing better insight on the protein regulation of RIT1, this work has crucial therapeutic implications. The protein abundance of RIT1 is important for its function, and our model suggests that USP9X inhibition could be an effective means of reducing RIT1 protein abundance and abrogating tumor growth. Overall, this work is poised to significantly impact the field of RIT1 biology and address a major unmet clinical need for the treatment of RIT1-driven diseases. Citation Format: Amanda K. Riley, Athea Vichas, Naomi T. Nkinsi, Phoebe C. Parrish, Shriya Kamlapurkar, Alice H. Berger. Identification of USP9X as a novel regulator of RIT1 protein abundance and as a potential therapeutic target in RIT1-driven lung cancer [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 2972.
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Portnoy, Vasiliy A., David A. Scott, Nathan E. Lewis, Yekaterina Tarasova, Andrei L. Osterman, and Bernhard Ø. Palsson. "Deletion of Genes Encoding Cytochrome Oxidases and Quinol Monooxygenase Blocks the Aerobic-Anaerobic Shift in Escherichia coli K-12 MG1655." Applied and Environmental Microbiology 76, no. 19 (August 13, 2010): 6529–40. http://dx.doi.org/10.1128/aem.01178-10.
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ABSTRACT The constitutive activation of the anoxic redox control transcriptional regulator (ArcA) in Escherichia coli during aerobic growth, with the consequent production of a strain that exhibits anaerobic physiology even in the presence of air, is reported in this work. Removal of three terminal cytochrome oxidase genes (cydAB, cyoABCD, and cbdAB) and a quinol monooxygenase gene (ygiN) from the E. coli K-12 MG1655 genome resulted in the activation of ArcA aerobically. These mutations resulted in reduction of the oxygen uptake rate by nearly 98% and production of d-lactate as a sole by-product under oxic and anoxic conditions. The knockout strain exhibited nearly identical physiological behaviors under both conditions, suggesting that the mutations resulted in significant metabolic and regulatory perturbations. In order to fully understand the physiology of this mutant and to identify underlying metabolic and regulatory reasons that prevent the transition from an aerobic to an anaerobic phenotype, we utilized whole-genome transcriptome analysis, 13C tracing experiments, and physiological characterization. Our analysis showed that the deletions resulted in the activation of anaerobic respiration under oxic conditions and a consequential shift in the content of the quinone pool from ubiquinones to menaquinones. An increase in menaquinone concentration resulted in the activation of ArcA. The activation of the ArcB/ArcA regulatory system led to a major shift in the metabolic flux distribution through the central metabolism of the mutant strain. Flux analysis indicated that the mutant strain had undetectable fluxes around the tricarboxylic acid (TCA) cycle and elevated flux through glycolysis and anaplerotic input to oxaloacetate. Flux and transcriptomics data were highly correlated and showed similar patterns.
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Dahan, Albert, Elise Sarton, Luc Teppema, Cees Olievier, Diederik Nieuwenhuijs, Hans W. D. Matthes, and Brigitte L. Kieffer. "Anesthetic Potency and Influence of Morphine and Sevoflurane on Respiration in μ-Opioid Receptor Knockout Mice." Anesthesiology 94, no. 5 (May 1, 2001): 824–32. http://dx.doi.org/10.1097/00000542-200105000-00021.
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Background The involvement of the mu-opioid receptor (muOR) system in the control of breathing, anesthetic potency, and morphine- and anesthesia-induced respiratory depression was investigated in mice lacking the muOR. Methods Experiments were performed in mice lacking exon 2 of the muOR gene (muOR-/-) and their wild-type littermates (muOR+/+). The influence of saline, morphine, naloxone, and sevoflurane on respiration was measured using a whole body plethysmographic method during air breathing and elevations in inspired carbon dioxide concentration. The influence of morphine and naloxone on anesthetic potency of sevoflurane was determined by tail clamp test. Results Relative to wild-type mice, muOR-deficient mice displayed approximately 15% higher resting breathing frequencies resulting in greater resting ventilation levels. The slope of the ventilation-carbon dioxide response did not differ between genotypes. In muOR+/+ but not muOR-/- mice, a reduction in resting ventilation and slope, relative to placebo, was observed after 100 mg/kg morphine. Naloxone increased resting ventilation and slope in both genotypes. Sevoflurane at 1% inspired concentration induced similar reductions in resting ventilation and slope in the two genotypes. Anesthetic potency was 20% lower in mutant relevant to wild-type mice. Naloxone and morphine caused an increase and decrease, respectively, in anesthetic potency in muOR+/+ mice only. Conclusions The data indicate the importance of the endogenous opioid system in the physiology of the control of breathing with only a minor role for the muOR. The muOR gene is the molecular site of action of the respiratory effects of morphine. Anesthetic potency is modulated by the endogenous mu-opioid system but not by the kappa- and delta-opioid systems.
