Nf1 loss promotes Kras ‐driven lung adenocarcinoma and results in Psat1‐mediated glutamate dependence
2019; Springer Nature; Volume: 11; Issue: 6 Linguagem: Inglês
10.15252/emmm.201809856
ISSN1757-4684
AutoresXiaojing Wang, Shengping Min, Hongli Liu, Nan Wu, Xincheng Liu, Tao Wang, Wei Li, Yuanbing Shen, Hongtao Wang, Zhongqing Qian, Huanbai Xu, Chengling Zhao, Yuqing Chen,
Tópico(s)Ferroptosis and cancer prognosis
ResumoResearch Article29 April 2019Open Access Source DataTransparent process Nf1 loss promotes Kras-driven lung adenocarcinoma and results in Psat1-mediated glutamate dependence Xiaojing Wang Corresponding Author Xiaojing Wang [email protected] orcid.org/0000-0001-6848-8688 Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Shengping Min Shengping Min Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Hongli Liu Hongli Liu Department of Gynecological Oncology, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Nan Wu Nan Wu Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Xincheng Liu Xincheng Liu Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Tao Wang Tao Wang Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Wei Li Wei Li Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Yuanbing Shen Yuanbing Shen Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Hongtao Wang Hongtao Wang Department of Immunology, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Zhongqing Qian Zhongqing Qian Department of Immunology, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Huanbai Xu Huanbai Xu Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated First People's Hospital, Shanghai, China Search for more papers by this author Chengling Zhao Chengling Zhao Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Yuqing Chen Corresponding Author Yuqing Chen [email protected] orcid.org/0000-0002-9783-1112 Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Xiaojing Wang Corresponding Author Xiaojing Wang [email protected] orcid.org/0000-0001-6848-8688 Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Shengping Min Shengping Min Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Hongli Liu Hongli Liu Department of Gynecological Oncology, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Nan Wu Nan Wu Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Xincheng Liu Xincheng Liu Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Tao Wang Tao Wang Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Wei Li Wei Li Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Yuanbing Shen Yuanbing Shen Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Hongtao Wang Hongtao Wang Department of Immunology, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Zhongqing Qian Zhongqing Qian Department of Immunology, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Huanbai Xu Huanbai Xu Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated First People's Hospital, Shanghai, China Search for more papers by this author Chengling Zhao Chengling Zhao Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Yuqing Chen Corresponding Author Yuqing Chen [email protected] orcid.org/0000-0002-9783-1112 Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China Search for more papers by this author Author Information Xiaojing Wang *,1,‡, Shengping Min1,‡, Hongli Liu2, Nan Wu1, Xincheng Liu1, Tao Wang1, Wei Li1, Yuanbing Shen1, Hongtao Wang3, Zhongqing Qian3, Huanbai Xu4, Chengling Zhao1 and Yuqing Chen *,1 1Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, Department of Respiration, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China 2Department of Gynecological Oncology, First Affiliated Hospital, Bengbu Medical College, Bengbu, Anhui Province, China 3Department of Immunology, Bengbu Medical College, Bengbu, Anhui Province, China 4Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated First People's Hospital, Shanghai, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 15105528215; Fax: +86 05523070260; E-mail: [email protected] *Corresponding author. Tel: +86 13695528585; Fax: +86 05523070260; E-mail: [email protected] EMBO Mol Med (2019)11:e9856https://doi.org/10.15252/emmm.201809856 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mutations to KRAS are recurrent in lung adenocarcinomas (LUAD) and are daunting to treat due