CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

Generation of Genetically Engineered Mouse LungOrganoidModels for Squamous Cell Lung Cancers Allowsfor the Study of Combinatorial Immunotherapy A C

Josephine Hai1, Hua Zhang1,2, Jin Zhou1, Zhong Wu1, Ting Chen1,2, Eleni Papadopoulos2,Catríona M. Dowling2, Val Pyon2, Yuanwang Pan2, Jie Bin Liu1, Roderick T. Bronson3, Heather Silver2,Patrick H. Lizotte1,4, Jiehui Deng1,2, Joshua D. Campbell1,5, Lynette M. Sholl6, Christine Ng7,Ming-Sound Tsao7, Cassandra Thakurdin2, Adam J. Bass1, and Kwok-Kin Wong1,2

ABSTRACT◥

Purpose: Lung squamous cell carcinoma (LSCC) is a deadlydisease for which only a subset of patients responds to immunecheckpoint blockade (ICB) therapy. Therefore, preclinical mousemodels that recapitulate the complex genetic profile found inpatients are urgently needed.

Experimental Design: We used CRISPR genome editing todelete multiple tumor suppressors in lung organoids derived fromCre-dependent SOX2 knock-in mice. We investigated both thetherapeutic efficacy and immunologic effects accompanying com-bination PD-1 blockade and WEE1 inhibition in both mousemodels and LSCC patient-derived cell lines.

Results:We show that multiplex gene editing of mouse lungorganoids using the CRISPR–Cas9 system allows for efficientand rapid means to generate LSCCs that closely mimic the

human disease at the genomic and phenotypic level. Usingthis genetically defined mouse model and three-dimensionaltumoroid culture system, we show that WEE1 inhibitioninduces DNA damage that primes the endogenous type IIFN and antigen presentation system in primary LSCC tumorcells. These events promote cytotoxic T-cell–mediated clear-ance of tumor cells and reduce the accumulation of tumor-infiltrating neutrophils. Beneficial immunologic features ofWEE1 inhibition are further enhanced by the addition ofanti–PD-1 therapy.

Conclusions: We developed a mouse model system to inves-tigate a novel combinatory approach that illuminates a clinicalpath hypothesis for combining ICB with DNA damage–inducingtherapies in the treatment of LSCC.

IntroductionThere are limited lung squamous cell carcinoma (LSCC) mouse

models that recapitulate the cooccurring human LSCC mutations ingenes encoding proteins operative in TP53, SOX2, PI3K, and P16(INK4a) pathways. The study of cancer genes in mouse models hastraditionally relied on genetically engineered strains made via genetargeting in embryonic stem cells. Suchmodels takemonths to years toestablish and require complicated breeding strategies when multiplegenetic alterations are needed. Moreover, unlike human lung adeno-carcinomas harboring EGFR-activating mutations or ALK fusions, for

which targeted inhibitors have achieved objective responses in up to80% cases, no targeted therapies currently exist for patients with LSCC.The extent to which LSCC mutations in these pathways contribute totumorigenesis, shape the tumor microenvironment, and affect ther-apeutic responses remains unclear. Here, we describe a new rapidapproach using a CRISPR–Cas9 genome multiediting system in lungorganoids derived from adult transgenic mice to generate an immu-nocompetent syngeneic mouse model that furthers rational immuno-therapeutic options for LSCC.

Targeting tumor immune suppression pathways represents a par-adigm shift in the treatment of lung cancer, which is the second mostcommon cancer type in the United States. Despite the promisingclinical activity of immune checkpoint–blocking antibodies againstprogrammed cell death protein 1/programmed death-ligand 1(PD-1/PD-L1) for non–small cell lung cancer (NSCLC), only a minority ofpatients (�20%) show a durable response (1). Thus, there is an urgentneed to improve objective response rates. One strategy is to combineanti-PD-1 (pembrolizumab) with chemotherapy, which has beenapproved for first-line treatment of patients with squamousNSCLC (2). Increasing evidence suggests that chemotherapy leads toimmunologic effects such as reduced T-regulatory cell activity,induced PD-L1 tumor expression, and enhanced cross-presentationof tumor antigens (3, 4). Chemotherapeutic efficacy relies on DNAdouble-stranded break (DSB) formation followed by inflammatorycytokine production to drive the killing of tumor cells over severaldivision cycles (5). Cyclin-dependent kinases 1 (CDK1) are funda-mental drivers of the cell-cycle G2–M checkpoint and are required forthe progression of various cancers (5, 6). We and others have previ-ously demonstrated that interference with the CDK1-negative regu-lator WEE1 via a selective small-molecule WEE1 kinase inhibitoractivates CDK1, which potently induces DSB formation due to loss of

1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Mas-sachusetts. 2Perlmutter Cancer Center, New York University Langone MedicalCenter, New York, New York. 3Rodent Histopathology, Harvard Medical School,Boston, Massachusetts. 4Belfer Center for Applied Cancer Science, Boston,Massachusetts. 5Department of Medicine, Boston University School of Medicine,Boston, Massachusetts. 6Department of Pathology, Brigham and Women'sHospital, Boston, Massachusetts. 7Princess Margaret Cancer Centre, UniversityHealth Network, Toronto, Ontario, Canada.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Kwok-Kin Wong, New York University Langone Med-ical Center, 550 First Avenue, New York, NY 10016. Phone: 617-632-6084;Fax: 617-632-7839; E-mail: kwok-kin.wong@nyumc.org; Adam J. Bass, Dana-Farber Cancer Institute, 450 Brookline Ave, Boston, MA 02215. Phone: 617-632-5707; E-mail: Adam_Bass@dfci.harvard.edu; and Josephine Hai,josephine.hai@utoronto.ca

Clin Cancer Res 2020;XX:XX–XX

doi: 10.1158/1078-0432.CCR-19-1627

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control at the G2–M checkpoint, leading to lung cancer cell death (6).Previous studies have also shown that CDK1 can activate STAT1signaling during mitosis, increasing proinflammatory cytokine pro-duction (5). We hypothesized that dual targeting of tumor cell–intrinsic (WEE1 inhibition) and immune cell–intrinsic (anti-PD-1)pathways may potentiate superior antitumor activity compared withmonotherapies.

Here, we show that CDK1 activation via WEE1 inhibition inducesDNA damage that primes the endogenous type I IFN and antigenpresentation system in primary mouse and human LSCC tumor cells.We show in two mouse models, including our novel organoid-derivedLSCCmodel, thatWEE1 inhibition can enhance the antitumor activityof anti–PD-1monotherapy by promoting cytotoxic NK-cell–mediatedclearance of tumor cells and decreasing immune-suppressive neutro-philic tumor infiltration.

Materials and MethodsGeneration of SOX2; Cas9 mice

All mice used in this study were housed in the pathogen-free animalfacilities inDana-Farber Cancer Institute (Boston,MA). TheRosa26R-lox-stop-lox-Sox2-IRES-GFP mice (hereafter referred as SOX2) havebeen described previously (7) and were generously gifted by Dr. KeithLigon's laboratory (Boston,MA). TheHipp11-lox-stop-lox-Cas9 (here-after referred as Cas9) mice were backcrossed to C57BL/6 background(Jackson Laboratory) and then bred with SOX2 to obtain a SOX2;Cas9colony. All breeding and care procedures were approved by the DanaFarber Animal Care and Use Committee (Protocol number: 09-073)and carried out in strict accordance with the recommendations in theGuide for the Care and Use of Laboratory Animals of the NIH(Bethesda, MD).