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Wei, George Zhang, Katherine A. Martin, Peter Yuli Xing, Ruchi Agrawal, Luke Whiley, Thomas K. Wood, Sophia Hejndorf, et al. "Tryptophan-metabolizing gut microbes regulate adult neurogenesis via the aryl hydrocarbon receptor." Proceedings of the National Academy of Sciences 118, no. 27 (July 1, 2021): e2021091118. http://dx.doi.org/10.1073/pnas.2021091118.
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While modulatory effects of gut microbes on neurological phenotypes have been reported, the mechanisms remain largely unknown. Here, we demonstrate that indole, a tryptophan metabolite produced by tryptophanase-expressing gut microbes, elicits neurogenic effects in the adult mouse hippocampus. Neurogenesis is reduced in germ-free (GF) mice and in GF mice monocolonized with a single-gene tnaA knockout (KO) mutant Escherichia coli unable to produce indole. External administration of systemic indole increases adult neurogenesis in the dentate gyrus in these mouse models and in specific pathogen-free (SPF) control mice. Indole-treated mice display elevated synaptic markers postsynaptic density protein 95 and synaptophysin, suggesting synaptic maturation effects in vivo. By contrast, neurogenesis is not induced by indole in aryl hydrocarbon receptor KO (AhR−/−) mice or in ex vivo neurospheres derived from them. Neural progenitor cells exposed to indole exit the cell cycle, terminally differentiate, and mature into neurons that display longer and more branched neurites. These effects are not observed with kynurenine, another AhR ligand. The indole-AhR–mediated signaling pathway elevated the expression of β-catenin, Neurog2, and VEGF-α genes, thus identifying a molecular pathway connecting gut microbiota composition and their metabolic function to neurogenesis in the adult hippocampus. Our data have implications for the understanding of mechanisms of brain aging and for potential next-generation therapeutic opportunities.
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Stanland, Lyla, Hazel Ang, Jacob Hoj, Yunqiang Chu, Patrick Tan, Kris Wood, and Micah Luftig. "Abstract 4949: Identification of novel regulators of response to PI3Ka inhibition in PIK3CA mutant gastric cancer." Cancer Research 83, no. 7_Supplement (April 4, 2023): 4949. http://dx.doi.org/10.1158/1538-7445.am2023-4949.
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Abstract PIK3CA, encoding the active alpha isoform of PI3K, is the second most commonly mutated gene in cancer leading to aberrant PI3K/AKT/mTOR signaling and increased protein translation, glucose metabolism, cellular proliferation, survival and migration. Some 4-25% of gastric cancers (GC) display activating PIK3CA mutations including 80% of the EBV-associated GC subset. Small molecules including pan-PI3K, dual PI3K/mTOR, and pan-AKT inhibitors have shown only moderate clinical success, primarily due to high toxicity and off target effects. Even isoform specific PI3K inhibitors, such as PI3Kα inhibitors, have displayed similar clinical problems. Importantly, PI3Kα mutation-selective inhibitors are on the horizon and will be key for treatment of PIK3CA mutant tumors. However, single agent drug resistance is still an anticipated clinical problem and both intrinsic and acquired resistance will affect success of new drugs. There has been a concerted effort to define mechanisms of resistance as well as identify synergistic drug combinations. In this study we aimed to identify mechanisms of sensitivity and resistance to the PI3Kα-specific inhibitor, BYL719, in PIK3CA mutant gastric cancers by using a CRISPR/Cas9-based screening approach. We found that loss of either NEDD9 or BCL-XL was synergistic with BYL719 and led to increased cell cycle arrest and apoptosis, respectively. In addition to identifying these sensitizer genes, the screening approach allowed us to model intrinsic resistance, and we discovered that knockout of the translation and transcription factor, CBFB, conferred resistance to BYL719. To model acquired resistance, we cultured cells in increasing concentrations of BYL719 over three months and generated clones that were resistant when