to the difficulties in KRAS oncoprotein inhibition. A possible resolution to this problem may lie with co-mutations to other genes that also occur in KRAS-driven LUAD that may provide alternative therapeutic vulnerabilities. Approximately 3% of KRAS-mutant LUADs carry functional mutations in NF1 gene encoding neurofibromin-1, a negative regulator of focal adhesion kinase 1 (FAK1). We evaluated the impact of Nf1 loss on LUAD development using a CRISPR/Cas9 platform in a murine model of Kras-mutant LUAD. We discovered that Nf1 deactivation is associated with Fak1 hyperactivation and phosphoserine aminotransferase 1 (Psat1) upregulation in mice. Nf1 loss also accelerates murine Kras-driven LUAD tumorigenesis. Analysis of the transcriptome and metabolome reveals that LUAD cells with mutation to Nf1 are addicted to glutamine metabolism. We also reveal that this metabolic vulnerability can be leveraged as a treatment option by pharmacologically inhibiting glutaminase and/or Psat1. Lastly, the findings advocate that tumor stratification by co-mutations to KRAS/NF1 highlights the LAUD patient population expected to be susceptible to inhibiting PSAT1. Synopsis KRAS-based molecular-targeted therapies have been largely unsuccessful. Defining mutations that co-occur with KRAS-mutant LUADs can provide alternative metabolic vulnerabilities to target these tumors. Approximately 3% of KRAS-mutant LUADs carry functional mutations in the NF1 gene. Nf1 loss is associated with Fak1 hyperactivation, Psat1 upregulation, and Kras-driven LUAD tumorigenesis in mice. Nf1-mutant LUAD cells are dependent upon increased glutaminolysis. This dependence can be therapeutically exploited through inhibition of glutaminase or Psat1. Human patients harboring KRAS;NF1-mutant LUAD tumors can be stratified as likely to respond to PSAT1 inhibition. Introduction Lung cancer remains the leading cause of cancer death globally, accounting for about 20% (1.6 million) of the total cancer deaths annually (Aggarwal et al, 2016; Hirsch et al, 2017). With a very high annual mortality-to-incidence ratio of 0.89 (Mao et al, 2016; Hirsch et al, 2017), the economic costs directly related to lung cancer are staggering. In the United States, annual lung cancer expenditures are approximately $13.1 billion and lost productivity due to premature lung cancer-related deaths is an estimated additional $36.1 billion (Mariotto et al, 2011; Howlader et al, 2015). In the E.U., lung cancer accounts for ~15% of the overall cancer costs (€18.8 billion; Luengo-Fernandez et al, 2013). Approximately 40% of lung cancers are adenocarcinomas (LUADs), usually originating from the peripheral lung tissue (Coroller et al, 2015). As can be expected, smoking has the strongest association to LUAD onset and prognosis (Network, 2014); nonetheless, LUAD is also the most common form of lung cancer in non-smokers (Gharibvand et al, 2017). Through a concerted effort of several multiplatform genomic profiling studies, the Kirsten rat sarcoma viral oncogene homolog gene (KRAS) was found to account for 25% of the molecular aberrations identified in various driver oncogenes in LUAD tumors (Tsao et al, 2016). However, KRAS-based molecular-targeted therapies have largely been unsuccessful, primarily due to the difficulties associated with directly inhibiting oncogenic KRAS (Downward, 2015). KRAS-mutant LUADs with distinct immune profiles and therapeutic susceptibilities have been distinguished based on co-occurring genomic alterations (Skoulidis et al, 2015). As such, characterizing and targeting other functionally relevant molecular aberrations in KRAS-mutant LUADs can be used as an alternative approach to managing these LUADs (Romero et al, 2017). To that end, putative loss-of-function mutations in the tumor suppressor NF1 (neurofibromin-1) have been identified in about 11% of all LUAD tumors and occur in approximately 3% of KRAS-mutant LUAD tumors (Network, 2014), suggesting that NF1 loss of function may play an important role in a subset of KRAS-mutant LUAD tumors. The NF1 gene encodes a GTPase-activating protein that regulates GTP-bound RAS' GTPase activity, thereby functioning as an "off" signal for RAS GTPase (Ratner & Miller, 2015). Thus, NF1 loss promotes the activity of RAS effector pathways with prominent roles