Genetic Aberrations in the CDK4 Pathway Are Associated ... · Translational Cancer Mechanisms and Therapy Genetic Aberrations in the CDK4 Pathway Are ... PD-1–humanized mouse (C57BL/6-hPD-1)

Translational Cancer Mechanisms and Therapy

Genetic Aberrations in the CDK4 Pathway AreAssociated with Innate Resistance to PD-1Blockade in Chinese Patients with Non-CutaneousMelanomaJiayi Yu1, Junya Yan1, Qian Guo1, Zhihong Chi1, Bixia Tang1, Bin Zheng2, Jinyu Yu1,Ting Yin1,Zhiyuan Cheng1, Xiaowen Wu1, Huan Yu1, Jie Dai1, Xinan Sheng1, Lu Si1, Chuanliang Cui1,XueBai1, LiliMao1, BinLian1, XuanWang1, XieqiaYan1, SimingLi1, Li Zhou1, KeithT. Flaherty3,Jun Guo1, and Yan Kong1

Abstract

Purpose: PD-1 checkpoint blockade immunotherapyinduces long and durable response in patients withadvanced melanoma. However, only a subset of patientswith melanoma benefit from this approach. The mechanismtriggering the innate resistance of anti–PD-1 therapyremains unclear.

Experimental Design: Whole-exome sequencing (WES)and RNA sequencing (RNA-Seq) analyses were performedin a training cohort (n ¼ 31) using baseline tumor biopsiesof patients with advanced melanoma treated with theanti–PD-1 antibody. Copy-number variations (CNVs) forthe genes CDK4, CCND1, and CDKN2A were assayedusing a TaqMan copy-number assay in a validation cohort(n ¼ 85). The effect of CDK4/6 inhibitors combinedwith anti–PD-1 antibody monotherapy was evaluated inPD-1–humanized mouse (C57BL/6-hPD-1) and human-ized immune system (HIS) patient-derived xenograft (PDX)models.

Results: WES revealed several significant gene copy-number gains in the patients of no clinical benefit cohort,such as 12q14.1 loci, which harbor CDK4. The associationbetween CDK4 gain and innate resistance to anti–PD-1therapy was validated in 85 patients with melanoma (P <0.05). RNA-Seq analysis of CDK4-normal cell lines andCDK4-normal tumors showed altered transcriptionaloutput in TNFa signaling via NF-kB, inflammatory response,and IFNg response gene set. In addition, CDK4/6 inhibitor(palbociclib) treatment increased PD-L1 protein levels andenhanced efficacy (P < 0.05) in the C57BL/6-hPD-1 mela-noma cell and the HIS PDX model.

Conclusions: In summary, we discovered that geneticaberrations in the CDK4 pathway are associated with innateresistance to anti–PD-1 therapy in patients with advancedmelanoma. Moreover, our study provides a strong rationalefor combining CDK4/6 inhibitors with anti–PD-1 antibodyfor the treatment of advanced melanomas.

IntroductionAs an immune checkpoint blockade, anti–PD-1/PD-L1 antibo-

dies have improved outcomes inmany cancer types. This approach,which has been approved for the treatment of metastatic melano-ma, non–small cell lung cancer (NSCLC), head and neck cancer,and urothelial carcinoma, may cause release of endogenous anti-

tumor immunity, eliminate tumor cells, and mediate durabletumor regression (1). Previous trials have revealed that 35%–

60% of patients with melanoma can achieve clinical response,based on RECIST, to anti–PD-1 mAb treatment (2, 3). Unfortu-nately, 40%–65% of patients have none or short-lived responses,and 43% of patients experience drug resistance within 3 years (3).On the basis of a phase II anti–PD-1 clinical trial that our centerreported at the American Society of Clinical Oncology (ASCO)meeting, the overall objective response rate (ORR) of a cohort ofpredominantly acral and mucosal melanoma was 20.7 overall,which is lower than that of the Caucasian population. Within acralandmucosal subtypes, the response rate was clearly lower than thatobserved in cutaneous melanomas (14.3% and 0%, respectively;ref. 4). It is vital to understand the resistance mechanisms ofimmunotherapy and to explore new strategies to overcome treat-ment resistance in various melanoma populations.

To date, most studies of anti–PD-1 therapeutic resistance havemainly concentrated on immune factors, such as PD-L1 expres-sion and tumor-infiltrating CD8þ T cells (5, 6). However, somaticgenetic factors may play equally vital roles, and several studieshave revealed that the mutational burden may also be associatedwith the response of immunotherapy (7, 8). Moreover, tumors

1Key Laboratory of Carcinogenesis and Translational Research (Ministry ofEducation/Beijing), Department of Renal cancer and Melanoma, Peking Uni-versity Cancer Hospital & Institute, Beijing, China. 2Cutaneous Biology ResearchCenter, Massachusetts General Hospital and Harvard Medical School, Charles-town, Massachusetts. 3Massachusetts General Hospital Cancer Center, HarvardMedical School, Boston, Massachusetts.

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

Corresponding Authors: Yan Kong, Peking University Cancer Hospital & Insti-tute, 52FuchengRoad, Beijing, 100142, China. Phone: 8610-8819-6317; Fax: 8610-8812-2437; E-mail: [email protected]; and Jun Guo, [email protected]

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with a high mutational burden, such as melanoma, NSCLC, andbladder cancer, have been correlated with superior clinicalresponses to immune checkpoint blockade compared withtumors with lower mutational rates (9, 10). This may be becausea greater number of genetic variants increase the probability ofstimulating tumor-specific antigens (neoantigens), leading tosuperior antitumor immune response during immunothera-py (11). There are significant differences in the incidence, genespectrum, and clinical manifestations between Caucasian andnon-Caucasian populations (12–14). For example, a whole-genome sequencing (WGS) study revealed that acral andmucosalmelanomas, which are the predominant subtypes amongnon-Caucasians, are dominated by somatic structural variants,whereas the burden of point and structuralmutations is not heavycompared with that of cutaneous melanoma (15). The majorsomatic variants include KIT, TERT, NRAS, EZH2, and CDK4 innon-Caucasians melanomas (16–20). Such distinct moleculardifferences may affect the immune microenvironment andresponsiveness to immunotherapy.