compared to parental cells. We found that in the clones with acquired resistance, CBFB was significantly down-regulated at the protein and RNA level suggesting a conserved mechanism of drug resistance. We found that CBFB loss led to up-regulation of the serine/threonine protein kinase PIM1, which can phosphorylate and activate several of the same downstream substrates as AKT thereby maintaining pathway activity in the presence of PI3Kα inhibition. Furthermore, a pan-PIM inhibitor re-sensitized both CBFB knockout and acquired resistant cells to BYL719. In the TCGA clinical dataset, we found that CBFB is over-expressed in gastric tumors, specifically in the EBV-associated subset, compared to normal tissue and is significantly over-expressed in PIK3CA mutant compared to wild type tumors. Consistently, we found that PIM1 expression displays the inverse phenotype. These data suggest that while CBFB loss is a key step in the development of PI3Kα inhibitor resistance, PIK3CA mutant gastric tumors may be good candidates for clinical success with BYL719 alone and resistance could be prevented with combination with a PIM kinase inhibitor. Citation Format: Lyla Stanland, Hazel Ang, Jacob Hoj, Yunqiang Chu, Patrick Tan, Kris Wood, Micah Luftig. Identification of novel regulators of response to PI3Ka inhibition in PIK3CA mutant gastric 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 4949.
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Costa, Emily, Corrin Wohlheiter, Samuel Tischfield, Alister Funnell, JT Poirier, Álvaro Quintanal Villalonga, Triparna Sen, and Charles Rudin. "Abstract 859: Characterizing SMARCA4/STK11/KEAP1 co-mutant lung adenocarcinoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 859. http://dx.doi.org/10.1158/1538-7445.am2022-859.
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Abstract Lung adenocarcinoma (LUAD), constituting 50% of non-small cell lung cancer, is classically defined by the presence of driver mutations in oncogenes such as KRAS, EGFR, and BRAF. While the development of driver-targeted therapies has significantly improved survival in subsets of patients with LUAD, studies examining tumor sequencing data and clinical outcomes have shown that intra-driver genetic and phenotypic heterogeneity underlie differential patient responses to these therapies. In particular, co-occurring alterations in the genes SMARCA4 (BRG1), STK11 (LKB1), and KEAP1 are predictive of exceptionally poor prognosis and worse overall survival in metastatic LUAD patients. SMARCA4 loss is also a hallmark of undifferentiated, highly chemoresistant NSCLC tumors. While these co-mutations most often appear in KRAS-driven tumors, their association with poor outcomes is maintained regardless of driver status. Studies exploring the molecular features of SMARCA4-deficient and STK11/KEAP1 co-mutant LUAD have yielded translational insights specific to these subsets, establishing a rationale to characterize the unique biology of SMARCA4/STK11/KEAP1 (BLK) triple-mutant LUAD. To this end, we are profiling BLK LUAD through a combination of genomic, transcriptomic, epigenomic, and phenotypic assays in in vitro models. We have generated paired isogenic in vitro models of BLK LUAD via CRISPR-Cas9 knockout. Phenotypic analyses of these cell lines, whose engineered genotypes model varying combinations of SMARCA4, KEAP1, and STK11 loss, have revealed growth rate differences in vitro, and pairwise comparisons of RNA-sequencing in these lines show downregulation of lung differentiation markers and upregulation of metastasis-associated genes concomitant with SMARCA4 loss. In conclusion, we have generated isogenic preclinical models showing molecular and phenotypic features consistent with observations reported in BLK LUADs. Further molecular and functional characterization of these models may reveal mechanistic insights into the biology of this subset of aggressive tumors. Citation Format: Emily Costa, Corrin Wohlheiter, Samuel Tischfield, Alister Funnell, JT Poirier, Álvaro Quintanal Villalonga, Triparna Sen, Charles Rudin. Characterizing SMARCA4/STK11/KEAP1 co-mutant lung adenocarcinoma [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 859.
