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Israel Cañadas, PhD

Dr. Canadas
About

Assistant Professor

Research Program

Education and Training

Educational Background

  • PhD, Biomedicine, Pompeu Fabra University (UPF), Barcelona, Spain, 2013
  • MS, Biomedical Research, Pompeu Fabra University (UPF), Barcelona, Spain, 2009
  • BS, Biology, Barcelona University (UB), Barcelona, Spain, 2007

Honors & Awards

  • 2018 Lung Cancer Research Foundation Annual Grant Recipient
  • Short-Term fellowship - Red Temática de Investigación Cooperativa en Cáncer (RTICC) (2014)
  • Summa cum laude distinction of Doctoral Thesis, 2013
  • PhD fellowship - Institut Hospital del Mar d’Investigacions Mèdiques (IMIM) (2009)
Research Profile

Research Program

Research Interests

Cañadas Lab: Targeting innate immunity in lung cancer

  • Study of tumor immunity and the tumor microenvironment in lung cancer
  • Deciphering mechanisms of Endogenous Retroviruses (ERV) silencing and reactivation at the epigenetic level.
  • Study of cytosolic nucleic acid-sensing pathways in the context of innate and adaptive antitumor immunity.
  • Development of novel functional ex vivo organotypic 3D culture platforms to identify and characterize mechanisms of response and resistance to immunotherapy using murine and patient derived tumor samples.

Lab Overview

Cañadas Lab

The Cañadas Lab studies tumor immunity and the tumor microenvironment in lung cancer with the ultimate goal of identifying new therapeutic opportunities to improve patient health.

We are primarily interested in delineating mechanisms of tumor resistance to immunotherapy, and in leveraging innate immune signaling pathways to break therapy resistance and restore immunogenicity.

Co-opting endogenous retroviral signaling as a lung cancer vulnerability

We recently identified a novel epigenetically regulated subclass of endogenous retroviruses (ERVs) that engages pathologic innate immune signaling in mesenchymal cancer subpopulations of Small Cell Lung Cancer (SCLC), with potentially important implications for cancer immunotherapy (Cañadas et al, Nat Med, 2018). Stimulated 3 Prime Antisense Retroviral Coding Sequences (SPARCS) are oriented inversely in 3’UTRs of certain interferon-inducible genes and silenced by EZH2. De-repression of these loci resulted in dsRNA generation following IFNs exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5’ LTR of the antisense ERV.

The overarching goal of this project is to study the biological mechanisms that connect the mesenchymal cancer resistant state to SPARCS de-repression in lung cancer, the impact on the tumor microenvironment, and co-opting this cell state to favor response to immune checkpoint blockade.