Aberrant cell-cycle progression is a hallmark of cancer cells.Cyclin-dependent kinase 4 (CDK4), or its close homolog CDK6,forms an active complex with Cyclin D that phosphorylates andinhibits the Rb protein, promoting the G1–S-phase transition ofthe cell cycle (21). Commonly dysregulated in most cancers, thisCDK4 pathway (p16Ink4A-Cyclin D-CDK4-Rb) is one of the dri-vers of melanoma (15, 22). Recent studies have shown thatgenetic alterations of CDK4 pathway components are frequentlyfound in acral melanoma, with a higher prevalence than incutaneous melanoma, suggesting that the CDK4 pathway maybe an effective therapeutic target in melanoma in the non-Caucasian population (13, 15, 20). Furthermore, recent studieshave demonstrated a correlation between the CDK4 pathway andtumor immunity. Zhang and colleagues found that cyclinD-CDK4 regulates PD-L1 protein abundance to affect cancerimmune surveillance (23). Deng and colleagues reported thatCDK4/6 inhibition enhances T-cell activation via derepression ofnuclear factor of activated T cell (NFAT) family proteins toaugment antitumor immunity (24). However, the above studiesare still at laboratory research stage. Explorations of clinical dataand preclinical studies are required.

In this study, we analyzed clinical samples in an effort toexplore factors that may influence innate sensitivity or resistanceto anti–PD-1 therapy and to screen druggable targets with thepotential to be combinedwith anti–PD-1 antibodymonotherapyfor treatment of advanced melanoma.

Materials and MethodsPatients and tissue samples

In this study, we obtained 31 tumor biopsies, 116 blood sam-ples, 24 normal control tissues, and 85 formalin-fixed, paraffin-embedded (FFPE) tumor tissues from baseline lesions of 116 pati-ents with advanced melanoma who were treated with an anti–PD-1antibody (JS001, Shanghai Junshi Biosciences, clinical trial ID:NCT02836795 and NCT03013101) at Peking University CancerHospital (Beijing, China) betweenMarch 2016 andDecember 2017.Informed consent for the use of materials in the study (includingarchived materials, as well as the establishment of cell lines andtumor models) was obtained from all patients enrolled in clinicaltrials. Informed written consent was obtained from each subject oreach subject's guardian. Clinical data, including sex, age, tumor side,tumor thickness, metastasis status, and clinical efficacy, were col-lected. Clinical efficacy was evaluated per RECISTv1.1 by the inde-pendent review committee (IRC) every 8 weeks. Diagnosis washistopathologically confirmed for all patients. Theflowchart describ-ing the total population of the study is shown in SupplementaryFig. S1. This study was approved by the medical ethics committeeof Peking University Cancer Hospital and Institute (Beijing, China)and was conducted according to Declaration of HelsinkiPrinciples. The institutional review board number is 2016KT50.

WESGenomic DNA was extracted from 31 matched tumor biopsies

and blood samples using DNeasy Blood & Tissue Kit (Qiagen).Exome capture was performed using SureSelect Human All ExonV6 (Agilent Technologies) according to the manufacturer's instruc-tions. The quantity of libraries was assessed using Qubit 2.0Fluoromete. The library quality and size were measured using a2100 Bioanalyzer high sensitivity DNA assay according tothe manual. For Illumina sequencing, qualified libraries wereapplied for 2 � 150 bp paired-end sequencing using the IlluminaHiSeq X-ten Platform (Illumina). All whole-exome capture,sequencing, and data analyses were performed at Shanghai Bio-technology Corporation. Bioinformatics analysis of WES resultscan be found in the Supplementary Materials and Methods.

RNA-SeqTotal RNA was isolated from 24 matched tumor biopsies and

normal control tissues using RNeasy Mini Kit (Qiagen) followingthe manufacturer's instructions. Strand-specific libraries wereprepared using the TruSeq Stranded Total RNA Sample Prepara-tion Kit (Illumina) according to the manufacturer's instructions.The purified libraries were quantified using a Qubit 2.0 Fluorom-eter (Life Technologies) and validated with an Agilent 2100Bioanalyzer (Agilent Technologies) to confirm the insert size andto calculate the mole concentration. Clustering was generatedwith cBot using the library diluted to 10 pmol/L and thensequenced using the Illumina HiSeq X-ten System (Illumina).Library construction and sequencing were performed at ShanghaiBiotechnology Corporation. Data analysis for gene expressionbased on RNA-Seq is described in the Supplementary Materialsand Methods.

Translational Relevance

Anti–PD-1 antibodies have received considerable attentionas effective therapies for advanced melanoma. However, themajority of the patients failed to response to this approach,due to genetic aberrations in the key signaling pathways. Here,based on the whole-exome sequencing and RNA-sequencingprofiling, we identified obvious CDK4, CCND1 gain, andCDKN2A loss in the patients with melanoma with resistanceto anti–PD-1 immunotherapy. Using the B16 cells–implantedC57BL/6-Pdcd1tm1(hPDCD1)/Bcgen (C57BL/6-hPD-1) andanti–PD-1–resistant melanoma-derived humanized immunesystem patient-derived xenograft mousemodel, we found thatCDK4/6 inhibitors activated the immune system. Importantly,combination of CDK4/6 inhibitors and anti–PD-1 antibodiessignificantly suppressed the tumor growth in both mousemodels.We conclude thatCDK4/6 canbe targeted in advancedmelanoma to improve the efficacy of immunotherapy.

Yu et al.

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QuantiGenePlex RNA assay (panel)All experiments were performed at Shanghai Biotechnology

Corporation following the usermanual ofQuantiGene Plex Assay(Panomics). Seedetails in SupplementaryMaterials andMethods.

DNA preparation and TaqMan copy-number assaysThe copy numbers of CDK4, CCND1, and CDKN2A were calcu-

lated using TaqMan Copy Number Assays (Applied Biosystems ofThermo Fisher Scientific); the Rnasep gene TaqMan probe was usedas a control. See details in Supplementary Materials and Methods.

FISHCDK4 gain, CCND1 gain, and CDKN2A loss were identified

using array-based comparative genomic hybridization. InterphaseFISH was performed using CDK4/CEN12q FISH Probe (FG0033,Abnova) with a CCND1/CEP11 amplification probe (Vysis) and aCDKN2A/CEP9 loss probe (Vysis). The imprint slides were cov-ered with 10mL of dual-probe hybridizationmixture containing apair of chromosome probes and subtelomere probes. The slidesand probes were denatured at 75�C for 5 minutes and hybridizedat 37�C for 16 hours. Post-hybridization washes were performedaccording to the manufacturer's protocol.