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Murimwa, Gilbert Z., Francesca Rossi, Henry K. Fleming, Zeynep Yazgan, Dina Alzhanova, Huocong Huang, and Rolf A. Brekken. "Abstract 3641: Loss of Smad4 induces SPP1 secretion and immunosuppressive myeloid cell formation in pancreatic ductal adenocarcinoma." Cancer Research 83, no. 7_Supplement (April 4, 2023): 3641. http://dx.doi.org/10.1158/1538-7445.am2023-3641.
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Abstract Up to 50% of pancreatic ductal adenocarcinomas (PDA) harbor mutations in canonical TGFß pathway genes such as SMAD4 and TGFBR2. SMAD4 mutations have been shown to drive resistance to chemotherapy, metastasis, and margin positivity following surgical resection. To characterize the unique tumor microenvironment of SMAD4 mutant PDA, we developed isogenic, orthotopic mouse models using CRISPR mediated knockout of Smad4 in a cell line derived from KPfC (KrasLSL-G12D;Trp53fl/fl;Pdx1Cre) mice, a genetically engineered mouse model of pancreatic cancer. To characterize the alteration of the secretome caused by the loss of Smad4, we performed proteomic analysis with conditioned media collected from Smad4 wildtype and mutant cancer cell clones. Interestingly, the loss of Smad4 resulted in the production of neuroendocrine related factors. In addition, gene ontology analysis with the secretome suggested that these factors were associated with neuronal processes. These data indicate that loss of Smad4 in PDA cancer cells may lead to a neuroendocrine signature change. In particular, we found one of the most up-regulated secreted factors in the mutant cells was SPP1, which has been shown to promote tumor progression by affecting cancer cell stemness and macrophage polarization. To validate the proteomic results, we established orthotopic models with Smad4 wildtype and mutant clones. We found that loss of Smad4 resulted in larger tumor growth. Moreover, immunohistochemistry showed a significant up-regulation of SPP1 in the cancer cells of Smad4 mutant tumors. To further profile the alteration of immune landscape, we stained for markers of myeloid cells and lymphocytes. We found that Smad4 mutant tumors had an immunosuppressive microenvironment characterized by increased M2 macrophage, neutrophil and MDSC infiltration. Altogether, our study suggests that loss of SMAD4 in PDA may induce a neuroendocrine signature with increased SPP1 secretion in cancer cells. The production of this unique cancer cell secretome may result in a microenvironment marked by immunosuppressive myeloid cells. Citation Format: Gilbert Z. Murimwa, Francesca Rossi, Henry K. Fleming, Zeynep Yazgan, Dina Alzhanova, Huocong Huang, Rolf A. Brekken. Loss of Smad4 induces SPP1 secretion and immunosuppressive myeloid cell formation in pancreatic ductal adenocarcinoma. [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 3641.
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Cai, Chufan, Jiayi Tu, Jeronimo Najarro, Rukang Zhang, Xue Gao, Hao Fan, Freya Zhang, et al. "Abstract 283: AHCYL1 is crucial for NRAS mutant-expressing melanoma tumor growth by governing ER calcium homeostasis." Cancer Research 83, no. 7_Supplement (April 4, 2023): 283. http://dx.doi.org/10.1158/1538-7445.am2023-283.