in oncogenesis, such as the RAS–MAPK pathway (Ratner & Miller, 2015). Interestingly, the NF1 protein has been proven to co-localize and interact with the tyrosine kinase focal adhesion kinase 1 (FAK1) in mammalian cells (Kweh et al, 2009). This is noteworthy, as FAK1 is upregulated in > 80% of solid tumors and serves as a protein scaffold for several important oncogenic binding partners involved in cancer cell survival, proliferation, invasiveness, and angiogenesis (Lenzo & Cance, 2017). Of relevance here, previous research has revealed that Fak1 activity is necessary for tumor progression in Kras;Cdkn2a-mutant and Kras;Lkb1-mutant murine models of LUAD (Konstantinidou et al, 2013; Gilbert-Ross et al, 2017), suggesting that Fak1 may be a critical oncogene in certain subsets of KRAS-mutant LUAD tumors. On this basis, we chose to target the Nf1-Fak1 axis in the loxP-STOP-loxP (LSL)-KrasG12D/+; Tp53flox/flox (p53) (referred to as KP) genetically engineered mouse model (GEMM) of human LUAD by employing a CRISPR/Cas9 platform (Romero et al, 2017). We show that loss of Nf1 is associated with Fak1 hyperactivation and upregulation of the glutamine-metabolizing enzyme phosphoserine aminotransferase 1 (Psat1) in mice. We also found that loss of Nf1 accelerates murine Kras-dependent LUAD tumorigenesis. Using analysis of the transcriptome and metabolome, we also demonstrate that tumors with mutation to Nf1 are reliant upon α-ketoglutarate (α-KG) production from glutamate via the glutaminase–Psat1 pathway. We also demonstrate that this metabolic vulnerability can be leveraged as a treatment strategy by pharmacologically inhibiting glutaminase and/or Psat1. Lastly, the work suggests that tumor stratification by co-mutations to KRAS/NF1 highlights the LAUD patient population expected to benefit from inhibiting PSAT1. Results Nf1 loss accelerates Kras;p53-mutant LUAD tumorigenesis As TP53 mutations show prognostic significance in KRAS-mutant LUAD cases and NF1-mutant LUAD tumors show significantly higher rates of TP53 mutation relative to KRAS-mutant LUAD tumors (Redig et al, 2016), we investigated the cooperative effect of silencing Nf1 and p53 expression on the oncogenicity of constitutively active Kras mutants. Using CRISPR-Cas9 technology, the KrasLSL-G12D/+; p53fl/fl (KP) murine model of Kras-driven LUAD was engineered to silence Nf1 and p53 expression (Fig 1A). Twenty weeks after intratracheal infection of KP mice with lentiviral vectors expressing sgRNAs against Nf1 (sgNf1.1, sgNf1.2, and sgNf1.3) or tdTomato (sgTom) as control, we observed significant increases in average and total tumor volume in KP mice infected with sgRNAs against Nf1 as compared to sgTom (Fig 1B and C). We also observed significant increases in tumor burden 21 weeks after infection with sgRNAs against Nf1 as compared to sgTom (Fig 1D). Histological grading analysis also revealed an overrepresentation of grade 3 and 4 tumors in sgNf1-infected KP mice compared to sgTom-infected KP mice (Fig 1E). Notably, the highest proportion of grade 4 tumors (which were completely absent in sgTom-infected KP mice) was observed in KP mice infected with sgNf1.3. Accordingly, the mitotic indices (as observed on pHH3-stained slides) were significantly greater for sgNf1-infected KP mice relative to sgTom-infected KP mice (Fig 1F). Comparative qPCR and immunohistochemical analysis of LUAD tumor sections 21 weeks after infection of KP mice confirmed significant Nf1 mRNA downregulation (Fig 1G), p-Fak1 protein upregulation (Fig 1H–J), and Psat1 mRNA upregulation (Fig 1K and L) in sgNf1-infected mice, with the most profound effects observed in the sgNf1.3-infected group. On the basis of this combined evidence, we selected sgNf1.3 as the sgRNA for Nf1 silencing in all further in vitro and in vivo experiments. Figure 1. Nf1 loss activates Fak1 and accelerates murine LUAD tumorigenesis A. Schematic of KrasLSL-G12D/+; p53fl/fl (KP) mice intratracheally infected with pSECC lentiviruses containing sgNf1 or control sgTom. Mouse tumor burden was tracked by micro-computed tomography (micro-CT) for 5 months post-infection, and lungs were finally harvested 21 weeks post-infection. Tumors were dissected out for immunohistochemical (IHC) staining and generation of the tumor-derived parental KP cell