IHCIHC analyses were performed using antibodies against CD4

(ab183685, Abcam), CD8 (ab217344, Abcam), B220(14-0452-82, eBioscience), FOXP3 (14-5773-80, eBioscience),and PD-L1 (SP142 and M4422, Spring), as described previ-ously (25). Staining intensity and percentage were indepen-dently scored by three pathologists. We used ImageJ softwareto quantify the IHC results.

ImmunofluorescenceImmunofluorescence (IF) analyses were performed using anti-

bodies against CD4 (ab196372, Abcam), CD8 (ab4055, Abcam),CD19 (ab134114, Abcam), and FOXP3 (ab20034, Abcam).

Immunology multiplex cytokine/chemokine profilingA panel of 38 cytokines, chemokines, and growth factors in

plasma was analyzed using MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel, Premixed 38 Plex, Immunol-ogy Multiplex Assay (HCYTMAG-60K-PX38, Merck). Measure-mentswereperformedusing aBio-PlexMAGPIXMultiplexReader(Bio-Rad Laboratories).

NanoString-based gene expression profilingTumor RNA samples were subjected to nCounter PanCancer

Immune Profiling Panel (NanoString Technologies) analysisconsisting of 770 human genes. See details in SupplementaryMaterials and Methods.

Cell lines and primary cell cultureA2058 (catalog No. CRL-11147), SK-MEL-5 (catalog No. HTB-

70), and WM-266-4 (catalog No. CRL-1676) cell lines wereobtained from ATCC. B16-OVA cell lines were a gift fromShanghai Junshi Biosciences. AMC-3 (acral primary melanomacell) cell lines were derived from a patient-derived xenograft (PDX)model. See details in Supplementary Materials and Methods.

Western blottingAll cells were harvested and lysed using the PhosphoSafe

Extraction Reagent (Millipore). Supernatants were collected bycentrifugation. Western blot analysis of protein complexes was

performed using antibodies against the following: PD-L1 (catalogNo. 13684, Cell Signaling Technology) andmouse PD-L1/B7-H1(MAB90781-100, R&D Systems).

AnimalsHumanized PD-1–knockin C57Bl/6 mice (C57BL/6-Pdcd1tm1

(hPDCD1)/Bcgen and C57BL/6-hPD-1) were purchased from Bio-cytogen Gene Biotechnology Co., Ltd. The C57BL/6-hPD-1 micewere developed by knocking-in human PDCD1 protein codingregion inserted into the ATG site of murine Pdcd1 gene, theexpression of endogenous murine Pdcd1 was replaced by theexpression of human full-length PDCD1 protein, and PD-1expression was detected by flow cytometry (SupplementaryFig. S2). NOD/SCID/IL2 receptor g chain null mice (NOD/SCID/IL2gnull, NSG) were obtained from the Vital River Labora-tory Animal Technology Co., Ltd. and bred and raised underspecific pathogen-free conditions. All animal care and experimen-tal procedures were carried out in accordance with the AnimalCare Ethics approved by the Medical Ethics Committee of theBeijing Cancer Hospital & Institute.

C57BL/6-hPD-1 mouse model and treatmentB16-OVA cells (1 � 105) were subcutaneously injected into

6-week-old C57BL/6-hPD-1 female mice. Six days later, the micewere daily treated with palbociclib (150 mg/kg body weight, bygastric gavage), the anti–PD-1 antibody (pembrolizumab 200 mg,every 3 days), or vehicle (sodium lactate buffer solution) for12 days. The CDK4/6 inhibitor, palbociclib (PD0332991, catalogNo. S1579) was purchased from Selleck Chemicals. Tumor sizeandmouseweightweremeasured every 3 days and tumor volumewas calculated using the following formula: volume ¼ length �width2/2. Samples of tumor and spleen were processed for FACSanalysis or IHC.

Humanized immune system PDX model and treatmentNSG mice were sublethally irradiated with 200 cGy. Human

peripheral blood monocular cells (PBMC) were isolated usingFicollHypaque centrifugation fromahealthy donor and transplant-ed (1 � 107) into the irradiated NSG mice via tail vein injection.

One week after PBMC transplantation, the fragments ofpatient-derived melanoma tissues were subcutaneously inoculat-ed into the PBMC-humanized NSG mice. When the tumor vol-ume reached approximately 70 mm3 and hCD45þ cells consti-tuted about 5% of the peripheral blood, the mice were treatedwith palbociclib (50 mg/kg, orally, daily dosing), the anti–PD-1antibody (200 mg, i.p., every 3 days), or vehicle (sodium lactatebuffer solution) for 21 days. Tumor size and mouse body weightwere measured twice a week, and tumor volume was calculatedusing the following formula: volume ¼ length � width2/2.Samples of tumor and spleen were processed for FACS analysisor IF. TheHumanized immune system (HIS) PDX experimentwasperformed at Yicon Biomedical Technology Inc. The similar HISPDX mouse model was described previously (26, 27).

FACSFITC-, PE-, PE-Cyanine7-, APC-, APC-eFluor-, PerCP-, or PerCP-

Cyanine5.5–conjugated anti-CD3, -CD4, -CD8, -CD19, -CD45, and-CD49bmAbswereused tostain lymphocytes (all fromeBioscience).

Statistical analysisThe statistical evaluation was conducted with IBM SPSS statis-

tical software (v20.0). t Test was used to analyze mean values fornormally distributed continuous variables, whereas the Mann–

CDK4 Aberrations in Anti–PD-1 Immunotherapy Resistance

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Whitney U test was applied to compare mean values for abnor-mally distributed continuous variables. Correlations between theaberration status and clinical parameters were evaluated by x2 testor Fisher exact test. For all statistical tests, P<0.05 (two-tailed test)was considered statistically significant.

ResultsPatient cohort, checkpoint blockade treatment, and tumor biopsies

To explore differential genetic aberrations associated with theresponse of advanced melanoma to anti–PD-1 immune

Figure 1.

Diversity of mutations and copy-number changes between patients in the CB and NCB cohorts.A,Mutation burden in the CB and NCB groups. B,Mutations insignificantly mutated genes and selected knownmelanoma driver genes of CB and NCB groups. C, Copy-number changes in selected melanoma-associatedgenes of CB and NCB groups.D and E, Percentage of samples with protein-affecting aberrations in candidate driver genes, as grouped by pathway: copy-number amplification (copy number > 3, red), copy-number deletion (copy number < 2, blue), structural variants (yellow). (Continued on the following page.)