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Abstract Cancer cells apply metabolic adaptations for proliferation and survival such as the Warburg Effect and the maintenance of redox balance, which are common for different cancer types. However, it remains elusive whether different cancer oncogenic backgrounds require oncogene-specific metabolic adaptations, which could represent unique cancer vulnerabilities for novel therapy development. Herein we report that, using a shRNA library designed to target a subset of genes related to cell metabolism, we identified adenosylhomocysteinase like 1 (AHCYL1) as a “synthetic lethal” partner of oncogenic NRAS mutations, which is selectively important for the proliferation and survival of NRAS mutant-expressing melanoma cells, but not for cells harboring BRAF mutations. AHCYL1 is an inhibitory binding partner of endoplasmic reticulum (ER) calcium channel protein inositol trisphosphate receptor (IP3R) by competing off its agonist inositol triphosphate (IP3) and consequently blocking ER calcium release. In consonance with our finding, AHCYL1 expression levels correlate with mutational status of NRAS but not with BRAF in diverse human melanoma cell lines. In addition, exogenous expression of mutant NRAS but not BRAF in immortal melanocytes MEL-ST cells results in upregulated AHCYL1 transcription and protein expression. Notably, these findings are consistent with the results of Cancer Genome Atlas (TCGA) database analysis, which reveal elevated AHCYL1 transcription in melanoma patients harboring NRAS mutations but not BRAF V600E mutation. Further transcription factor screening studies suggested that the transcription of AHCYL1 is regulated by activating transcription factor 2 (ATF2), which is supported by TCGA database analysis results showing that ATF2 transcription level is selectively higher in NRAS-mutant melanoma patients. Furthermore, AHCYL1-deficiency by shRNA-mediated knockdown and CRISPR/Cas9-mediated knockout significantly reduces in vitro cell proliferation and in vivo tumor growth potential of NRAS mutant-expressing melanoma cells, but not for control melanoma cells expressing BRAF V600E mutant. Mechanistically, AHCYL1 deficiency in NRAS mutant-expressing melanoma cells triggers calcium leakage from the ER, leading to sustained activation of the ER unfolded protein response (UPR) that subsequently attenuates cell proliferation and initiates apoptosis. Altogether, these findings suggest a positive feedback mechanism by which mutant NRAS enhances AHCYL1 expression that contributes to NRAS mutation-dependent melanoma transformation and tumor growth. The AHCYL1-IP3R axis may represent a novel therapeutic target for treatment of NRAS mutant-expressing tumors including melanoma. Citation Format: Chufan Cai, Jiayi Tu, Jeronimo Najarro, Rukang Zhang, Xue Gao, Hao Fan, Freya Zhang, Jiacheng Li, Michele Ciboddo, Shannon Elf, Rong Wu, Jing Chen. AHCYL1 is crucial for NRAS mutant-expressing melanoma tumor growth by governing ER calcium homeostasis [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 283.
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Kazmi, Hasan Raza, Xuan Mo, Bo Zhou, Sara Preston-Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, Jonathan Soboloff, Salim Merali, and M. Raza Zaidi. "Abstract 5652: A novel anti apoptotic function of CTLA4 in melanoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5652. http://dx.doi.org/10.1158/1538-7445.am2022-5652.
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Abstract Cytotoxic T Lymphocyte Antigen 4 (CTLA4) is an important immune checkpoint protein and has been utilized as an immunotherapeutic target against melanoma. Our previous studies have shown that CTLA4 is expressed at low levels in primary human melanocytes but is upregulated in mutant-BRAF/NRAS melanoma cells and tissues. Despite predominantly intracellular localization of CTLA4, its intracellular function remains highly contradictory and unsubstantiated. We found that ectopic expression of Ctla4 in mouse melanoma cell lines substantially promoted lung colonization in allograft model systems in syngeneic mice. Moreover, similar results were observed in immunocompromised recipient mice, suggesting an intracellular cell-autonomous tumorigenic role of Ctla4 in melanoma. These finding led us to investigated intracellular functions of CTLA4 and its potential roles in melanomagenesis. Through the Ingenuity Pathway Analysis of our proteomics data, we found apoptosis as a major affected pathway in Ctla4-expressing mouse melanoma cells. We treated the cells (B2905-Ctla4-ee and EV control) with doxorubicin to induce apoptosis, followed by assessment by Annexin V assay. We found that Ctla4 expressing mouse melanoma cells were significantly resistant to doxorubicin-induced apoptosis. In reciprocal experiment, we generated (by CRISPR-Cas9) CTLA4-knockout A2058 human melanoma cells that exhibit high endogenous CTLA4 expression and we found that knockout cells showed significantly increased apoptosis than the parental cells. Moreover, we also observed significant upregulation of anti-apoptotic proteins (Bnip3, Birc6, Pak1, Mcl-1, Survivin, Rac1/Cdc42, Bcl-2, and Bnip3l) and downregulation of pro-apoptotic proteins (p53 and Bad) in CTLA4-expressing human melanoma cell lines. CTLA4-expressing cells also showed significantly higher invasion in Matrigel-coated transwell assay. These findings lead us to conclude that CTLA4 plays a significant role in regulating apoptosis and promoting melanoma progression in BRAF/NRAS mutant cells. Citation Format: Hasan Raza Kazmi, Xuan Mo, Bo Zhou, Sara Preston-Alp, Scott Gross, Hassaan Wajeeh, Carmen Merali, Jonathan Soboloff, Salim Merali, M. Raza Zaidi. A novel anti apoptotic function of CTLA4 in melanoma [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 5652.