Isolation and culturing of epithelial organoids from murinetrachea and lung

After rinsing the dissected mouse lung with Hank's Salt (HBSS)supplemented withGibco antibiotic–antimycotic, we opened themainbronchi, placed the tissue in 1mL of dispase and incubated the tissue ina thermomixer for 15 minutes at 37�C. We next peeled the epitheliafrom the submucosa and incubated the epithelia in 1 mL of 0.25%trypsin-EDTA at 37�C for 10 minutes, briefly vortexed, rinsed thetissue with 8 mL of soybean trypsin inhibitor and repeated thetrypsinization and wash cycle once more. We filtered the dissociatedtissue through a 40-mmcell strainer using PBS and centrifuged the cellsat 200 � g for 5 minutes before resuspending the cells in organoidmedia (advanced DMEM/F12 media supplemented with Glutamax,0.15 mmol/L HEPES, Gibco antibiotic–antimycotic, N2 Supplement,B27 supplement, 1 mmol/L N-Acetylcysteine, 50 ng/mL human EGF,and 3% conditioned media from L-WRN cells containing Wnt3a,Noggin, and R-spondin). Using a 1:1 ratio of organoid media and

growth factor reduced basement membrane matrix (Matrigel, Corn-ing), epithelial organoids weremaintained for successive passages (>30passages) by hanging drop method. For subsequent passages, orga-noids were dissociated by incubation in 0.25% trypsin. The trypsiniza-tion process disrupted the spherical organoids into cell aggregates,which were then embedded in fresh Matrigel.

Cell culture and reagentsHuman embryonic kidney (HEK293T) cells, BEAS2B cells, and

LSCC cell lines (NCI-H157, NCI-H226, and NCI-H520) wereobtained from the ATCC. ATCC cells were cultured in either DMEMand RPMI1650 supplemented with 10% FBS and antibiotics. Theprimary cultures of normal human bronchial epithelial HBE4 cellswere previously derived (Supplementary Table S1) and cultured inkeratinocyte serum free (KSF) medium (Gibco). Patient-derivedxenografts were established from resected primary lung cancer withoutprior systemic therapy. Lung squamous carcinoma was confirmed byhistology and p40 IHC, a diagnostic marker for carcinoma of squa-mous lineage. All patients provided written consent and all patient-derived xenograft (PDX) cell lines (PDX139, 267, and 277) werepreviously derived using protocols approved by the University HealthNetwork Human Research Ethics and Animal Care Committee (8).KRAS, EGFR, and PIK3CA mutation status for PDX cell lines weredetected using the Sequenom OncoCarta panel v1.0 as describedpreviously (8) and on Supplementary Table S1. All cell lines wereauthenticated by short-tandem repeat fingerprinting andMycoplasmatested by PCR.

LentiCRISPRv2 vector was obtained from Addgene (#52961). Thecre-recombinase gene was first subcloned to replace the SpCas9 geneunder the EFS promoter. Guide RNAs (gRNA) against mouse PTEN,CDKN2A/p16, and TP53 and E. coli LacZ gene were cloned into ourmodified lentiCRISPRv2-Cre vector and sequence verified (Supple-mentary Table S2). Lentiviral pLKO.1 vector containing shRNAstargeting WEE1 (TRCN0000226425 and TRCN0000226426) werepurchased from Sigma-Aldrich. Transient transfections and viruspreparation in HEK293T cells were performed using Fugene Reagents(Promega) as per themanufacturer's protocol. Lentivirus was preparedby transfecting two packaging plasmids into 293T cells using protocolsfrom The RNAi Consortium (TRC; Broad Institute).

Organoids were isolated by digesting the Matrigel with 0.25%trypsin-EDTA in culture plates for 5–10 minutes at 37�C and washedtwice with PBS. Once organoids were dissociated, cells were pelletedand resuspended in 250 mL lentiviral solution. Spinoculation wasperformed by transferring the suspension onto a 48-well plate andcentrifuging the plate at 600 � g for 1 hour at 32�C. Plates were thenincubated at 37�C for 6 hours before washing the suspensionwith freshmedia and pelleting the cells to be embedded in fresh Matrigel mediamixture by hanging drop method. Stable organoid cultures wereselected by puromycin antibiotics (1–2 mg/mL).

Drugs were obtained commercially: AZD1775 (HY-10993, Med-ChemExpress), PD0166285 (HY-13925; MedChemExpress), Wee1_II(CAS 622855-50-9, Calbiochem), BX-795 (SML0694, Sigma-Aldrich),Carboplatin (NYU Pharmacy), InVivoMAb anti-mouse IFNAR-1(clone MAR1-5A3, Bio X Cell) and anti-mouse CD8a (clone 53-6.7,Bio X Cell). Anti-mouse IgG and anti-mouse PD-1 (29F.1A12) weregenerously gifted by Dr. Gordon Freeman Laboratory (DFCI, Boston,MA).

JH715 and JH716 mouse modelsOrganoids (2 � 106) were suspended in media, mixed with growth

factor reduced Matrigel (Corning) at 1:1 ratio, and subcutaneously

Translational Relevance

Considering the absence of targeted therapies for lung squamouscell carcinoma (LSCC), the generation of clinically relevant pre-clinical mouse models is urgently needed. Here, we generated anovel mouse model that represents the most frequent aberrationsfound in human LSCC, serving as an invaluable tool for developingbetter immunotherapeutic combinatorial strategies to treatpatients.

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injected into female nudemice (Nu/Nu; Charles River Laboratories) inafinal volumeof 100mL. Length andwidthweremeasured twice aweekusing calipers. Tumor volumes were calculated using the formula(Length�Width2)/2. All primary tumor cell lineswere harvested fromapproximately 150–300 mm3 tumors, minced and digested in 4%collagenase for 30minutes to 1 hour, then filtered through a 70mmol/Lcell strainer. Cell lines derived from mouse primary tumors weregrown in advanced DMEM/F12 media supplemented with Glutamax(Invitrogen), with 10% FBS (Thermo Fisher Scientific) and antibiotics.Serial passage of tumor cell lines in vivo was performed by sub-cutaneous implantation into the flank of female C57BL/6 mice(Jackson Laboratory) and harvested as described above. All miceincluded in the survival analysis were euthanized when criteria fordisease burden or targeted end points were reached.

Animal treatment studiesC57BL/6 and DBA/2J mice were obtained from Jackson Laboratory

and subcutaneously inoculated with JH716 (3 � 106) or KLN205(0.5� 106) cells in the right flank of 7- to 8-week-old mice. Mice wereexamined every 3–4 days, and tumor length and width were measuredusing calipers. Tumor volumes were calculated using the formula(Length�Width2)/2.When tumor size reached an approximatemeanof 150 or 300 mm3, mice were randomly stratified to treatmentarms: vehicle, AZD1775 (20 mg/kg), anti-PD-1 (200 mg), anti-CD8a(200 ug), carboplatin (40 mg/kg), or combination therapies. JH716mice were treated up to 30 days, while KLN205 mice were treatedup to 16 days. All manipulations were performed under sterile con-ditions in a laminar flow hood, in accordance with proceduresapproved by the DFCI Animal Care and Use Committee. At sacrifice,portions of tumors were snap-frozen and stored in liquid nitrogen orwere fixed in 10% buffered formalin for routine histopathologicprocessing.

IHC and histologySamples resected from mice were fixed in 10% formalin, and then

stored in 70% ethanol before processing and embedding. IHC wasconducted on 5-micron sections. Tissue sections were deparaffinized,rehydrated, and boiled for 15 minutes with a pressure cooker in10mmol/L pH6.0Citrate buffer. Slideswere incubated in 3%hydrogenperoxide for 30minutes and then blocked in serum for 1 hour. Primaryantibodies were added to the section and incubated overnight at 4�C(Supplementary Table S3). Representative sections of human andmouse lung tumors were reviewed by independent pathologistsincluding a rodent pathologist with expertise in mouse lung pathology(Roderick T. Bronson). Patient slides were obtained from the BrighamandWomen's Hospital (Boston, MA) Department of Pathology underthe Institutional Review Board protocol 2014P001715 and reviewedby a pathologist (Lynette M. Sholl).