Tumor cell heterogeneity is a key determinant of cancer progression and drug resistance, which is often mediated by mesenchymal cell subpopulations (1, 2). While these subclones can secrete growth factors, chemokines and cytokines that can influence the tumor microenvironment, the immune signaling networks that fuel this pro-tumorigenic state remain incompletely defined. In a manuscript recently published in Nature Medicine, I identified a novel epigenetically regulated subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in mesenchymal cancer subpopulations (3). Using a novel approach of ex vivo culture of patient-derived organotypic tumor spheroids (PDOTS) (4) I determined the translational relevance of these findings, with potentially important implications for cancer immunotherapy. Because of their well-defined nature, I used the phenotypically distinct H69M and H69AR Small Cell Lung Cancer (SCLC) mesenchymal subclones (2) to uncover this novel mechanism of dysregulated innate immune signaling as compared with parental neuroendocrine H69 cells. These analyses uncovered Stimulated 3 Prime Antisense Retroviral Coding Sequences (SPARCS) as a novel subclass of ERVs oriented inversely in 3’UTRs of certain interferon-inducible genes and silenced by EZH2. De-repression of these loci in mesenchymal cancer subpopulations results in dsRNA generation following IFNexposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5’ LTR of the antisense ERV. I found that viral sensing by MAVS and STING fuels activation of TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop (Figure 1). SPARCS induction across specific human tumors and cell lines from TCGA and CCLE databases, respectively, was tightly associated with a mesenchymal	AXL/MET positive cell state, STING expression and MHC class 1 antigen presentation. This SPARCShigh/STINGhigh state was also associated with downregulation of chromatin modifying enzymes, including EZH2 and multiple SWI/SNF components. Correlation of SPARCS expression with genomic     alterations   across TCGA tumors revealed co-associations with multiple markers on chromosome 3p, including BAP1, PBRM1 and SETD2 alterations. SPARCS high tumors were marked by immune infiltration, but also exhibited multiple features of tumor immune suppression including checkpoint gene expression and myeloid cell infiltration. SPARCS expression was observed across distinct cancer histologies in TCGA, and was strongly enriched in Renal Cell Carcinoma (RCC), Lung Adenocarcinoma (LUAD), head/neck squamous (HNSC) and glioblastoma (GBM). Of note, IFNtreatment of patient-derived organotypic tumor spheroids (PDOTS) (4) with de-repressed SPARCS markedly enhanced CXCL10 production and sensitized them to PD-1 blockade (3). These findings may have important therapeutic implications for drug combinations with immune checkpoint blockade. For example, therapies that hyperactivate JAK signaling, target immune suppressive cytokines or block specific chromatin regulators could alter SPARCS physiology to favor response. The overarching goal of my research is to study the biological mechanisms that connect the mesenchymal cancer resistant state to SPARCS/STING de-repression, the impact on the tumor microenvironment, and co-opting this cell state to favor response to immune checkpoint blockade. Outlined below are two immediate aims of my research, as well as future translational directions.  1)	Systematically identify regulators of SPARCS/STING expression and genetic dependencies specific to the SPARCShigh/STINGhigh cancer state. First, I propose to use CRISPR-Cas9 genome-wide gene-knockout screens to identify regulators of SPARCS using a SPARCSlow/STINGlow cancer cell line (3). We will generate a GFP-based reporter for a cell surface SPARCS containing gene which can be monitored by flow cytometry. GFP positive cells will be   sorted to screen for genes that regulate SPARCS expression. In parallel, we will engineer a STING-GFP reporter, which can be measured by intracellular flow cytometry, and compare hits that induce STING expression in these cells. This will systematically characterize chromatin and other regulators that govern SPARCS and STING expression, and may identify key enzymes to target to induce their expression. Second, to systematically identify cancer dependencies associated with the  SPARCShigh/STINGhigh cell state, we will use CRISPR-Cas9 genome-wide gene-knockout screens in SPARCShigh/STINGhigh cancer cell lines. We will compare genes that are depleted in control, IFN(SPARCS activation) or poly-dAdT (STING activation) conditions. These analyses will allow us to identify synthetic lethal interactions with the SPARCShigh/STINGhigh cell state. Dissecting these genetic dependencies will provide detailed insights into the vulnerabilities that are associated with SPARCS and STING expression, and provide additional therapeutic targets that may synergize with those identified above, which induce this state.  2)	Develop combination therapies that alter SPARCS/STING physiology to favor response to PD-1 blockade using NSCLC PDOTS. Recently, we utilized ex vivo culture of PDOTS to demonstrate that IFNtreatment of human Non-Small Cell Lung Cancer (NSCLC) samples with de-repressed SPARCS markedly enhanced CXCL10 production and sensitized them to PD-1 blockade (3). This suggested that hyperactivation of IFN signaling associated with the SPARCShigh state can be directly tested from patient samples and may promote sensitivity to PD-1 blockade. Moreover, based on recent evidence indicating a role for endogenous STING pathway signaling in the generation of spontaneous immune responses against tumors (5) and our previous data showing a tight correlation between SPARCShigh tumors and STING expression (3), we hypothesize that therapeutic STING pathway activation may potentiate the effect of immunotherapies in this cancer cell state. Thus, since our previous data showed that SPARCS expression was strongly enriched in NSCLC, the goal of this aim is to use NSCLC PDOTS to extend our initial findings by evaluating ex vivo whether the efficacy of immunotherapy might be increased by enhancing IFNor STING signaling in the SPARCShigh state. We will evaluate ex vivo the efficacy of IFNand/or STING agonists as single agent or in combination with PD-1 blockade in SPARCShigh and SPARCSlow tumors using NSCLC PDOTS. In parallel, based on the regulators   and genetic dependencies discovered in Aim 1, we will identify drugs to target this SPARCShigh/STINGhigh  cancer state and evaluate them ex vivo using the PDOTS platform in human tumors. This will allow us to perform integrated analysis of immune cell profiles, cytokine secretion, RNAseq and ex vivo killing to evaluate the efficacy of immunotherapies in the context of IFN/STING signaling hyperactivation as well as  potential  novel therapeutic targets in the mesenchymal cancer resistant state.  