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checkpoint blockade, we assembled a test cohort of 116 patientswith advanced melanoma who were treated with this therapy.Among them, 31 patients were classified as a training cohort and85 patients as a validation cohort. The clinical pathologic para-meters of the patients are shown in Supplementary Table S1.Clinical efficacy was evaluated per RECIST 1.1 by the IRC every8 weeks. Clinical benefit (CB) cohort included patients with acomplete response (CR) or partial response (PR) according toRECIST 1.1 (i.e., tumor shrinkage > 30% from baseline) or stabledisease (SD) if they had any objective reduction in tumor burdenlasting at least 6 months. No clinical benefit (NCB) were definedas those experiencing progressive disease according to RECIST 1.1or SD lasting �6 months and were discontinued from immuno-therapy within 3 months (28, 29). As of November 30, 2018,among 116 evaluable patients with at least three IRC evaluationsfor clinical efficacy, one achieved CR, 21 PR, and 42 SD.

CDK4 pathway aberrations associate significantly with NCB toPD-1 blockade

To investigate genetic variation associated with innate resistanceto anti–PD-1 therapy, genomic DNA extracted from tumors andmatched peripheral blood cells of 31-sample cohort were subjectedto WES, including 15 patients of CB cohort and 16 NCB. Themeansequencing depth in the training cohort was 338-fold for tumorand 135-fold for matched blood. At least 93.27% of exome-widetarget bases were covered to a depth of more than 10 �. The WESdemonstrated that the overall nonsynonymous mutation burdenwas moderate in the training cohort with a median number of 90

(ranging from 6 to 3,068). The tumors of patients in the CB andNCB groups showed similar mutation burdens (Fig. 1A). Theoverall mutations and copy-number changes in selected knownmelanoma driver genes are shown in Fig. 1B and C. Frequencies ofalteration in various key genes and signaling pathways between CBand NCB cohort are shown in Fig. 1D–F. Alterations in the cell-cycle pathway occurred more frequently in NCB cohort than CBcohort. The copy-number alterations affecting antigen-presentationmachinery and HLA class I alleles were frequent in tumors of bothgroups, whereas mutations were relatively uncommon (Fig. 1G).

According to GISTIC2.0 output of single cohort of samples,WES revealed several significant gene copy-number gains in theNCB group, such as 5p15.33, 7p22.3, 7q36.1, and 12q14.1 loci,which harbor CDK4, TERT, CARD11, and EZH2. In addition,several significant gene copy-number losses were observed in theNCB group, such as 9q21.3 and 10q23.31 loci, which harborCDKN2A and PTEN (Fig. 1H and I).

Confirmation of CDK4 pathway aberration associated withinnate resistance to anti–PD-1 therapy

To validate the findings from WES, we investigated CNVs inCDK4, CCND1, andCDKN2A in an independent cohort (n¼ 85).On the basis of data of 85 patients, we found a negative correlationbetween CDK4 copy-number gain and anti–PD-1 response, more-over, a positive correlation between CDK4 copy-normal and anti–PD-1 response (P¼0.013; Fig. 2A; Table 1). AlthoughpatientswithCCND1 copy-number gain and CDKN2A copy-number loss tendto be nonresponsive to anti–PD-1 therapy, the difference was not

Figure 1.

(Continued. ) F, Frequency of aberrations in pathways as a percentage of CB and NCB groups. SWI/SNF, SWItch/sucrose nonfermentable nucleosomeremodeling complex. G, Alterations in HLA alleles and antigen presentation machinery in CB and NCB groups. H and I,According to GISTIC2.0 output, there weresignificant copy-number changes in the CB and NCB groups.






statistically significant (P ¼ 0.643, 0.318, respectively; Fig. 2A;Table 1). Among 32 patients with acral subtype melanoma,CCND1 copy-number gain was associated with a lack of responseto anti–PD-1 therapy (P ¼ 0.043); among 13 patients withunknown primary subtype melanoma, CDKN2A copy-numberloss was associated with nonresponse to anti–PD-1 therapy(P ¼ 0.021; Table 1). We also performed FISH to confirm theobserved CDK4 gain, CCND1 gain, and CDKN2A loss (Fig. 2B).

Furthermore, we performed QuantiGenePlex RNA analysis toinvestigate differential RNA expression levels of CDK4, CCND1,and CDKN2A between the CB andNCB groups. On the basis of 34tumor biopsy specimens, the RNA expression levels of CDK4,CCND1, and CDKN2A in the NCB group were higher than thosein the CB group (Fig. 2C; 0.28 vs. 0.09, P¼ 0.03; 1.27 vs. 0.32, P¼0.05; 0.033 vs. 0.018, P ¼ 0.2, respectively). Similar results werefound in 42 FFPE tumor tissue specimens (Fig. 2D). The tumor

Figure 2.

Correlation between CDK4 pathway aberrations and CB of anti–PD-1 therapy in melanoma. A, Genetic aberrations in the CDK4 pathway and response toanti–PD-1 therapies based on sample. CB, n¼ 33; NCB, n¼ 52. B, FISH analysis of CNVs in CDK4, CCND1, and CDKN2A. C, RNA expression levels of CDK4, CCND1,and CDKN2A in tumor biopsy specimens of NCB and CB groups according to data from the QuantiGenePlex RNA assay. CB, n¼ 22; NCB, n¼ 12. D, RNAexpression levels of CDK4, CCND1, and CDKN2A in FFPE tumor tissue specimens from NCB and CB groups according to the QuantiGenePlex RNA assay(CB, n¼ 16; NCB, n¼ 26; � , P < 0.05).