Tumor-infiltrating immune cell isolation and flow cytometryanalysis

Tumor-bearing mice were sacrificed and tumors were minced anddigested in collagenaseD andDNase I inHank's Balanced Salt Solution(HBSS) at 37�C for 30 minutes. After incubation, the digested tissuewas filtered through a 70-mm cell strainer to obtain single-cell suspen-sions. Separated cells were treated with 1� RBC Lysis Buffer (BioLe-gend) to lyse red blood cells. Live cells were determined by LIVE/DEAD fixable Aqua Dead Cell Stain Kit (Molecular Probes). Fcreceptors were blocked by incubation with the anti-mouse CD16/CD32 (Fisher Scientific). Cells were stained with antibodies listed inSupplementary Table S3 followed by fixation/permeabilization (Bio-

Legend). Cells were read on LSR Fortessa (BD Biosciences) andanalyzed with Flowjo Software (Tree Star) using gating strategiesshown in Supplementary Fig. S9.

Cytokine profiling of tumor lysates and conditioned mediaMultiplex assays were performed on the EMD Millipore mouse

cytokine/chemokine 32-plex panel (MCYTOMAG-70K) utilizing thebead-based immunoassay approach. Conditioned media from ex vivothree-dimensional (3D) cultures were assayed neat. Mouse tumorswere lysed in RIPA lysis buffer and protein levels were normalized inassays. Fold changes (FC) relative to the vehicle-treated tumors/Matrigel alone media were calculated and plotted as log2FC. Lowerand upper limits of quantitation (LLOQ/ULOQ) were imputed fromstandard curves for cytokines above or below detection.

Ex vivo culture systemsMouse tumoroids were processed and loaded onto microfluidic

device as described previously (9). Live/Dead dual labeling wasperformed by loading microfluidic device with AO/PI Staining Solu-tion (CS2-0106, Nexcelom). After 20-minute dye incubation at roomtemperature, images were captured on a Nikon Eclipse 80i fluores-cence microscope equipped with Z-stack (Prior) and CoolSNAP CCDCamera (Roper Scientific). Image capture and analysis was performedusingNIS-Elements AR software. Live and dead cell quantificationwasperformed by measuring total cell area of each dye.

For 3D cell viability assays, tumoroids (40–100 mm) were filtered bycell strainers and embedded in 1:1 growth factor reduced Matrigel:media and seeded in 96-well spheroid microplates (Thermo FisherScientific) forming a hanging drop. Cell viability was measured at7 days using CellTiter-Glo 3D (Promega) and the data were repre-sented as the mean luminescence reading of 5–10 technical replicatesrelative to controls. Assays were repeated three times.

Ethics approvalAll animal experiments were performed in accordance with pro-

cedures approved by the DFCI Animal Care and Use Committee.

ResultsGeneration of CRISPR-mutant organoids gives rise to lungsquamous carcinoma

SOX2 is the most frequently amplified gene in LSCC (7). Lu andcolleagues previously demonstrated that SOX2 acts as an oncogene intransgenic mouse models conditionally overexpressing SOX2 alone inthe lung; however, penetrance in this model was only 50% andpathology of resulting tumors were lung adenocarcinoma (7). Rea-soning that multiple genetic alterations are required to potentiateLSCC, we developed a transgenic mouse to enable combined over-expression of SOX2 with deletion of distinct tumor suppressors,starting by crossing mice with inducible SOX2 and Cas9 genes (twocopies, þþ, each; Fig. 1A). The SOX2 transgene at the Rosa26 locusand the Cas9 at the Hipp11 (H11) locus were both preceded by astopper cassette (LSL) allowing the expression to be controlled via cre-recombinase in vitro and in vivo (Fig. 1A).

Given that LSCC is thought to originate from the basal progenitorcells, we next surgically harvested the tracheal bronchial epitheliumfrom adult SOX2;Cas9 mice and derived organoid cultures after tissuedissociation (Fig. 1A). To determine the efficiency of conditionallyoverexpressing SOX2, we treated organoids with lentivirus containingcre-recombinase in vitro. As shown in Fig. 1B and C, treatment withcre induced protein expression of SOX2 and SOX2-fused GFP, as seen

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Figure 1.

Generation of CRISPR-mutant organoids that give rise to lung squamous carcinoma.A,Generation of cre-inducible SOX2þþCas9þþmice. TheRosa26R-lox-stop-lox-Sox2-IRES-GFP mice (SOX2) were bred with H11-lox-stop-lox-Cas9 mice (Cas9) to obtain a SOX2;Cas9 colony. The SOX2 and Cas9 gene at Rosa26 and H11 loci,respectively, are preceded by a stopper cassette allowing the expression to be controlled via cre recombinase. Organoids were derived from the tracheal bronchialtissue of mouse lungs where human LSCC most frequently occurs. B, Immunoblot analyses indicate that SOX2 protein levels are elevated compared with controlswhen transfected with lentivirus containing cre. C, IHC analyses demonstrate that transfected organoids express basal cell–specific markers, such as cytokeratin5 (CK5), p63, andEpCAM.Organoidsdisplayedundifferentiated epithelial phenotype in vitro. GFP reporter activity is detectedonly after cre-mediated recombinationof the LoxP sites as demonstrated by treating organoids with lentivirus containing Cre. D, Integrated view of the top 10 most frequent genomic aberrations in TheCancer Genome Atlas (TCGA) data in LSCC tumors. E, Three different genotypes of mutant organoids were generated using three lentiviral constructs containingsgRNAs targeting Pten, p53, p16, and LacZ control. F, Representative images depicting transfected organoids. G, Representative chromatogram sequences ofPten (c.12C>T), p53 (c.719delT/c.718dupA), and p16 (c.295dupA) loci in triple-mutant organoids. H, Immunoblot analyses demonstrate decreased levels of targetproteins relative to control. I, Twomillion CRISPR-mutant organoids were implanted into nude mice to assess tumorigenicity. P value was determined by unpaired ttest (� , P < 0.05). J, Tumors were assessed sequentially to evaluate tumor progression. Representative H&E-stained sections of harvested tumors. K, RepresentativeH&E-stained sections showing the difference between tumors derived from sgp53;sgp16 and sg53;sgp16;sgPten implanted organoids. L, Immunoblot analysiscompared SOX2 expression in human ATCC LSCC cell lines with our mouse SCC cells. M, IHC analyses showed that SCC tumors from triple-mutant organoidsresembled the human LSCC, expressing the classic squamous markers, p63 and cytokeratin 5, seen in human disease and the hallmarks of squamous differentiation,such as keratin deposition. Scale bar, 100 mm.

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by immunoblot and IHC analyses. Transfected organoids express basalcell–specific markers such as cytokeratin 5 (CK5), p63, and EpCAM(Fig. 1C).