3)	Co-opting SPARCShigh/STINGhigh state to target neo-antigens and deliver STING agonists via antibody conjugates (Future Directions). Recent studies demonstrated that tumor-specific mutations may generate altered cell surface peptide- HLA complexes (neoantigens) that can be recognized by T cells. These neoantigens are relevant therapeutic targets since they are tumor specific and T cells recognizing them are not affected by central T cell tolerance (6). Since our previous work showed that SPARCS ERVs expression was tightly associated with MHC class I upregulation across tumors in TCGA, I propose to evaluate SPARCS and/or other ERVs as a potential neoantigens displayed specifically on the cell surface of drug resistant mesenchymal cancer subclones. Through a collaboration with Reinherz laboratory (DFCI) we are performing computational predictions using adaptive algorithms trained with various forms of empirical data to identify SPARCS neoantigens that are capable of binding host HLA alleles. Then, we will use Mass Spectrometry (MS) technology to physically detect these SPARCS epitopes with the sensitivity of a high avidity T cell using SPARCShigh cell lines and tumors (7). This will elucidate novel neoantigens as potential biomarkers in cancer immunotherapy and provide an incentive to develop peptide- or RNA-based immunotherapeutic vaccines to selectively enhance T cell reactivity against this mesenchymal resistant cancer state. While recent studies have demonstrated that direct activation of STING in the tumor microenvironment using STING agonists induced a potent anti-tumor response and immunity in mouse models (8), the potency of these agonists remains a challenge because limitations related with delivery and stability (9). STING agonists show a relatively short half-life and have to be delivered by intratumoral injection at high doses to achieve efficacy. In addition, recent studies showed that STING hyperactivation might have negative effects on T cells activating cell stress and death pathways (10). Since our previous data revealed that cell lines   poised to induce high level of SPARCS expression were strongly associated with an AXL/MET positive mesenchymal cell state, as well as increased STING protein levels (3), the use of antibodies specific for the SPARCShigh/STINGhigh state conjugated to STING agonist cyclic dinucleotides may potentially solve the current STING agonists concerns and guide its delivery to specifically target drug-resistant mesenchymal subpopulations. Thus, I propose the development of antibody-drug conjugate (ADC) comprising an antibody specific for a membrane marker of the SPARCShigh/STINGhigh state (i.e. AXL, MET, CD44, SPARCS genes) linked to a STING agonist. We have also initiated collaborations to develop these ADC compounds and we will evaluate its activity as single agent or in combination with immune checkpoint inhibitors using the PDOTS platform. Integrated analysis will allow us to determine whether direct engagement of STING pathway in mesenchymal subclones will result in effective anti-tumor immunity and, ultimately, determine the efficacy of this strategy as a potential way to improve immunotherapy effects in heterogeneous tumors.  Summary Overall, this project aims to identify and characterize biological mechanisms by which intratumor heterogeneity may influence the tumor microenvironment and response to therapy, providing insights into tumor immunology and inform clinical strategies to improve immunotherapies. Based on our previous findings, I will perform functional approaches to target the mesenchymal drug resistant state to favor response to immune checkpoint blockade. Ultimately, the PDOTS platform will allow us to determine the potential translational relevance of novel therapeutic combinations that may influence responsiveness to immune checkpoint therapy. The proposed strategy addresses a key unmet need in the field in an effort to overcome resistance to immunotherapy because of intratumor heterogeneity.  References 1.	Tabassum DP. & Polyak K. Tumorigenesis: it takes a village. Nat Rev Cancer. 2015;15(8):473-83. 2.	Cañadas I. et al. Targeting epithelial-to-mesenchymal transition with Met inhibitors reverts chemoresistance in small cell lung cancer. Clin Cancer Res. 2014;20(4):938-50. 3.	Cañadas I, et al. Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses. Nat Med 2018 Aug;24(8):1143-1150. 4.	Jenkins RW, Aref AR, Lizotte PH, Ivanova E, Stinson S, Zhou CW, et al. Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids. Cancer Discov. 2018;8(2):196-215. 5.	Spranger S, Gajewski TF. Impact of oncogenic pathways on evasion of antitumour immune responses. Nat Rev Cancer. 2018;18(3):139-47. 6.	Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy. Science. 2015;348(6230):69- 74. 7.	Keskin, DB, Reinhold, BB, Zhang, GL, Ivanov, AR, Karger, BL, Reinherz, EL. Physical detection of influenza A epitopes identifies a stealth subset on human lung epithelium evading natural CD8 immunity. Proc Natl Acad Sci USA 2015; 112, 2151-2156. 8.	Corrales L, Glickman LH, McWhirter SM, Kanne DB, Sivick KE, Katibah GE, et al. Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep. 2015;11(7):1018-30. 9.	Mullard A. Can innate immune system targets turn up the heat on 'cold' tumours? Nat Rev Drug Discov. 2018;17(1):3-5. 10.	Larkin B, Ilyukha V, Sorokin M, Buzdin A, Vannier E, Poltorak A. Cutting Edge: Activation of STING in T Cells Induces Type I IFN Responses and Cell Death. J Immunol. 2017;199(2):397-402.