line. B. Quantification by micro-CT of mean tumor volumes in KP mice derived from 50 randomly selected tumors at 8, 12, 16, and 20 weeks post-infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 18 mice, 21 mice, 21 mice, and 20 mice, respectively). C. Quantification by micro-CT of total tumor volume per mouse in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 8, 12, 16, and 20 weeks post-infection (n = 18 mice, 21 mice, 21 mice, and 20 mice, respectively). D. Depictions and quantitation of total tumor burden (total tumor area/total lung area) in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 at 21 weeks after infection (n = 18 mice, 21 mice, 21 mice, and 20 mice, respectively). E. Distribution of histological tumor grades in KP mice after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each) at 21 weeks after infection. F. Assessment of the mitotic index of tumor cells by phosphorylated-histone H3 (pHH3)-positive nuclei density in KP mice LUAD tumors at 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each). G. Nf1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each). H. Representative hematoxylin and eosin (H&E) and p-Fak1 immunohistochemical (IHC) staining of LUAD tumor sections 21 weeks after infection with control sgTom (grade 1 depicted), sgNf1.1 (grade 3 depicted), sgNf1.2 (grade 3 depicted), or sgNf1.3 (grade 3 depicted) (n = 50 tumors each). H&E scale bars (low-magnification top row = 100 μm, high-magnification bottom row = 250 μm); p-Fak1 IHC scale bars (low-magnification top row = 250 μm, high-magnification bottom row = 500 μm). I. Quantification of p-Fak1 IHC signals in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each). J. Quantification of p-Fak1 IHC signals in sgNf1.3 LUAD tumor sections analyzed by tumor grade (n = 50 tumors). K. Quantification of Psat1 mRNA expression in LUAD tumor sections 21 weeks after infection with control sgTom, sgNf1.1, sgNf1.2, or sgNf1.3 (n = 50 tumors each). L. Quantification of Psat1 mRNA expression in sgNf1.3 LUAD tumor sections analyzed by tumor grade (n = 50 tumors). Data information: P-values are reported in Appendix Table S3. In bar charts and line graphs, data presented as means with error bars representing standard deviations (SDs). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean. Download figure Download PowerPoint Using CRISPR-Cas9 technology, murine KrasLSL-G12D/+; p53fl/fl (KP) cells were engineered to silence Nf1 using sgNf1.3 (KPΔNF1) and Fak1 using sgFak1.1 (KPΔFAK1; Appendix Fig S1A and B). We generated subcutaneous and orthotopic transplants of non-recombinant KP and recombinant KPΔNF1 cells to answer whether Nf1 inactivation confers a selective growth advantage in vivo. From day 10 post-implantation into nude mice, we observed a consistent increase in subcutaneous tumor volumes for mice with KPΔNF1 tumors (Appendix Fig S1C and D) as well as an ~4-fold increase in subcutaneous tumor masses for mice with KPΔNF1 tumors (Appendix Fig S1E and F). Ki-67, a cellular proliferation marker, was significantly more enriched in nuclei from subcutaneous KPΔNF1 tumors (Appendix Fig S1G and H). We also observed a consistent increase in tumor growth for mice with orthotopic KPΔNF1 tumors (Appendix Fig S1I and J). In vitro, cumulative population doublings were found to progressively increase in KPΔNF1 cells (Appendix Fig S1K). Nf1 loss also accelerates Kras-mutant;p53-WT murine LUAD tumorigenesis The previous experiments demonstrated that Nf1 loss accelerates Kras;p53-mutant LUAD tumorigenesis. However, the effects of Nf1 loss on Kras-mutant;p53-WT LUAD tumorigenesis remain unknown. Therefore, we investigated p53's possible involvement in NF1-mediated KRAS-LUAD. p53 protein expression was pharmacologically induced in the patient-derived KRAS-mutant/NF1-mutant/TP53-WT LUAD cell lines PDKN1 and PDKN2, the human KRAS-mutant/NF1-WT/TP53-WT LUAD cell line SW1573, and the murine Kras-mutant/Nf1-WT/p53-WT LUAD clones LKR10 and LKR13 by incubating them with a DNA intercalator doxorubicin for 6 h (Fig 2A, Appendix Fig S2A). Western blot analysis validated NF1 upregulation and downregulated FAK1 activation following transduction of doxorubicin-treated PDKN1 and PDKN2 cell lines with an NF1-expression vector (Fig 2B and C; Appendix Fig S2B and C). Moreover, we