Using the Consortium of Genomic Analysis (TCGA) data fromLSCC cohort, we next selected the top three tumor suppressor genes(TP53, PTEN, and CDKN2A/p16) most frequently comutated withSOX2 amplification in human LSCC (Fig. 1D and E). As shownin Fig. 1E, we constructed lentiviral expression vectors with sgRNAstargeting one to three distinct tumor suppressors to establish differentgenotypes of CRISPR-mutant organoids, herein defined as control(sgLacZ), double-mutant (sgp53;sgp16), and triple-mutant (sgPten;sgp53;sgp16). We recognized that alternative splicing of exon 1 inCDKN2A could lead to two isoforms, p16 and p19(ARF); therefore, wespecifically designed our guide to disrupt both isoforms. Lentiviralinfection of organoids did not alter the morphology of the organoidcultures compared with controls, but we did observe a modest growthadvantage in the double- and triple-mutant organoids (Fig. 1F; Sup-plementary Fig. S2A). To reflect a realistic microanatomy of the tissueand to avoid potential clonal effects, our organoids were comprised ofmixed multipotent progenitor cell types under puromycin selection.Sanger and whole-exome sequencing of organoids confirmed muta-tions in the targeted genes (Fig. 1G; Supplementary Fig. S2B; Sup-plementary Table S2) and immunoblotting confirmed that targetedproteins were indeed downregulated comparedwith control organoids(Fig. 1H). Furthermore, given that Pten is a negative regulator of PI3Ksignaling, we assessed whether loss of Pten resulted in upregulation ofthe PI3K pathway. As shown in Fig. 1H, immunoblotting showedincreased phosphorylation of AKT at serine 473, indicating AKTpathway activation.

We next implanted transduced organoid cells into flanks of immu-nodeficient nude mice (Nu/Nu) and sequentially harvested tumorsover a course of 96 days to evaluate tumor latency and diseaseprogression (Fig. 1I and J). After 12 weeks in vivo, none of the lacZcontrol organoids formed tumors. The double-mutant organoidsinitially grew small tumors, but this growth eventually stalled. Inearly-stage cases, the triple-mutant organoids formed cilia, a featureseen in lung epithelia, and at week 3, tumors grew significantly andprogressively larger compared with controls (Fig. 1J). Hematoxylinand eosin (H&E) staining confirmed that all the double-mutanttumors were benign, while triple-mutant tumors harvested after day68 formed well-differentiated squamous cell carcinomas (Fig. 1Jand K). SOX2 protein levels in triple-mutant tumor cell lines wereindeed elevated relative to human LSCC tumor cell lines (Fig. 1L). Wealso observed some cases of adenosquamous cell carcinomas and well-differentiated adenocarcinomas arising from the triple mutants atearlier harvests (Supplementary Fig. S2C). We and others have pre-viously observed that LKB1-deficient ADC can progressively trans-differentiate into SCC inmousemodels (10, 11), but further studieswillbe needed to understand whether tumors are arising frommultipotentcells that transition between ADC and SCC or through selectiveredifferentiation within squamous carcinoma.

To confirm the phenotype of the LSCC triple-mutant tumors, wefurther performed IHC for markers used clinically to distinguishhuman lung SCC from ADC. Similar to the human SCC samples,the LSCC triple-mutant tumors displayed positive staining of p63,CK5, and SOX2 (Fig. 1M; Supplementary Fig. S2D). Triple-mutanttumors recapitulated the hallmarks and pathology of human LSCC:squamous cells grew in solid configurations with aberrant nuclearmorphology, infiltrating neutrophils, and invading stroma withprominent keratin pearls (Fig. 1K and M). We also confirmed thatour LSCC model recapitulates features of the human disease

transcriptionally utilizing bulk RNAseq. Prior TCGA studieson human LSCC have shown significant alternations in innateimmune transcriptional networks, including HLA gene complexregulation, that are not observed in KRAS oncogene–driven lungadenocarcinoma (11–13). Intriguingly, we found significant enrich-ment for innate immune and infectious disease gene sets, includingviral infection response and antigen processing, in SCC tumorsrelative to the well-established KRASG12C;TP53 lung adenocarcino-ma model (Supplementary Table S5; Supplementary Fig. S3A).

Cytokine profiling of conditioned media from ex vivo 3D culturingof double- and triple-mutant tumors after 72 hours revealed increasedlevels of neutrophil regulators (CXCL1, G-CSF, and GM-CSF) in SCCtumors compared with benign double-mutant tumors, supporting ourobservation of neutrophil infiltration (Supplementary Fig. S3B).

We generated a stable LSCC cell line (JH716) and orthotopicallyimplanted these lines back into littermates (Cas9;SOX2) via intratra-cheal injection to generate immunocompetent LSCCmodels. Immuneprofiling analysis demonstrated a similar percentage of tumor lym-phocyte infiltration compared with our KRAS-driven geneticallymodified mouse models, but higher levels of myeloid CD11bþ

cell populations (Supplementary Fig. S3C). LSCC phenotype wasindeed preserved in tumors derived from these models and seriallypassaged in vivo to generate subsequent cell lines such as JH716-18(Supplementary Fig. S3D). Taken together, these data indicate thatthe triple-mutant tumors (SOX2, TP53, PTEN, and CDKN2A/p16)strongly resembled human LSCC.

WEE1 inhibition induces DSB-stimulated type I IFN signalingand expression of genes encoding antigen-presenting MHCclass I in mouse- and patient-derived lung cancer cells

Given the recent clinical response and FDA-approval for thecombination of anti–PD-1 with chemotherapy as a first-line treatmentof patients with NSCLC, we reasoned that the DNA repair machineryis an underlying critical component that triggers anti–PD-1 responsein lung cancer. We previously demonstrated that a selective small-molecule WEE1 kinase inhibitor potently induces DNA DSB forma-tion leading to cell death in KRAS-mutant lung adenocarcinomas. Wetherefore first assessed the tumor-intrinsic cytotoxic impact of WEE1inhibition (AZD1775) in our new squamous models (JH716) andpatient-derived xenograft LSCC tumor cells (PDX139 and PDX267)along with matched normal mouse fibroblasts (JH716F) or nontu-morigenic human bronchial epithelial (HBE4 and BEAS2B) cellsderived from primary lung tissues (Fig. 2A and B). AZD1775 reducedcell proliferation in all lines tested but to a greater extent in tumor cellscompared with normal cells (IC50s: 330 nmol/L vs. >1310 nmol/L). Asexpected, confocal microscopy confirmed that AZD1775 inducedDSBformation as indicated by high levels ofDSBmarker gH2AX in treated-JH716 and PDX267 cells, supporting previous studies demonstratingcell death due to accumulation of DNA damage from mitotic catas-trophe (Fig. 2C). Immunoblot analyses confirmed downstream targetengagement (reduced phosphorylation of CDK1 at the inhibitorytyrosine 15 site and gH2AX upregulation in a dose- and time-dependentmanner) inmurine JH716 and humanPDX267 cells treatedwith vehicle (DMSO) or AZD1775 (Fig. 2D; Supplementary Fig. S4Aand S4B).

Inhibition of CDK1 has been shown to block mitotic entry andSTAT1 activation (5). To investigate whether WEE1 inhibitionincreases STAT1 signaling by inducing CDK1 activation, wemeasuredSTAT1 phosphorylation at tyrosine 701 (pSTAT1 Y701) in mousetumor cells treated with AZD1775 over a 24-hour time course.Activation of pSTAT1 Y701 was observed in the treated AZD1775

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cells after 24 hours, and this was correlated with reduced phosphor-ylation of CDK1 on the inhibitory residue Y15 and elevated gH2AX,suggesting that WEE1 inhibition may be promoting DSB-inducedSTAT1 activation viaCDK1 activation (Fig. 2E). Given that STAT1 is acritical downstream transcription factor for type I IFN signaling, wenext examined the expression of IFN-stimulated genes (ISG), such asIFNB1, OAS1, CCL5, IFITM1, IFIT3, CDKN1A, CCL2, ISG54, andIFNA13. Consistent with STAT1 activation, expression of ISGs wassignificantly upregulated in both AZD1775-treatedmouse and humanLSCC lines (JH716, H157, and H520; Fig. 2F and G; SupplementaryFig. S5A and S5B) compared with controls. Similar trends wereobserved when cells were treated with additional small-molecule

WEE1 inhibitors, Wee1_II (on JH716 cells; Fig. 2F), and PD0166285(on H157 and H226 cells; Fig. 2G and H) suggesting that these were“on-target” effects. In addition, H226 and H157 cells were transducedwith lentivirus containing shRNA against WEE1 to significantlydownregulate WEE1 transcript levels by 51%–93% compared withcells transfected with control vector (Fig. 2I). RNA from cells wasextracted 3 days after transfection to assess ISG expression levels.WEE1 downregulation significantly correlated with elevated ISGs asshown in Fig. 2I and Supplementary Fig. S5C.