Ex vivo organotypic cultures

We are developing novel functional ex vivo organotypic 3D culture platforms using surgically resected murine and patient-derived tumor samples that will allow us to validate promising therapeutic combinations in an ex vivo system that incorporates features of the tumor microenvironment and models the dynamic response to immune checkpoint blockade (Jenkins et al, Cancer Discovery, 2018). Using this sophisticated approach, we aim to identify and characterize novel mechanisms of response and resistance to immunotherapy.  

Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive and lethal lung malignancy with a 5-year overall survival of less than 5%. While having one of the highest mutational burdens because of its strong association with tobacco smoking, SCLC is often characterized by a reduced antigen presentation and an immunosuppressive tumor microenvironment. Therefore, despite promising advances in the use of immunotherapy, only a fraction of SCLC patients respond to these therapies.

SCLC is composed of phenotypically different cells with either neuroendocrine or mesenchymal features. Of note, the mesenchymal compartment enhances chemoresistance and metastatic potential of neuroendocrine cells. We and others have shown that activation of MET and/or RAS signaling can fuel this mesenchymal phenotype, resulting in chemoresistance and increased tumorigenesis in a well-established SCLC model (Cañadas et al, Clin Cancer Res, 2014). In addition, we recently showed an enhanced innate immune signaling in these SCLC mesenchymal subpopulations because of the de-repression of a novel epigenetically regulated subclass of ERVs (Cañadas et al, Nat Med, 2018).

Phospho-TBK1 Immunohistochemistry Brain metastisis (Small Cell Lung Cancer Patient)Using SCLC as a model, we aim to identify and characterize biological mechanisms by which intratumor heterogeneity may influence the tumor microenvironment and response to therapy, providing insights in tumor immunology and informing clinical strategies to improve immunotherapies in SCLC.