validated Nf1 downregulation and Fak1 activation following transduction of doxorubicin-treated SW1573, LKR10, and LKR13 clones with sgNf1.3 (Fig 2D–F; Appendix Fig S2D–F). Figure 2. Nf1 loss accelerates tumor growth in p53-independent manner A. Western blotting analysis of p53 expression in various p53/TP53-WT cell lines incubated with the DNA-intercalating agent doxorubicin (DOXO, 0.2 μg/ml for 6 h) to induce p53 stabilization (n = 3 biological replicates). Full experimental data provided in Appendix Fig S2A. B, C. Western blotting analysis confirming NF1 upregulation and p-FAK1 downregulation in (B) DOXO-treated PDKN1 cells and (C) DOXO-treated PDKN2 cells transduced with PGK-NF1 (n = 3 biological replicates). Full experimental data provided in Appendix Fig S2B and C. D. Western blotting analysis confirming NF1 downregulation and p-FAK1 upregulation in DOXO-treated SW1573 cells transduced with sgNf1.3 (n = 3 biological replicates). Full experimental data provided in Appendix Fig S2D. E, F. Western blotting analysis confirming Nf1 downregulation and p-Fak1 upregulation in (E) DOXO-treated LKR10 and (F) DOXO-treated LKR13 clones transduced with sgNf1.3 (n = 3 biological replicates). Full experimental data provided in Appendix Fig S2E and F. G. FAK1 target gene expression in DOXO-treated PDKN1 cells transduced with PGK-control or PGK-NF1 (n = 3 biological replicates). H, I. Subcutaneous tumor volumes of DOXO-treated PDKN1 and PDKN2 cells transduced with PGK-control or PGK-NF1 (n = 18 each). J–L. Subcutaneous tumor volumes of (J) SW1573, (K) LKR10, and (L) LKR13 cells transfected with sgTom or sgNf1.3 (n = 18 each). M. Depictions and quantitation of KrasG12D/+; p53+/+ (K-only) autochthonous tumor burden (total tumor area/total lung area) in pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). N. Analysis of tumor grades in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). O. Quantification of Ki-67-positive nuclei per mm2 in K-only autochthonous tumors derived from pSECC-sgTom (n = 18 mice) or pSECC-sgNf1.3 mice (n = 21 mice). Data information: P-values are reported in Appendix Table S3. In bar charts and line graphs, data presented as means with error bars representing standard deviations (SDs). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean. Source data are available online for this figure. Source Data for Figure 2 [emmm201809856-sup-0003-SDataFig2.zip] Download figure Download PowerPoint We examined the effects of Nf1 overexpression and loss across multiple p53-WT models. First, overexpression of NF1 in the doxorubicin-treated PDKN1 cell line led to repression of FAK1 activity as evidenced by downregulation of the FAK1 target genes PSAT1, AREG, and PEG10 (Golubovskaya et al, 2009; Fig 2G). Moreover, overexpression of NF1 decreased subcutaneous tumor volumes for doxorubicin-treated PDKN1 and PDKN2 cell lines (Fig 2H and I). Second, silencing NF1/Nf1 in doxorubicin-treated SW1573 cells as well as doxorubicin-treated LKR10 and LKR13 clones resulted in significantly greater subcutaneous tumor volumes across all three models (Fig 2J–L). Third, we examined the effects of Nf1 silencing in KrasG12D/+; p53+/+ (K-only) autochthonous tumors. sgNf1.3 mice displayed a significantly greater tumor burden (Fig 2M), an overrepresentation of grade 2 and 3 tumors (Fig 2N), and nuclear Ki-67 enrichment in their K-only autochthonous tumors (Fig 2O) relative to sgTom mice. Note that these grading results for K-only tumors are lower than those observed in the KrasLSL-G12D/+; p53fl/fl (KP) tumors from sgNf1.3 mice (Fig 1D), which can likely be attributed to the tumor-suppressive effects of p53 expression in the K-only autochthonous tumors. To summarize, Nf1 loss exacerbates Kras-driven lung adenocarcinogenesis even in the background of WT p53 expression. Nf1 loss enhances Fak1 activation in vitro We analyzed Fak1 activation in engineered KPΔNF1 and KPΔFAK1 cells. Immunoblot and qRT-PCR analyses revealed that Fak1, phosphorylated Fak1 (p-Fak1), and its target genes (Psat1, Areg, and Peg10; Golubovskaya et al, 2009) were significantly upregulated in KPΔNF1 cell lines (Appendix Fig S3A and B). Because phosphatidylinositol -4,5-bisphosphate (PIP2) is a Fak1 activator, recombinant and non-recombinant KP cells were incubated in 10 μM PIP2 