Importantly, we observed increased expression of genes encodingantigen-presenting mouse major histocompatibility complex (MHC)class I molecules (H2K1 and B2M), peptide transporters (TAP1 and

Figure 2.

WEE1 inhibition induces DNA damage, activating STAT1 signaling and stimulating expression of type I IFN and antigen presentation genes in murine and patient-derived LSCC cell lines.A,Dose–response curves of murine JH716 tumor cells andmatched fibroblast cells treated for 3 dayswith AZD1775.B,Dose–response curvesof patient-derived xenograft LSCC cells (PDX139 and PDX267) and normal epithelial lung cells (HBE4 andBEAS2B) treated for three dayswith AZD1775.C,Detectionof gH2AX and DAPI in JH716 (left) and PDX267 (right) cells by immunofluorescence after 48-hour treatment with vehicle (DMSO) or AZD1775 (300 and 600 nmol/L,respectively). Representative foci-containing cells at high-power magnification are shown. D, To assess downstream target engagement (pCDK1 at tyrosine 15 andgH2AX) in murine JH716 and patient-derived LSCC PDX267 cells, immunoblot analyses were performed on whole-cell lysates 48 hours after treatment with vehicle(DMSO) or AZD1775 (600 nmol/L). E,Representative immunoblot for activation of STAT1 signaling at indicated times after 600 nmol/L AZD1775 treatment on JH716cells. F–H, qRT-PCR analysis was used to measure mRNAs of type I IFN-stimulated genes (ISG) and antigen presentation genes in murine LSCC JH716 cells (F) andhuman LSCC H157 (G) and H226 cells (H) treated with small-moleculeWEE1 inhibitors (AZD1775,Wee1_II, or PD0166285) for 24 hours. I,WEE1 transcript levels weredownregulated in H226 cells using two different shRNAS targeting WEE1 and a pLKO. empty vector control. mRNA of ISGs were measured by qRT-PCR analysis3 days after transduction. J, Expression of H-2Kb on JH716 cells treated with AZD1775 (600 nmol/L) or PD0166285 (600 nmol/L) for 3 days. Quantificationof mean fluorescence intensity (MFI) is shown on the right. FMO indicates fluorescence minus one. Data are shown as mean �SEM. P values were determined byKruskal–Wallis test (n ¼ 4 biological replicates; � , P < 0.05; �� , P < 0.01; ��� , P < 0.001; ���� , P < 0.0001).

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TAP2), peptide cleavers (ERAP1), and transporter-MHC interactors(TAPBP) in JH716 cells after WEE1 inhibition (Fig. 2F, right).AZD1775 treatment in vitro also significantly increased expressionof orthologous genes in human lung cancer lines (H157, PDX277, andH520; Fig. 2G, right; Supplementary Fig. S5A and S5B). Flow cyto-metry analysis showed that 72-hour treatment of AZD1775 orPD0166285 at 600 nmol/L significantly elevated expression of H-2Kb(MHC-I) by approximately 2-fold but did not change PD-L1 or I-A/I-E (MHC-II) expression levels on the surface of JH716 tumor cells(Fig. 2J; Supplementary Fig. S5D). As further support, the JH715tumor cell line was generated from another of the cohort of 8malignant tumors in Supplementary Fig. S2C. We confirmed JH715cells to be a separate clonal population as it contains a different cut sitein p53 (c.809G>A/p.Arg270His) compared with the earlier-usedJH716 cell line. Consistent with our results in JH716 cells, we alsoobserved increased expression of H-2Kb upon AZD1775 treatmentin vitro in JH715 cells (Supplementary Fig. S5E).

Combined immune checkpoint blockade and WEE1 inhibitionreduces tumor growth and prolongs survival

Given that our in vitro data demonstrated that WEE1 inhibitioncan increase expression of genes encoding antigen presentationmolecules on lung squamous carcinoma models, we reasoned thatcombining AZD1775 with anti–PD-1 may enhance tumor cellkilling by stimulating T-cell activity. To test this ex vivo, we firstcultured fresh tumoroids from subcutaneous implants of JH716cells and another lung SCC mouse model, KLN205, in 3D micro-fluidic culture systems. Our previous studies demonstrated thattumoroids retain the autologous tumor-infiltrating immune cells inthis short-term culture system (9, 14). We observed that bothsingle-agent AZD1775 and anti–PD-1 treatment were insufficientto induce significant cell death; however, combined anti–PD-1 andAZD1775 synergistically induced cell death in both JH716- andKLN205-derived lung squamous tumoroids by 6-day cotreatment(Fig. 3A and B).

To further investigate whether the observed cell death is medi-ated by type-I IFN signaling, we performed a CellTiter-Glo 3Dcell viability assay using neutralizing antibodies specific for mouseIFN-a/b receptor subunit 1 (IFNAR) and a TBK1 small-moleculeinhibitor (BX-795), a regulator of IFN transcription (15). Theaddition of anti-IFNAR and BX-795 to combined AZD1775 andimmune checkpoint blockade (ICB) therapy (triple combination)significantly reduced cell death of JH716 tumoroids treated withAZD1775 and ICB therapy (P < 0.0001; Fig. 3C).

We next examined the effects ofWEE1 inhibition on tumor burdenin vivo using a low-AZD1775 dose to minimize potential off-targeteffects while achieving target engagement (Fig. 3D). AZD1775 alonewas not sufficient to reduce tumor growth in JH716 mice (Fig. 3E;Supplementary Fig. S6A and S6B). However, combined AZD1775 andanti–PD-1 treatment led to significant tumor regression comparedwith single agents or vehicle-treated control mice, showing 97% tumorgrowth inhibition and nearly 60% (3/5) complete tumor remissions(Fig. 3E). JH716 tumors were sensitive to anti–PD-1 single-agenttreatment with 59% tumor growth inhibition, but no complete regres-sions were observed. Depleting anti-CD8a was administrated 7 daysand 1 day prior to first anti–PD-1 dose and at day 14 during treatment.Therapeutic efficacy of the combination treatment was partially lostwhen CD8þ T cells were depleted, suggesting that while the tumorelimination phenotype is partly T-cell–dependent, there are otherimmune modulators contributing to complete tumor elimination(Fig. 3E,n¼ 7).We further validatedwhether AZD1775 complements

PD-1 blockade in another myeloid-enriched syngeneic mouse model,KLN205, that is refractory to anti–PD-1 therapy and has lower tumorimmune infiltrate (CD45þ cells) relative to JH716 tumors (Supple-mentary Fig. S6C). Similar to JH716 tumors, we observed KLN205models were unresponsive to AZD1775 or anti–PD-1 alone butshowed significant tumor growth inhibition in the combined treat-ment relative to anti–PD-1 or AZD1775 monotherapy, although wedid not observe complete regressions (Fig. 3F).