Table 1. Correlation of CDK4 pathway aberrations with a CB of anti–PD-1 therapy in melanoma

CDK4 Aberration CCND1 Aberration CDKN2A AberrationGain Normal Pa Gain Normal Loss Pa Loss Normal Pa

CB (%)CB 16 (18.8) 17 (20) 0.013 14 (16.5) 8 (9.4) 7 (8.2) 0.643 18 (21.2) 15 (17.6) 0.318NCB 39 (45.9) 13 (15.3) 30 (35.3) 11 (12.9) 15 (17.6) 34 (40) 18 (21.2)

Acral subtype (%)CB 6 (18.8) 3 (9.4) 0.398 3 (9.4) 1 (3.1) 5 (5.9) 0.043 6 (18.8) 3 (9.4) 0.638NCB 18 (21.2) 5 (5.9) 17 (20) 4 (12.5) 2 (6.3) 15 (17.6) 8 (25)

Mucosal subtype (%)CB 1 (7.6) 1 (7.6) 0.154 1 (7.6) 0 (0) 1 (7.6) 0.538 0 (0) 2 (15.3) 0.192NCB 11 (84.6) 0 (0) 3 (23) 3 (23) 5 (38.4) 7 (53.8) 4 (30.8)

CSD subtype (%)CB 3 (17.6) 4 (23.5) 0.646 4 (23.5) 2 (11.8) 1 (5.9) 0.581 5 (29.4) 2 (11.8) 0.354NCB 4 (23.5) 6 (35.3) 5 (29.4) 4 (23.5) 1 (5.9) 5 (29.4) 5 (29.4)

Non-CSD subtype (%)CB 3 (30) 3 (30) 0.452 2 (20) 2 (20) 2 (20) 0.262 5 (50) 1 (10) 0.667NCB 3 (30) 1 (10) 3 (30) 0 (0) 1 (10) 3 (30) 1 (10)

Unknown primary subtype (%)CB 3 (23.1) 6 (46.2) 0.217 4 (30.7) 3 (23.1) 2 (15.4) 0.657 2 (15.4) 7 (53.8) 0.021NCB 3 (23.1) 1 (7.7) 2 (15.4) 0 (0) 2 (15.4) 4 (30.7) 0 (0)

Abbreviations: CSD, melanomas on skin with chronic sun-induced damage; non-CSD, melanoma on skin without sun-induced damage.aP values of the x2 test or Fisher exact test were used for evaluating the CB.

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biopsies in Fig. 2C were mainly metastatic lesions, while most ofthe FFPE tumor tissue specimens in Fig. 2D were primary mela-nomas (Supplementary Table S2).

Correlation of CDK4 pathway aberrations with PD-L1 proteinexpression in patients with melanoma

Because PD-L1 expression has been demonstrated to partial-ly predict the efficacy of PD-1 mAb, we investigate the asso-ciation between CDK4 pathway aberrations with PD-L1 proteinexpression. We performed IHC to detect levels of PD-L1 proteinexpression, in which PD-L1 � 5% was defined as positivestaining (Fig. 3A). The overall PD-L1–positive rate was23.3%. Patients with greater levels of PD-L1 expression by IHChad higher response rates (P ¼ 0.024), with a response rate forPD-L1–positive versus -negative patients was 64.7% versus33% (Supplementary Table S3). Moreover, patients with CDK4gain showed a lower PD-L1–positive rate than those with aCDK4-normal status (20.8% vs. 28%; P ¼ 0.492), although thedifference was not significant (Supplementary Table S3).

CDK4 gain status in melanoma influences immune geneexpression

To further explore the consequences of CDK4 gene copy-number gain, the tumor biopsies of 24 patients treated withPD-1 mAb were subjected to RNA-Seq analysis. After performingdifferentially expressed analysis between CDK4 gain tumors andCDK4-normal tumors, and gene set enrichment analysis (GSEA)for differentially expressed genes, we found that three gene sets,including TNFa signaling via NF-B, inflammatory response, andIFNg response, were enriched in CDK4-normal tumors (P < 0.05;q < 0.25; Fig. 3B–D). As for cell lines, compared with the cell linewith CDK4 gain (SK-MEL-5), the CDK4-normal cell line (A2058)highly expressed genes enriched in TNFa signaling via NF-kB andIL6/JAK/STAT3 signaling pathway (P < 0.05 but q > 0.25; Fig. 3E).

Furthermore, we performedNanoString-based gene expressionprofiling and Immunology Multiplex cytokine/chemokine pro-filing. Among 55 patients, NanoString-based gene expressionprofiling revealed high expression levels of IL17A, TNFRSF10C,CCL28, MICB, LICRB3, CREB1, NOTCH1, and IL6 among thetumor specimen of patientswithCDK4 gain (Fig. 3F). ImmunologyMultiplex cytokine/chemokine profiling was performed to verifythese results, and patients' tumorswithCDK4 gain hadhigher levelsof IL17A and IL6 in plasma than did patients with a CDK4-normalstatus (59.7 vs. 33.4, P¼ 0.016; 34.9 vs. 27.8, P¼ 0.038), as well aslower levels of eotaxin (260.0 vs. 339.2; P ¼ 0.044; Fig. 3G).

Effects of combined CDK4/6 inhibitors and anti–PD-1 on thegrowth of melanoma in the C57BL/6-hPD-1 model

To further explore the therapeutic effects of CDK4 inhibitors incombination with PD-1, we performed in vivo experiments.C57BL/6-hPD-1 mice were subcutaneously injected with B16-OVA cells. Six days later, themice were treated with either vehicle,palbociclib, the anti–PD-1 antibody, or a combination of palbo-ciclib and the antibody for 12 days. According to tumor measure-ments over the treatment period, there were significant reductionsin growthwith the combined therapy of palbociclib and the anti–PD-1 antibody compared with monotherapy or the control (P <0.05; Fig. 4A).

Histology of the B16-OVA melanoma tumors revealedincreased presence of B cells, CD4þ T cells, CD8þ T cells, andFoxP3þ cells in the palbociclib-alone group aswell as in the grouptreated with the combination therapy, compared with PD-1

antibody–alone group or the control group (Fig. 4B). FACSanalysis was performed to detect immune cells infiltrating intothe tumors after therapy, with an increase in the leukocyte contentin the palbociclib alone or combined treatment group. In addi-tion, there was significant increase in B cells, T cells, cytotoxic T(Tc) cells, Th cells, NK (natural killer) cells, and NKT cells in thepalbociclib alone–treated tumors and combined treatmenttumors (Fig. 4C). However, there was no difference in the pro-portion of immune cells among the four treatment groups.

Effects of combined CDK4/6 inhibitors and anti–PD-1treatment on the growth of melanoma in the HIS PDX model

The results for B16-OVA mouse tumors prompted us to assesshowpalbociclib affects tumor growth andantitumor immunity ina patient-derived melanoma model. Thus, we generated the HISPDX model, in which tumors from patients with advancedmelanomawere engraftedwith the human PBMCs into irradiatedNSG mice. This patient is in the NCB cohort, with CDK4 gain.Tumor growth curves for each treatment group demonstrated theimproved efficacy of combining PD-1 blockade with the CDK4/6inhibitor in this model, compared with the monotherapy or thecontrol group (P < 0.05; Fig. 5A).