for 6 h. We observed that upregulation of p-Fak1 [Appendix Fig S3C; with concomitant upregulation of its target genes Psat1, Areg, and Peg10 (Appendix Fig S3D–F; Golubovskaya et al, 2009)] only occurs in PIP2-stimulated parental KP cells and is further upregulated in PIP2-stimulated KPΔNF1 clones. Nf1 loss enhances Kras-mutant fermentation and glutamine dependence Having better established the role of NF1 loss in KRAS-dependent LUAD, discovery of metabolic susceptibilities in Kras;Nf1-mutant LUAD cells that can be therapeutically exploited was pursued next. To answer whether these cells exhibit the marked glutamine dependence typical of many cancer cells, we assessed metabolic changes associated with glutamine restriction/depletion in vitro. Analyses of glucose/lactate levels (Fig 3A) and glutamine levels (Fig 3B) reveal that both the fermentation and the utilization of glutamine are significantly enhanced in KPΔNF1 cells. Incidentally, inhibition of glycolysis (by incubating cells in 2DG) was mildly cytotoxic to Kras-mutant cells but extremely cytotoxic to Kras;Nf1-mutant cells (Appendix Fig S4A). An isotopomer of glucose in which every carbon was 13C-labeled (referred to as [U13C]) was used as a metabolic tracer in KPΔNF1 cells. They showed a lowered input into the Krebs cycle and its intermediates of 13C derived from [U13C] glucose (Appendix Fig S4B–D). Restriction of glutamine uptake by treatment with γ-l-glutamyl-p-nitroanilide (GPNA, a small-molecule inhibitor of the glutamine transporter SLC1A5 that is expressed in 74% of LUADs; Hassanein et al, 2013; Fig 3C) or decreased glutamine supplementation (Fig 3D) significantly decreased KPΔNF1 cell viability and proliferation, respectively. Taken together, KPΔNF1 cells display enhanced fermentation and heightened glutamine dependence. Figure 3. Nf1-silenced cells are sensitive to reduced glutamine levels A. Glucose consumption and lactate excretion in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). B. Glutamine consumption in KP and KPΔNF1 clones normalized by cell count (n = 4 biological replicates). C. Relative viability of KP and KPΔNF1 cells after 72 h of GPNA treatment assayed by cell-titer glo (relative luminescent units; n = 4 biological replicates). D. Cumulative population doublings of KP and KPΔNF1 cells cultured with 2.0 or 0.5 mM glutamine (n = 4 biological replicates). E. Two patient-derived KRAS;NF1-mutant LUAD cell lines (PDKN1, PDKN2), as well as the control patient-derived KRAS-mutant/NF1-WT LUAD cell line (PDK), were passaged for 14 population doublings prior to collection. The heatmap displays the most significantly upregulated genes in the PDKN1 and PDKN2 cell lines (relative to the PDK control), with the degree of absolute fold-change upregulation depicted by color as indicated in the legend. F, G. Quantification of the expression of the six key altered genes in cultured PDKN1 and PDKN2 cells (passaged for 14 population doublings) vs. their respective original patient tumor samples using qPCR (n = 4 biological replicates). H. Western blotting analysis of Psat1 expression in KP and KPΔNF1 cells infected with sgTom or sgPsat1 following selection. GAPDH was used as a loading control. I. Cumulative population doublings of KP and KPΔNF1 cells after transduction with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates). J–L. Cumulative population doublings of patient-derived KRAS-mutant LUAD cell lines that are either (J, K) NF1-mutant (PDKN1 and PDKN2) or (L) NF1-WT (PDK) after selection with sgTom- or sgPsat1-containing vectors (n = 4 biological replicates). Data information: P-values are reported in Appendix Table S3. In bar charts and line graphs, data presented as means with error bars representing standard deviations (SDs). For boxplots, whiskers indicate the minimum and maximum values, the upper and lower perimeters represent the first and third quartiles, the midline represents the median value, and the x symbol represents the mean. Source data are available online for this figure. Source Data for Figure 3H [emmm201809856-sup-0004-SDataFig3H.pdf] Download figure Download PowerPoint Nf1 loss enhances expression of the glutamine-metabolizing enzyme Psat1 We performed a transcriptomic microarray s
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