Given the potent antitumor effect of combined AZD1775 and ICBtherapy, we next evaluated how this comparedwith chemotherapy andICB dual therapy, which is currently FDA-approved for NSCLCs.Using a secondary cell line derived from parental JH716 tumors(denoted JH716-18; Supplementary Fig. S3D), we repeated an inde-pendent efficacy study on JH716-18 tumors starting at a volumeof approximately 300 mm3 (n ¼ 10–13/treatment arm) with theaddition of the chemotherapy (carboplatin) and a combination arm(carboplatin and anti–PD-1). Survival analysis up to terminal tumorvolume (20 mm length) showed significant prolonged survival ofmice (with 5/13 complete tumor regressions) in the AZD1775 andanti–PD-1 combination group compared with single-agent–treatedarms (Fig. 3G). Similarly, combined carboplatin and anti–PD-1 alsoachieved significant survival benefit with 6/13 complete tumor regres-sions, suggesting that WEE1 inhibition may have comparable antitu-mor immunity as chemotherapy in our JH716-18 models. Hemato-logic toxicity assays and body weight analyses showed that bothcombination therapies were well-tolerated in mice (SupplementaryFig. S7A and S7B). We also observed that combiningWEE1 inhibitionwith ICB therapy markedly extended survival in treated KLN205 mice(Fig. 3H). Multiplexed cytokine profiling of JH716-18 tumors after1 week of treatment revealed that cotreatment with AZD1775 andanti–PD-1 downregulated levels of G-CSF, GM-CSF, CXCL2, andCXCL1, which are neutrophil chemoattractants, and increased levelsof CCL5, which may enhance NK- and T-cell recruitment, relative tovehicle controls (Fig. 3I).

Combined ICB and WEE1 inhibition reduces tumor-associatedneutrophil infiltration and enhances NK recruitment in tumors

We next sought to determine howWEE1 inhibition and combinedtreatment may affect the quantity and subsequent recruitment oftumor-associated immune cell populations. Using flow cytometry, weanalyzed the tumor-infiltrating lymphoid and myeloid cell popula-tions at 14 days posttreatment in JH716 mice (Fig. 4A–I) and 9 daysposttreatment in KLN205 mice (Fig. 4J–Q). In JH716 tumor-bearingmice, we observed a significant increase in CD8þ tumor-infiltratinglymphocytes (TIL) in anti–PD-1–treated tumors compared withAZD1775 alone or vehicle control (Fig. 4B). Combined treatmentdid not further elevate CD8þ TIL levels relative to anti–PD-1, but didsignificantly increase tumor-infiltrating NK (CD45þ/CD3�/CD49bþ)cells relative to vehicle controls (Fig. 4C). We also observedincreased Ki67þ-proliferating NK cells in tumors in the combinationgroup compared with controls (Fig. 4D). In addition to its effects onthe lymphoid populations, combined therapy significantly decreasedthe CD11bþ cells, including the tumor-associated neutrophils(CD11bþ/MHCII�/Ly6Gþ/Ly6Clo; TANs) compared with AZD1775and isotype controls (Fig. 4E and F). However, combined therapydid not significantly change the proportions of tumor-associatedmonocyte (CD11bþ/MHCIIþ/F4/80�/Ly6Cþ), macrophage (CD11bþ/MHCIIþ/F4/80þ/Ly6C�; TAMs) or dendritic cell (CD11bþ/MHCIIþ/F4/80�/Ly6C�/CD11cþ) populations in the tumor (Fig. 4G–I).In KLN205 tumor-bearing mice, we observed significantly increas-ed CD8þ TILs in the combination-treated tumors compared with

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anti–PD-1 and isotype controls (Fig. 4J). In addition, we found signif-icantly more proliferating NK cells in the combination group relative tothe controls (Fig. 4K and L). In fact, AZD1775 alone increased NKinfiltration (Fig. 4K). Consistent with JH716 tumors, combinedtherapy had no significant effects on inflamed monocytes or den-dritic cells, but significantly reduced TANs in KLN205 tumorsrelative to isotype controls (Fig. 4N–Q). Taken together, these datasuggest that combining WEE1 inhibition with ICB therapy

enhances antitumor activity by reducing tumor-associated neutro-phils in myeloid-enriched tumors, which is correlated with higherlevels of cytotoxic CD8þ T and NK cells.

DiscussionIn vivo interrogation of the function of genes implicated in tumor-

igenesis is limited by the resource-intensive need to generate and cross

Figure 3.

Combined ICB and WEE1 inhibition reducestumor growth and prolongs survival.A,Quan-tification results of live (AO¼green) anddead(PI ¼ red) analysis of LSCC tumoroids (JH716and KLN205) cultured in 3D microfluidic cul-ture at day 0, day 3, and day 6 followingtreatment of AZD1775 (300 nmol/L) alone orin combinationwith PD-1 antibody (10mg/mL)as indicated (n ¼ 3 biological replicates).B, Representative images of deconvolutedfluorescence microscopy show live/deadstained JH716 cells at day 6 after indicatedtreatment. Statistical analysis was calculatedby comparing each group with the combina-tion group treated at day 6 by one-wayANOVA. C, CellTiter-Glo 3D cell viabilityassays using neutralizing antibodies specificfor mouse IFNa/b receptor subunit 1 (IFNAR;40 mg/mL) and a TBK1 small-molecule inhib-itor (BX-795; 1 mmol/L) was performed onJH716 tumoroids treated with AZD1775(300 nmol/L) and anti–PD-1 (10 mg/mL) for7 days. Quantification of mean luminescencewas normalized to vehicle controls as shown.P values were determined by one-wayANOVA (n¼ 3 biological replicates). D, Ther-apy regimen (PO, per os; IP, intraperitoneal;QD, once a day; QW, once a week).Mean tumor volume of subcutaneous JH716(n¼ 5–7/group; E) and KLN205 (n¼ 7/group;F) implants in mice treated with vehicle,AZD1775, anti–PD-1, or the combination whentumor burden reached approximately 150mm3. Survival analysis was performed on anindependent JH716-18 tumor efficacy studyat approximately 300 mm3 consisting ofn ¼ 10–13 animals per group (G) and KLN205tumors consisting of n ¼ 7 animals per groupfrom F (H). I, Heatmap of intratumoral cyto-kine profiles from JH716-18 tumors treatedfor 1 week are expressed as log2 foldchange (L2FC) relative to vehicle controls(n ¼ 5–7 tumors/group). Data are shownas mean � SEM (�, P < 0.05; �� , P < 0.01;��� , P < 0.001; ���� , P < 0.0001). A log-rankMantel–Cox test was used for Kaplan–Meieranalysis. Scale bar, 100 mm.

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Figure 4.

Phenotyping of tumor-associated immune cell populations after treatment in JH716 and KLN205 mouse models. A, Therapy regimen and flow cytometry analysistime points (PO, per os; QD, once a day; IP, intraperitoneal). Mice were subcutaneously inoculated with JH716 (3� 106) or KLN205 (0.5� 106) cells and treated withvehicle, AZD1775 (20 mg/kg), anti–PD-1 (200 mg), or combination therapy for 14 and 9 days, respectively. Quantification by flow cytometry of CD8þ T cells (B),Natural killer cells (NK;C), Ki67þ-proliferatingNK cells (D), CD11bþmyeloid cells (E), Tumor-associated neutrophils (TAN; CD11bþ/MHCII�/Ly6Gþ/Ly6Clo; F), Tumor-associatedmacrophages (TAM;CD11bþ/MHCIIþ/F4/80þ/Ly6C�;G), Tumor-associatedmonocytes (CD11bþ/MHCIIþ/F4/80�/Ly6Cþ;H), Tumor-associateddendriticcells (CD11bþ/MHCIIþ/F4/80�/Ly6C�/CD11cþ; I), in CD45þ TILs from dissociated JH716 tumors. J–Q, Flow cytometry analysis of dissociated KLN205 tumors. Eachdata point represents one mouse (n ¼ 4–5 mice/group). P values were determined by one-way ANOVA analyses. Data are shown as mean � SEM.

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mice bearing multiple, distinct engineered inducible mutant alleles.Furthermore, these models require tissue-specific cre-drivers, whichcan pose challenges for squamous cancers, because tamoxifen-inducible cre recombinase can lead to premature study terminationswhen mice develop skin lesions before developing tumors (16).