The effects of combined treatment on B cells, Tc cells, Th cells,and regulatory T cells (Treg) in HIS PDX model tumors weregreater than those of monotherapy (Fig. 5B). We performedimmuno-phenotyping analysis by FACS in these tumors aftertherapy. Tumor-bearing mice treated with palbociclib and com-bined therapy exhibited an increase in immune cells, T cells, Tccells, and Th cells of tumor (Fig. 5C).

CDK4/6 inhibitor treatment positively regulates PD-L1 proteinexpression and influences immune gene expression

The above in vivo experiments showed that CDK4 inhibitorscan activate antitumor immunity and synergistically improve thetherapeutic effect of PD-1 mAb. We conducted the following ex-periments to explore related mechanisms. SK-MEL-5 cell line(withCCND1 gain plusCDKN2A loss) and the primary acralmela-noma line AMC-3 cell line (with CDK4 gain plus CDKN2A loss),which the two cell lines have been proven to be sensitive to palbo-ciclib (20), were treatedwithpalbociclib for 24hours.Western blotassays showed that palbociclib treatment increased PD-L1 proteinlevels in the SK-MEL-5 cell line, AMC-3 cell line, C57BL/6-hPD-1,and HIS PDX murine models (Supplementary Fig. S3A).

RNA-Seq analysis revealed that palbociclib treatment enhancedimmune gene expression in the samples of SK-MEL-5 cell line,AMC-3 cell line, and C57BL/6-hPD-1 murine model, includinggenes related to the IL6/JAK/STAT3 signaling pathway (P < 0.05but q > 0.25; Supplementary Fig. S3B).

DiscussionAnti–PD-1 therapy has revolutionized treatment for advanced

melanoma andmany other cancer types. However, only a minor-ity of patients benefit frommonotherapy, especially small, amongthe Asian melanoma population (4, 30, 31). According to datafrom a large-scale prospective anti–PD-1 clinical study publishedby our center at ASCO, the ORR of chronic sun-induced damage(CSD), non-CSD, mucosal, and acral melanomas was 35.3, 33.3,0, and 14.3, respectively (4). Overall, the anti–PD-1 antibodyappeared to be similarly efficacious for CSD and non-CSD in aChinese population comparedwith aCaucasian population, withgreater effects than themucosal and acral subtypes (4). The lack of






Figure 3.

Correlation of CDK4 pathway aberrations with immune gene expression. A, Representative images of melanoma tumor cells with PD-L1 expression.B, Differential expression of genes between the cohort of CDK4-gain group and CDK4-normal group. C, Differential expression of genes likely to be associatedwith PD-1 mAb treatment was reported between the cohort of CDK4-gain group and CDK4-normal group.D, GSEA was performed on RNA-Seq from tumorbiopsy samples from patients with CDK4-gain and CDK4-normal. Enrichment plots show differential expression of TNFa signaling via NF-kB, inflammatoryresponse, and IFNg response gene set in the CDK4-normal tumors. E, GSEA was performed on RNA-Seq from tumor cell line with CDK4-gain and CDK4-normal.Enrichment plots show differential expression of TNFa signaling via NF-kB, and IL6/JAK/STAT3 signaling pathway gene set in CDK4-normal cell line (SK-MEL-5).F, Heatmap representation of differentially expressed genes (P < 0.05) between CDK4-gain and CDK4-normal tumors based NanoString analysis. G,Differentialexpression of cytokines, chemokines, and growth factors in plasma between CDK4-gain and CDK4-normal tumors based on Immunology Multiplex cytokine/chemokine profiling. CDK4 normal, n¼ 38; CDK4 gain, n¼ 47. The two independent sample t test was used to determine significance.

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predictors of a response, mechanisms of therapeutic resistance,and effective combination therapies remains a challenge fortreatment of patients with mucosal and acral subtypes. Here, wepresent the role of specific genetic aberrations in determiningresponses of advanced melanoma to anti–PD-1 therapy. Wedemonstrated that genetic aberrations in the CDK4 pathway wereassociated with the innate resistance to anti–PD-1 therapy inpatients with advanced melanoma. Gene expression analysis

showed that CDK4-normal tumors and cells exhibited enrichedtranscriptional output in TNFa signaling via NF-kB, inflamma-tory response, and IFNg response gene set. Moreover, our in vivostudy provided a rationale of combining CDK4/6 inhibitors withanti–PD-1 antibody for the treatment of advanced melanomas.

Our results showed a similar tumormutation rate in theCB andNCB groups. Several studies have noted that the overall mutationload correlates with clinical responses to anti–PD-1 therapy in

Figure 4.

Effects of combined CDK4/6 inhibitors andanti–PD-1 treatment on the growth of melanoma inthe C57BL/6-hPD-1 model. A, Sensitivity of theC57BL/6-hPD-1 PDXmodel to combined PD-1blockade and CDK4/6 inhibitors in vivo. n¼ 5 pergroup. B, Histology of melanomawith enhancedpresence of B cells, CD4þ T cells, CD8þ T cells, andFoxP3þ cells in tumors of C57BL/6-hPD-1 PDXmodels treated with palbociclib or combinedtherapy. H&E, hematoxylin and eosin. C, The IHCresults of B cells, CD4þ T cells, CD8þ T cells, andFoxP3þ cells were presented as mean� SE.D, FACS analysis of immune cells frommelanomashowed increased leukocytes in anti–PD-1antibody, palbociclib, and combined treatmentgroups, with significant increases in B cells, T cells,Tc cells, Th cells, NK cells, and NKT cells inpalbociclib alone–treated and combinedtreatment groups. Error bars indicate SD,and P values were determined byStudent–Newman–Keuls (� , P < 0.05;�� , P < 0.01; �� , P < 0.001; ns, not significant).






NSCLC, colon cancer, andmelanoma (32–34), with a statisticallysignificant difference in the median, and the range of mutationburden in tumors among CB significantly overlaps with the rangein NCB (35). Nonetheless, there are still patients with a highmutation load with NCB (33). Moreover, previous researchindicates high mutational load does not associate with tumorresponse to anti–PD-1 therapy but correlates with improvedpatient survival of metastatic melanoma. Therefore, additional

genomic and immunologic features are likely to contribute to theresponse patterns of anti–PD-1 therapy.

We performed WES of 31 pretreated advanced melanomatumors and found significant CDK4 gain and CDKN2A loss inthe NCB group, and the association between CDK4 gain andinnate resistance to anti–PD-1 therapy was validated in anothermelanoma cohort (n¼ 85), moreover, we also found that CBwasassociated with CDK4 normal in the validation cohort. We found

Figure 5.