Lab Staff

Xueying Ma, MS, MB

Sr. Scientific Associate

Room: R362
215-214-1581

Takahiko Murayama, PhD

Postdoctoral Associate

Room: R362
215-214-1581

Rahul M. Prasad, PhD

Postdoctoral Associate

Room: R362
215-214-1581
Publications

Selected Publications

Jerby-Arnon L, Shah P, Cuoco MS, Rodman C, Su MJ, Melms JC, Leeson R, Kanodia A, Mei S, Lin JR, Wang S, Rabasha B, Liu D, Zhang G, Margolais C, Ashenberg O, Ott PA, Buchbinder EI, Haq R, Hodi FS, Boland GM, Sullivan RJ, Frederick DT, Miao B, Moll T, Flaherty KT, Herlyn M, Jenkins RW, Thummalapalli R, Kowalczyk MS, Cañadas I, Schilling B, Cartwright ANR, Luoma AM, Malu S, Hwu P, Bernatchez C, Forget MA, Barbie DA, Shalek AK, Tirosh I, Sorger PK, Wucherpfennig K, Van Allen EM, Schadendorf D, Johnson BE, Rotem A, Rozenblatt-Rosen O, Garraway LA, Yoon CH, Izar B, Regev A. A Cancer Cell Program Promotes T Cell Exclusion and Resistance to Checkpoint Blockade. Cell. 2018 Nov 1;175(4):984-997.e24. doi: 10.1016/j.cell.2018.09.006.

Aref AR, Campisi M, Ivanova E, Portell A, Larios D, Piel BP, Mathur N, Zhou C, Coakley RV, Bartels A, Bowden M, Herbert Z, Hill S, Gilhooley S, Carter J, Cañadas I, Thai TC, Kitajima S, Chiono V, Paweletz CP, Barbie DA, Kamm RD, Jenkins RW. 3D microfluidic ex vivo culture of organotypic tumor spheroids to model immune checkpoint blockade. Lab Chip. 2018 Sep 5. doi: 10.1039/c8lc00322j.

Cañadas I, Thummalapalli R, Kim JW, Kitajima S, Jenkins RW,  Christensen CL,  Campisi C,  Kuang Y, Zhang Y, Gjini E, Zhang G, Tian T, Sen DR, Miao D, Imamura Y, Thai T, Piel B, Terai H, Aref AR, Hagan T, Koyama S, Watanabe M, Baba H, Adeni AE, Lydon CA, Tamayo P, Wei Z, Herlyn M, Barbie TU, Uppaluri R, Sholl LM, Sicinska E, Sands J, Rodig S, Wong KK, Paweletz CP, Watanabe H, Barbie DA.  Tumor  innate  immunity  primed   by   specific   interferon-stimulated   endogenous   retroviruses. Nat Med. 2018 Aug;24(8):1143-1150.

Jenkins RW, Aref AR, Lizotte PH, Ivanova E, Stinson S, Zhou CW, Bowden M, Deng J, Liu H, Miao  D, He MX, Walker W, Zhang G, Tian T, Cheng C, Wei Z, Palakurthi S, Bittinger  M, Vitzthum  H, Kim JW, Merlino A, Quinn M, Venkataramani C, Kaplan JA, Portell A, Gokhale PC, Phillips B, Smart A, Rotem A, Jones RE, Keogh L, Anguiano M, Stapleton L, Jia Z, Barzily-Rokni M, Cañadas I, Thai TC, Hammond MR, Vlahos R, Wang ES, Zhang H, Li S, Hanna GJ, Huang W, Hoang MP, Piris A, Eliane      JP, Stemmer-Rachamimov AO, Cameron L, Su MJ, Shah P, Izar B, Thakuria M,  LeBoeuf  NR,  Rabinowits G, Gunda V, Parangi S, Cleary JM, Miller BC, Kitajima S,  Thummalapalli  R,  Miao  B, Barbie TU, Sivathanu V, Wong J, Richards WG, Bueno R, Yoon CH, Miret J, Herlyn M, Garraway LA,

Van Allen EM, Freeman GJ, Kirschmeier PT, Lorch JH, Ott PA, Hodi FS, Flaherty KT, Kamm RD, Boland GM, Wong KK, Dornan D, Paweletz CP, Barbie DA. Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids. Cancer Discov. 2018 Feb;8(2):196-215.

Jenkins RW, Thummalapalli R, Carter J, Cañadas I, Barbie DA. Molecular and Genomic Determinants of Response to Immune Checkpoint Inhibition in Cancer. Annu Rev Med. 2018 Jan 29;69:333-347.

Yang S, Imamura Y, Jenkins RW, Cañadas I, Kitajima S, Aref A, Brannon A, Oki E, Castoreno A, Zhu Z, Thai T, Reibel J, Qian Z, Ogino S, Wong KK, Baba H, Kimmelman AC, Pasca Di Magliano M, Barbie DA. Autophagy Inhibition Dysregulates TBK1 Signaling and Promotes Pancreatic Inflammation. Cancer Immunol Res. 2016 Jun;4(6):520-30.