Since the Clevers group described the successful derivation ofepithelial stem cell–based organoid long-term cultures from singleLgr5þ stem cells in 2009, a plethora of different tissue organoidculture methods have been developed (17). Emerging data suggestthat 3D organoid cultures can recapitulate cellular composition,structure, and signaling characteristics better than traditional two-dimensional culture (18). Here, we demonstrate a successful orga-noid culture approach where LSCC is induced by CRISPR–Cas9–based multiplex editing of TP53, PTEN, and CDKN2A (p16) tumorsuppressor genes in lung epithelial cells derived from a SOX2-conditionally expressing transgenic mouse. Subsequent transplan-tation to immunocompetent mice reproduced LSCC that pheno-typically mimic the human disease. This system enabled rapidin vivo characterization of driver mutations essential to inducesquamous differentiation and enabled further interrogation ofimmunotherapeutic combinations on a genetically defined preclin-ical model. Given the capacity to generate organoid models from avariety of distinct squamous tissues (19, 20), we believe this generalapproach will prove robust for generating squamous cell modelswith varied genomic features, which will powerfully enable bothfundamental mechanistic and preclinical studies.

There is a clear need to develop immune-competent murinemodelsof lung and other SCCs. Historically, squamous NSCLC treatment wasmostly limited to cytotoxic chemotherapy due to the lack of targetableaberrations. In the past 5 years, clinical trials have demonstrated thatinhibitors of PD-1 are effective in the treatment of squamous andnonsquamous NSCLC (21–23). In the CheckMate 017 trial, the overallsurvival for previously treated patients with squamous cell NSCLCwassignificantly better with nivolumab than with docetaxcel, regardless ofPD-L1 expression levels (21).

Most recently, the landmark KEYNOTE-407 trial has revealedefficacious antitumor responses in metastatic, squamous NSCLCtreated first-line with pembrolizumab and platinum-based chemo-therapy combination, leading to FDA approval of the treatment (2).Accumulating evidence suggests that chemotherapy can stimulateimmunosurveillance in several ways by acting on cancer cells to: (i)increase antigen presentation, (ii) induce danger signals that stim-ulate innate immune responses, or (iii) increase susceptibility oftumor cells to be recognized and killed by immune effector cells.Cisplatin, etoposide, and paclitaxel have all been shown to inducebreast cancer cells to produce IFNb, which in turn operates as anautocrine factor to stimulate MHC class I expression (24). Similar tothe chemotherapeutics' reliance on DSB formation to drive tumorcell killing, we and others have previously shown that selectiveinhibition of WEE1, a negative regulator of a CDK1, leads to potentDSB formation due to exacerbated mitotic catastrophe in deregu-lated cancer cells (25). As such, we hypothesized that selectiveinhibition of WEE1 may similarly elicit tumor cell immunogenicity.Indeed, we show that AZD1775 and WEE1i treatment in vitropotently increases expression of antigen-processing and presenta-tion genes in both mouse and patient-derived LSCC cell lines, whichcorrelates with DSB as measured by increased gH2AX foci forma-tion in tumor cells.

Harding and colleagues previously showed that CDK1i preventedSTAT1 activation in PARPi-treated BRCA1-mutated ovarian cancercells, suggesting that mitotic entry is a gateway to inflammatory

signaling for diverse DSB-inducing stimuli (5). Consistent with this,we demonstrate that activation of CDK1 by AZD1775 treatment notonly induces STAT1 phosphorylation at Y701 in a time-dependentmanner, but also increases ISG expression in tumor cells, likelyexplaining their enhanced capacity for antigen presentation (24).Along similar lines, Goel and colleagues recently showed thatCDK4/6 inhibition induced cell-cycle arrest, which resulted in globalupregulation of an IFN-driven transitional program and viral mimicryon breast cancer cells (26). AlthoughCDK1 is best known for its role incontrolling the G2–M cell-cycle checkpoint, a novel function of thekinase in immune response has emerged. For instance, Cribier andcolleagues identified that CDK1 phosphorylates SAMHD1 at residueThr592, directly inhibiting its antiviral restriction activity (27).SAMHD1 is a deoxynucleotide triphosphate (dNTP) triphosphohy-drolase that reduces the intracellular pool of dNTPs to restrict HIV-1synthesis (27). Depletion of SAMHD1 leads to enhanced antigenpresentation and chronic type I IFN secretion upon HIV-1 infectionand facilitates T-cell responses in coculture models (28). Furtherstudies will be necessary to carefully interrogate whether SAMHD1or other interacting partners of CDK1 may be mediating the IFNresponse upon AZD1775 treatment, rendering the tumor microenvi-ronment more permissive to T- or NK-cell infiltration.

Nearly all humans with LSCCs have a history of tobacco use thatdrives squamous metaplasia and the development of an inflammatorymicroenvironment and chronic immune suppression (29). TANs andTAMs comprise a significant proportion of the inflammatory infil-trates in both mouse tumor models and human cancers (29). Here, wefound that our orthotopic JH716 tumors were indeed enriched fortumor-infiltrating CD11bþ myeloid cells and inflammatory cytokinescompared with a well-established KRASG12D lung adenocarcinomamouse model (Supplementary Fig. S3C). Our in vivo efficacy studiesdemonstrate that cotreatment with anti–PD-1 and AZD1775 signif-icantly reduces tumor growth and TAN infiltration in JH716 andKLN205 tumors. We found that treatment with AZD1775 in JH716mice resulted in reduced levels of neutrophil chemoattractant G-CSF.Notably, the addition of anti–PD-1 therapy with AZD1775 furtherreduced intratumoral levels of G-CSF and CXCL2. As type I IFNs havebeen shown to downregulate G-CSF expression and G-CSF is knownto be a major regulator of neutrophil development and mobilizationand CXCL2 secretion, we speculate that reduced neutrophil chemoat-tractants may be partly due to DSB-stimulated type I IFN signalingupon AZD1775 treatment (30).

Previous studies have shown that TANs can suppress T-cellresponses and inhibit NK-cell function, establishing a protumormicroenvironment that facilitates metastatic formation (31). Consis-tent with these results, we observed an inverse correlation betweenTAN abundance and increased CD8þT andNK cells in our JH716 andKLN205 tumors treatedwith dual therapy. In agreement with our data,De Henau and colleagues showed that targeting myeloid cells viaPI3Kg inhibition enhanced immunotherapeutic effect of anti–PD-1 inresistant tumors (32). Given that emerging data suggest that neutro-phils contribute to anti–PD-1 resistance, further studies are needed todelineate the mechanistic basis of how combined anti–PD-1 andAZD1775 reduce TAN infiltration.

Previous studies have demonstrated that CDK inhibition preventsdeath of terminally differentiated neurons, suggesting that noncyclingcells such as neutrophils may require activation of CDK for apoptoticinduction (33, 34). In contrast, others have shown that humanneutrophils express CDK1, CDK2, and CDK5, and that specificinhibitors of CDKs (namely, R-roscovitine) induce caspase-dependent apoptosis (35). CDK activity is clearly important to

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neutrophil survival (34, 35). Thus, it would be interesting to examinewhether AZD1775-induced DNA damage directly mediates neutro-phil homeostasis by either deregulating signaling of its traffickingreceptors (i.e., CXCR2) or apoptotic response.