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Qian, Yu, Irene Guijarro, Ana Galan-Cobo, Minghao Dang, Alissa Poteete, Fahao Zhang, Qi Wang, et al. "Abstract 2160: MCT4 blockade reverses lactate-mediated immunosuppression in LKB1-deficient NSCLC." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2160. http://dx.doi.org/10.1158/1538-7445.am2022-2160.
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Abstract Genomic alterations that result in loss of function of the tumor suppressor serine/threonine kinase STK11/LKB1 occur in 20-30% of lung adenocarcinomas. We previously observed that STK11/LKB1 mutations are genomic drivers of primary resistance in lung adenocarcinoma. Moreover, LKB1-mutant NSCLCs exhibit higher hypoxia and glycolysis rates, resulting in enhanced production and secretion of lactate. Accordingly, we hypothesize that high production of lactate of LKB1 mutant tumor may contribute to its immunologically cold phenotype and that blockade of the lactate pathway may potentiate the efficacy of immune checkpoint blockade (ICB) therapies. We characterized the immune landscape of LKB1 mutant clinical samples and performed scRNAseq analysis in KRAS mutant (K) and KRAS mutant LKB1 knockout (KL) syngeneic murine models. To evaluate inhibition of lactate metabolism as a therapeutic strategy, we knocked out the lactate transporter SLC16A3/MCT4 and characterized the impact on the tumor microenvironment (TME), and response to ICB. Clinical analysis of LKB1 mutant NSCLC patients from the MD Anderson’s ICON and PROSPECT cohorts suggested that LKB1 mutant tumors showed reduced immune cell infiltration, restricted T cell function, and enhanced M2-like macrophages phenotypes. Moreover, in preclinical models, LKB1 mutant tumors showed enhanced glycolysis and upregulation of MCT4 expression in a variety of human and murine cell lines. Deletion of MCT4 dramatically reduced glycolysis, energy production, and cell proliferation. By scRNAseq, we identified distinct immune subclusters modulated by LKB1 mutation. Hypofunctional T cells and M2-like macrophages were abundant in LKB1 mutant tumors, while these populations were significantly reduced in KL tumors with MCT4 KO. The conditioned medium from KL cells impaired T cell activation and decreased T cell killing, IFNγ production and glycolysis rate. Moreover, conditioned medium from KL cells induced M2-associated genes expression, as well as CD206+ expression in both peritoneal macrophages and Raw264.7 cells. These effects were at least in part MCT-dependent, as medium from MCT4 KO cells induced the opposite effects on T cells and macrophages, and the effects could be reversed by introducing exogenous lactate, suggesting that blockade of lactate transport reactivated T cells and reversed M2 polarization. Importantly, MCT4 KO in LKB1-mutant tumors sensitized tumors to anti-PD1 immunotherapy in syngeneic murine tumors and promoted long-term anti-tumor immunity. Collectively, our data indicate that LKB1 mutant tumors enhanced lactate secretion into the TME and this results in decreased T cell cytotoxic potential as well as higher pro-tumor M2 polarization, leading to resistance to immunotherapy. These data suggest that therapeutic inhibition of MCT4 is a promising strategy to overcome immunotherapy resistance in NSCLC patients harboring LKB1 mutant tumors. Citation Format: Yu Qian, Irene Guijarro, Ana Galan-Cobo, Minghao Dang, Alissa Poteete, Fahao Zhang, Qi Wang, Jing Wang, Edwin Parra, Ferdinandos Skoulidis, Ignacio Wistuba, Svena Verma, Taha Merghoub, Linghua Wang, Jedd Wolchok, Alexandre Reuben, John Heymach. MCT4 blockade reverses lactate-mediated immunosuppression in LKB1-deficient NSCLC [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 2160.