Sánchez-Martín FJ, Bellosillo B, Gelabert M, Dalmases A, Cañadas I, Vidal J, Martinez A, Argilés     G, Siravegna G, Arena S, Koefoed K, Visa L, Arpí O, Horak  ID, Iglesias M, Stroh  C, Kragh  M, Rovira  A, Albanell J, Tabernero J, Bardelli A, Montagut C. The first-in-class anti-EGFR antibody  mixture Sym004 overcomes cetuximab-resistance mediated by EGFR  extracellular  domain  mutations  in colorectal cancer. Clin Cancer Res 2016 Jul 1;22(13):3260-7.

Cañadas I, Taus A, Villanueva X, Arpí O, Pijuan L, Rodríguez Y, Menéndez S, Mojal S, Rojo F, Albanell J, Rovira A, Arriola E. Angiopoietin-2 is a negative prognostic marker in small cell lung cancer. Lung Cancer 2015 Nov;90(2):302-6.

Casadevall D, Gimeno J, Clavé S, Taus A, Pijuan L, Arumí M, Lorenzo M, Menéndez S, Cañadas I, Albanell J, Serrano S, Espinet B, Salido M, Arriola E. MET expression and copy number heterogeneity in nonsquamous non-small cell lung cancer (nsNSCLC). Oncotarget 2015; 30;6(18):16215-26.

Arena S, Bellosillo B, Siravegna G, Martínez A, Cañadas I,  Lazzari  L,  Ferruz  N,  Russo  M,  Misale S, González I, Iglesias M, Gavilan E, Corti G, Hobor S, Salido M, Sánchez J, Dalmases A,  Bellmunt J, De Fabritiis G, Rovira A, Di Nicolantonio F, Albanell J,  Bardelli  A,  Montagut  C.  Emergence of  multiple  EGFR  extracellular  mutations  during  cetuximab  treatment  in  colorectal cancer. Clin Cancer Res 2015; 21(9):2157-66

Cañadas I, Taus A, González I, Villanueva X, Gimeno J, Pijuan L, Domine M, Sánchez-Font A, Vollmer I, Menéndez S, Arpí O, Mojal S, Rojo F, Rovira A, Albanell J, Arriola E. High circulating hepatocyte growth factor levels associate with epithelial to mesenchymal transition  and  poor  outcome in small cell lung cancer patients. Oncotarget 2014; 5(14): 5246-5256.

Cañadas I, Rojo F, Taus A, Arpí O, Arumí M, Pijuan L, Menéndez S, Zazo S, Domine M, Salido M, Mojal S, García de Herreros A, Rovira A, Albanell J, Arriola E. Targeting epithelial to mesenchymal transition with Met inhibitors reverts  chemoresistance  in  small  cell  lung  cancer.  Clin Cancer Res 2014; 20(4): 938-950.

Salido M, Pijuan L, Galvan AB, Gimeno J, Cañadas I, Rodríguez M, Rojo F, Albanell J, Solé F, Arriola E.  ALK  status  in  a  primary  lung  tumour  and  metachronous  metastases.  Histopathology  2012; 60(5): 843-845.

Arriola E*, Cañadas I*, Arumí M, Domine M, López-Vilariño JA, Arpí O, Salido M, Menéndez S, Grande E, Hirsch FR, Serrano S, Bellosillo B, Rojo F, Rovira A, Albanell  J. MET  phosphorylation predicts poor  outcome  in  small  cell  lung  carcinoma  and  its  inhibition  blocks  HGF-induced  effects  in MET mutant cell lines. Br J Cancer 2011; 105(6): 814-823.
*equal contribution.

Salido M, Pijuan L, Martínez-Avilés L, Galvan AB, Cañadas I, Rovira A, Zanui M, Martínez A, Longarón R, Solé F, Serrano S, Bellosillo B, Wynes MW, Albanell J, Hirsch FR, Arriola  E. Increased  ALK Gene Copy Number and Amplification are Frequent in  Non-small Cell Lung  Cancer. J Thorac  Oncol 2011; 6(1): 21-27.

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