In addition to the reduction of TANs, we also observed thatAZD1775 combined with ICB therapy led to increased NK-cellinfiltration. Our cytokine profiling data showed elevated intratu-moral CCL5 levels in cotreated JH716 samples, which may partiallyexplain the heightened NK-cell influx. Recently, B€ottcher andcolleagues demonstrated that cDC1 accumulation in mouse tumorsoften depends on NK cells that produce cDC1 chemoattractantsCCL5 that augment immunotherapeutic response (36). Indeed, wealso observed a positive correlation between intratumoral CCL5chemokine levels and NK infiltrates. However, while we observed atrend of increased tumor-associated DCs (CD11bþ/MHCIIþ/F4/80�/Ly6C�/CD11cþ) compared with controls in JH716 tumors, thisdid not reach significance. Further NK-cell depletion studies areneeded to delineate whether NK cells are directly mediating theantitumor effects of combined therapy or indirectly stimulatingrecruitment of cDC1. It is also tantalizing to speculate that we maybe observing NK-cell–mediated antitumor immunity due to WEE1inhibitor–mediated DNA damage and subsequent STING and typeI IFN pathway activation, as has been recently shown (37). Such amechanism would be consistent with our observed NK-cell accu-mulation and increased proliferation in our AZD1775-treatedtumor models.

We have pioneered the development of ex vivo genomic editingof mouse 3D organoid cultures to generate a rapid, novel LSCCmouse model that exhibits characteristic histopathology and bio-marker expression similar to human LSCC. We generated two celllines, JH715 and JH716, harboring SOX2;PTEN;CDKN2A;TP53alterations. JH715 cells differ from JH716 by the TP53 cut site,but also exhibited increased expression of H-2Kb upon AZD1775treatment in vitro, which we believe supports the WEE1-specificimmunologic mechanism described in significant depth utilizingJH716 cells. We have also applied this system to explore differentgenotypes of human LSCC, but have observed that single or doublealterations of TP53, CDKN2A, or KDM6A in concert with SOX2overexpression was not sufficient to form LSCC tumors, thereforelimiting generation of additional mouse cells from these specificgenotypes (Fig. 1I; Supplementary Fig. S8A–S8D). Our workillustrates the challenges in understanding which cooperating muta-tions potentiate LSCC, because not all genotypes lead to LSCC inmice. Consistent with our data, Bern's group has also demonstratedthat various combinations of genetic lesions can determine differentcell fates and showed that SOX2;PTEN;CDKN2A transgenic micegave rise to LSCC tumors in 7–9 months with a 73% penetrance,while FGFR;PTEN;CDKN2A mice had 19% SCC penetrance (38).Sequencing of JH716 tumors also revealed mutations in genes notknown to drive LSCC (data not shown). We believe that thecombined effects of deleting genes in the DNA damage repair/cell-cycle machinery during long term in vivo culturing leads toaccumulation of passenger mutations that help facilitate rapidLSCC tumorigenesis. Yet another possibility is the potential ofCas9-induced immunity or off-target effects, which continues tobe actively investigated in this field because the full impact ofpreexisting immunity has yet to be fully determined (39).

Using this mouse model, we explored a rational therapeuticcombination to enhance response to PD-1 blockade by stimulatingimmunosurveillance with a selective DSB-inducing agent. Wedemonstrated for the first time that CDK1 activation via WEE1

inhibition can induce DSB-stimulated type I IFN signaling andantigen presentation in mouse and patient-derived LSCC tumors.Combined WEE1 and PD-1 inhibition significantly induced anti-tumor efficacy both ex vivo and in vivo, consistent with theefficacious response observed in mice treated with carboplatin andanti-PD1 cotreatment. Although we did observe some inhibitoryactivity in nontransformed cells treated with AZD1775, we believethat cancer cells with high replicative stress are more vulnerable,and therefore we speculate that selectively activating CDK1 byAZD1775 may minimize the off-target side effects associated withchemotherapy while augmenting antitumor immunity. The clinicalchallenge will be to assess dose-limiting toxicity associated with thepromiscuous nature of kinase inhibitors such as AZD1775. In fact,phase I trials (NCT00648648) of AZD1775 and chemotherapyshowed responding patients with advanced solid tumors weremildly enriched for p53 mutations (response rate of 21% and12% in p53-mutant and p53 wild-type, respectively), supportingthe notion that patient selection may be necessary to obtain themaximum benefit from AZD1775 and ICB combination thera-py (40). Yet another possibility is that relapsed p53-mutant tumorsrelying on G2–M checkpoint may be more responsive to AZD1775and ICB combination therapy than tumors with intact G1 check-point. Future studies may also evaluate whether triple combinationssuch as AZD1775, PARPi, and ICB therapies targeting both G1,G2, and immune checkpoints may reach improved efficacy. Ourobservations illuminate a clinical hypothesis for combining anti-PD1 with DNA damage–inducing therapies in the treatment ofLSCC. As with all preclinical model systems, prospective clinicaltrials will be needed to substantiate the hypothesis of our work.

Disclosure of Potential Conflicts of InterestL.M. Sholl is an employee/paid consultant for AstraZeneca, LOXO Oncology,

Foghorn Therapeutics, and EMD Serono. A.J. Bass reports receiving commercialresearch grants fromNovartis, Bayer, andMerck. K.-K.Wong is a founder and equityholder of G1 Therapeutics and has consulting/sponsored research agreements withAstraZeneca, Janssen, Pfizer, Array,Novartis,Merck, Takeda,Ono, Targimmune, andBristol-Myers Squibb. No potential conflicts of interest were disclosed by the otherauthors.

Authors’ ContributionsConception and design: J. Hai, A.J. Bass, K.-K. WongDevelopment of methodology: J. Hai, J. Zhou, Z. Wu, T. Chen, J.B. Liu,A.J. BassAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Hai, H. Zhang, J. Zhou, Z. Wu, E. Papadopoulos,C.M. Dowling, V. Pyon, Y. Pan, H. Silver, P.H. Lizotte, M.-S. Tsao, C. Thakurdin,K.-K. WongAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Hai, H. Zhang, C.M. Dowling, Y. Pan,R.T. Bronson, H. Silver, P.H. Lizotte, J. Deng, J.D. Campbell, L.M. Sholl,K.-K. WongWriting, review, and/or revision of the manuscript: J. Hai, H. Zhang, P.H. Lizotte,L.M. Sholl, A.J. Bass, K.-K. WongAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): J. Hai, T. Chen, Y. Pan, J.B. Liu, C. NgStudy supervision: J. Hai, A.J. Bass, K.-K. WongOthers (did the pathology for this study): R.T. Bronson

AcknowledgmentsThe authors thank Mei Zhang (BWH) for help with IHC work, Yanxi Zhang

(DFCI) and Xiaoen Wang (DFCI) for technical animal assistance, Dr. ElenaIvanova (Belfer Center) for technical help and protocols for staining 3D cultures,and Dr. Israel Canadas (DFCI) for thoughtful discussions about innate pathwaysignaling. The authors thank Drs. Joe Wang, Limei Ouyang, and Elinor Sunfrom the BGI Group for whole-exome sequencing and RNAseq analysis.

Immunotherapy for Lung Squamous Cell Carcinoma

AACRJournals.org Clin Cancer Res; 2020 OF11

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This work was supported by the NCI [U01 CA233084-01 (co-principalinvestigator), CA213333 (principal investigator), R01 CA219670-A1 (co-principal investigator), CA216188-01A1 (co-principal investigator), CA222218-01A1 (co-investigator), CA205150 (co-investigator), CA201049 (co-investigator),CA197329 (co-investigator), CA140594 (principal investigator), CA166480(co-investigator), P01 CA098101 (principal investigator), and CA154303(co-investigator)] to K.-K. Wong and CA187119 to A.J. Bass.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 17, 2019; revised November 22, 2019; accepted March 19, 2020;published first March 24, 2020.

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Published OnlineFirst March 24, 2020.Clin Cancer Res   Josephine Hai, Hua Zhang, Jin Zhou, et al.   Study of Combinatorial ImmunotherapyModels for Squamous Cell Lung Cancers Allows for the Generation of Genetically Engineered Mouse Lung Organoid

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