Effects of combined CDK4/6inhibitors and anti–PD-1 therapyon the growth of melanoma in theHIS PDXmodel. A, Sensitivity ofthe HIS PDXmodel to combinedPD-1 blockade and CDK4/6inhibitors in vivo. n¼ 5 per group.B, Histology of melanomawithenhanced presence of B cells,CD4þ T cells, CD8þ T cells, andFoxP3þ in tumors of HIS PDXmodels treated with palbociclibor combined therapy. H&E,hematoxylin and eosin. C, The IFresults of B cells, CD4þ T cells,CD8þ T cells, and FoxP3þ cellswere presented as mean� SE.D, FACS analysis of immune cellsfrommelanoma showedincreased leukocytes in anti–PD-1antibody, palbociclib, andcombined treatment groups, withsignificant increases in immunecells, T cells, Tc cells, and Th cellsin palbociclib alone–treatedand combined treatmentgroups. Error bars indicate SD,and P values were determinedby Student–Newman–Keuls(� , P < 0.05; ��, P < 0.01;�� , P < 0.001; ns, not significant).

Yu et al.





CCND1 copy-number gain to be associated with a lack ofresponse to anti–PD-1 therapy among 32 patients with acralsubtype melanoma, and among 13 patients with unknown pri-mary subtype melanoma, CDKN2A copy-number loss was asso-ciated with anti–PD-1 therapy nonresponse. Thus, the geneticaberrations in the CDK4 pathway are associated with innateresistance to PD-1 blockade. According to SNP array analyses of46 melanoma cell lines and data from The Cancer Genome Atlas,Leonardelli and colleagues found that because of associated JAK2allele losses, melanoma harboring allelicCDKN2A deletions maybe more prone to develop resistance to immunotherapy. Asdrivers of somatic copy-number alterations,CDK4 amplificationshave been verified in a series of cancers, such as melanoma (20),NSCLC (36), and urothelial carcinoma (37). In addition, PTENloss is associated with the resistance to immunotherapy (38, 39).We found PTEN loss significantly occurred in the NCB group, butthe correlation between PTEN loss and resistance to PD-1 block-ademay require a large sample of cohorts for validation. Recently,several selective inhibitors of CDK4/6, such as palbociclib, ribo-ciclib, and abemaciclib, have been approved by the FDA for thetreatment of metastatic breast cancer and are in clinical trials forseveral other indications (40). Indeed, a series of preclinicalexperiments indicate the feasibility of using CDK4 as an antitu-mor target of melanoma (20, 41), and a recent clinical case reportshowed that two patients withmetastatic melanoma with geneticaberrations in the CDK4pathway achieved tumor control for over6 months with palbociclib treatment (42).

To further explore the relevant mechanisms triggered by CDK4gain, RNA-Seq was performed to investigate alterations inducedby amplification of CDK4 pathway components, in which CDK4-normal cell lines and CDK4-normal tumors displayed alteredtranscriptional output in immune signaling pathways, such asTNFa signaling via NF-kB pathways, inflammatory response, andIFNg response gene set, differential expression of these genes mayimpact CDK4-normal patients' response to anti–PD-1 therapy.Luoto and colleagues reported glioblastoma cases with focalamplification of CDK4 that presented negative adaptive immuneresponses that were associated with a lower macrophage andCD4þ T-cell component (43). In addition, recent studies haverevealed essential roles of the noncanonical NF-kB pathway inregulating different aspects of immune function (44–46). Forexample, several members of the TNF receptor (TNFR) superfam-ily regulate the generation of immunosuppressive Tregs by con-trolling the development of medullary thymic epithelial cells(mTEC), which are known to mediate activation of both thecanonical and noncanonical NF-kB pathways (47). Moreover,recent studies have shown that IFNg is a key driver of PD-L1expression, and the deficiency of the IFNg signaling pathway hasbeen linked with anti–PD-1 therapy resistance (48, 49). Nano-String-based gene expression profiling of patients with CDK4 gain exhibited altered output in IL17A, TNFRSF10C, CCL28,MICB, LICRB3, CREB1, NOTCH1, and IL6. These molecules canprovide clues for combination therapy. Furthermore, Immunol-ogy Multiplex cytokine/chemokine profiling confirmed higherlevels of IL17A and IL6, as well as lower levels of Eotaxin inpatients with CDK4 gain. NF-kB pathways modulate the effectorfunction of differentiated T cells via Th17 cells, which secreteIL17A (50). Several studies have also demonstrated that NF-kBsignaling plays a role in regulating the development of NKT cells,as indirectly mediated via mTEC regulation (51). Emerging evi-dences also show that CREB is able to regulate immune responses

via inhibition of NF-kB activity. It regulates macrophage, T, and Blymphocytes (such as Th17) survival and induces transcription ofimmune-related genes (52, 53). Furthermore, IL17A and IL6mayaffect expression of PD-L1. Ma and colleagues showed thattargeting IL17A inhibits PD-L1 expression in tumor cells,decreases the percentage of Tregs in tumor-infiltrating lympho-cytes, promotes IFNg secretion by CD4þ and CD8þ T cells, andexhibits a synergistic antitumor effect with the anti–PD-1 anti-body in estrogen receptor (ER)-negative breast cancer (54). Kimand colleagues also reported that PD-1 is overexpressed in IL17A-producing T cells in patients with psoriasis (54). Tsukamoto andcolleagues found that IL6 blockade upregulated expression of PD-L1 on melanoma cells and that IL6 blockade promoted infiltra-tion of IFNg-producing CD4þ T cells into tumor tissues, exerting asynergistic antitumor effect with the anti–PD-1 antibody (55). Aprevious study also demonstrated that activated EGFR regulatesPD-L1 expression via the IL6/JAK/STAT3 pathway inNSCLC (56).

Several studies have demonstrated that CDK4/6 inhibitors alterthe tumor immune microenvironment, which may enhance theeffects of immuno-oncology agents (57, 58). In fact, previousstudies have shown that CDK4/6 inhibitors enhance immune cellinfiltration and that secretion of chemokine (C-C motif) ligand 5(CCL5) is upregulated in palbociclib-treated melanoma cells,possibly promoting T-cell infiltration (59). CDK4/6 inhibitorsalso increase tumor infiltration and activation of effector T cellsvia derepression of NFAT family proteins and their targetgenes (24), inactivating immunosuppressive cells such as mye-loid cells and Tregs (60, 61), and enhancing tumor antigenpresentation. Moreover, another study demonstrated that cyclinD-CDK4 promoted proteasome-mediated degradation of PD-L1proteins via Cul3SPOP in melanoma (B16) and colon carcinoma(MC38) murine model, which is a critical factor for response toanti–PD-1 therapy (23). The research also found that CDK4/6inhibition could augment the response to PD-1 blockade in coloncarcinoma (MC38 andCT26)murinemodel (23). On the basis ofpromising preclinical data, three clinical trials are currently under-way for FDA-approved CDK4/6 inhibitors in combination withanti–PD-1/PD-L1 antibodies and aromatase inhibitors in thetreatment of ERþ breast cancer (NCT03147287, NCT02778685,and NCT03294694). In another clinical trial, the combination ofCDK4/6 inhibitors with anti–PD-1/PD-L1 antibodies is currentlybeing investigated for advanced solid tumors (NCT02791334),including melanoma.

To further explore whether inhibition of the CDK4 pathway canactivate antitumor immunity, we performed a series of experimentsin vitro and in vivo. Our data confirm cooperative antitumor effectsof CDK4/6 inhibitor (palbociclib) and the anti–PD-1 antibody inmousemelanomamodels. Furthermore, we performed a simulatedcombined dosing regimen of patients resistant to anti–PD-1 ther-apy and achieved effectiveness. We also demonstrated that palbo-ciclib treatment increased PD-L1 protein levels and the quantity ofB cells, T cells, and NK cells in a melanoma cell line and mousemodel. Furthermore, RNA-Seq analysis revealed that palbociclibtreatment enhanced immune gene expression in a melanoma cellline and mouse model, including genes related to the T-cellreceptor signaling pathway, leukocyte transendothelial migration,and NK-cell–mediated cytotoxicity. On the basis of these findings,CDK4/6 inhibitors may synergize with anti–PD-1 immune check-point blockade in the treatment of advanced melanoma.

There are some limitations in our study as well as potentialperspectives. Because of limited follow-up time, we did not






have complete progression-free survival (PFS) and overallsurvival (OS) data, and we will continuously monitor theefficacy readouts of the patients (PFS and OS) for futureanalyses. In addition, the sample size was relatively small. Asthe PD-1 mAb was approved by the China Food and DrugAdministration in 2018, we will include more patients for dataanalysis in future studies. Furthermore, we did not investigatewhether various genetic aberrations in the CDK4 pathway incancer cells may influence the response to anti–PD-1 treatmentin melanoma mouse models. In the future, we will generateB16 stable cell lines ectopically expressing CDK4 and injectthem into C57BL/6-hPD-1 mice and establish additional HISPDX models with various genetic aberrations in the CDK4pathway to assess their sensitivity to anti–PD-1 treatment.

Our study demonstrated thatCDK4pathway genetic aberrationscan serve as genomic biomarkers for predicting the response toanti–PD-1 therapy in advanced melanoma. We also revealed thatCDK4/6 inhibition enhances antitumor immunity and improvessusceptibility to anti–PD-1 therapy in melanoma, providing animportant rationale for developing precision combination therapyand carrying out clinical trials for advanced melanoma.

Disclosure of Potential Conflicts of InterestK.T. Flaherty reports receiving commercial research grants from Novartis

and Sanofi, holds ownership interest (including patents) in Clovis Oncol-ogy, Strata Oncology, Vivid Biosciences, Checkmate Pharmaceuticals, X4Pharmaceuticals, PIC Therapeutics, Fount Therapeutics, Shattuck Labs, Apri-city, Oncoceutics, Fog Pharma, Tvardi, and xCures, and is a consultant/advisory board member for Clovis Oncology, Strata Oncology, Vivid Bios-ciences, Checkmate Pharmaceuticals, X4 Pharmaceuticals, PIC Therapeutics,Sanofi, Amgen, Asana Biosciences, Adaptimmune, Fount Therapeutics,Aeglea, Array Biopharma, Shattuck Labs, Tolero, Apricity, Oncoceutics, FogPharma, Neon, Tvardi, xCures, Monopteros, Novartis, Genentech, Bristol-Myers Squibb, Merck, Takeda, Verastem, Boston Biomedical, Pierre Fabre,Cell Medica, and Debiopharm. No potential conflicts of interest weredisclosed by the other authors.

The Editor-in-Chief of Clinical Cancer Research is an author on this article. Inkeeping with AACR editorial policy, a senior member of the Clinical CancerResearch editorial team managed the consideration process for this submissionand independently rendered the final decision concerning acceptability.

Authors' ContributionsConception and design: Y. Kong, B. Zheng, J. GuoDevelopment of methodology: Y. Kong, J. Yu, J. GuoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Kong, J. Yu, J. Yan, Q. Guo, Z. Chi, B. Tang,J. Yu, T. Yin, Z. Cheng, X. Wu, H. Yu, J. Dai, X. Sheng, L. Si, C. Cui, X. Bai,L. Mao, B. Lian, X. Wang, X. Yan, S. Li, L. Zhou, J. GuoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Kong, K.T. Flaherty, J. GuoWriting, review, and/or revision of the manuscript: Y. Kong, J. Yu, B. Zheng,K.T. Flaherty, J. GuoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Kong, J. GuoStudy supervision: Y. Kong, J. Guo

AcknowledgmentsWe would like to thank Yicon Biomedical Technology Inc. (Beijing, China)

for humanized immune system patient-derived xenograft model experiments.We would like to thank American Journal Experts for English language editing.This work was supported by grants from Natural Science Foundation of China(81672696 and 81772912), Fostering Young Scholars of Peking UniversityHealth Science Center, Beijing Baiqianwan Talents Project, and Beijing Munic-ipal Administration of Hospitals' Ascent Plan (DFL20181101), Clinical Med-icine Plus X-Young Scholars Project (Peking University), the FundamentalResearch Funds for the Central Universities.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 7, 2019; revised May 18, 2019; accepted July 30, 2019;published first August 2, 2019.

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2019;25:6511-6523. Published OnlineFirst August 2, 2019.Clin Cancer Res Jiayi Yu, Junya Yan, Qian Guo, et al. Non-Cutaneous MelanomaInnate Resistance to PD-1 Blockade in Chinese Patients with Genetic Aberrations in the CDK4 Pathway Are Associated with

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