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. Author manuscript; available in PMC: 2023 Nov 1.
Published in final edited form as: Ann Thorac Surg. 2021 Sep 27;114(5):1842–1852. doi: 10.1016/j.athoracsur.2021.08.054

Inhibition of CDK4/6 Overcomes Primary Resistance to PD-1 Blockade in Malignant Mesothelioma

Hee-Jin Jang a, Cynthia Y Truong a, Eric M Lo a, Hudson M Holmes a, Daniela Ramos a, Maheshwari Ramineni b, Ju-Seog Lee c, Daniel Y Wang d, Massimo Pietropaolo e, R Taylor Ripley a, Bryan M Burt a, Hyun-Sung Lee a
PMCID: PMC8957629  NIHMSID: NIHMS1745335  PMID: 34592265

Abstract

Background:

Despite the profound number of malignant pleural mesothelioma (MPM) patients now treated with PD-1 blockade, insight into the underpinnings of rational therapeutic strategies to treat resistance to checkpoint immunotherapy remains unrealized. Our objective was to develop a novel therapeutic approach to overcome primary resistance to PD-1 blockade in MPM.

Methods:

We generated a transcriptome signature of resistance to PD-1 blockade in MPM patients treated with nivolumab (four responders and four non-responders). We used the TCGA MPM cohort (N=73) to determine what genomic alterations were associated with the resistance signature. We tested whether regulation of identified molecules could overcome resistance to PD-1 blockade in an immunocompetent mouse malignant mesothelioma model.

Results:

Immunogenomic analysis by applying our anti-PD-1 resistance signature to the TCGA cohort revealed that deletion of CDKN2A was highly associated with primary resistance to PD-1 blockade. Under the hypothesis that resistance to PD-1 blockade can be overcome by CDK4/6 inhibition, we tested whether CDK4/6 inhibitors could overcome resistance to PD-1 blockade in subcutaneous tumors derived from Cdkn2a(−/−) AB1 malignant mesothelioma cells, which were resistant to PD-1 blockade. The combination of daily oral administration of CDK4/6 inhibitors (abemaciclib or palbociclib) and intraperitoneal anti-PD-1 treatment markedly suppressed tumor growth, compared with anti-PD-1 or CDK4/6 inhibitor alone.

Conclusions:

We identified a therapeutic target, CDK4/6, to overcome primary resistance to PD-1 blockade through comprehensive immunogenomic approaches. These data provide a rationale for undertaking clinical trials of CDK4/6 inhibitors in more than 40% of patients with MPM who demonstrate loss of CDKN2A.

Keywords: mesothelioma, CDKN2A, CDK4/6 inhibitor, immune checkpoint inhibitors, systems immunology

Immune checkpoint inhibitors (ICIs) have dramatically changed the treatment approach to various advanced cancers, resulting in durable clinical responses in many cases.1,2 Programmed Cell Death 1 (PD-1) inhibitors have recently shown encouraging clinical activity with good tolerability in patients with advanced malignant pleural mesothelioma (MPM) previously treated with chemotherapy. In these single-agent trials, objective response rates, defined as complete response (CR) or partial response (PR), ranged from 18 to 29%; and rates of stable disease (SD) were 23 to 52%, indicating a favorable impact of these drugs on durable clinical benefit.3–6 However, similar to patients with other tumor types treated with anti-PD-1 therapy, most patients with MPM do not respond to PD-1 inhibition, highlighting an unmet need to develop novel therapeutic approaches to augment the efficacy of immune checkpoint treatment.

Anti-PD-1 monoclonal antibody blocks the interaction between PD-1 on activated T cells and its ligands, which is thought to reinvigorate T cells to kill cancer cells.7 However, the therapeutic efficacy of PD-1 checkpoint inhibition is limited by primary and acquired resistance.8,9 Resistance to ICIs may be classified into two categories: (1) primary resistance, which generally refers to patients who do not respond at all to ICIs and instead progress; and (2) acquired resistance, referring to those who have an initial response to ICIs followed eventually by disease progression. The mechanisms responsible for primary resistance continue to be characterized as ineffective T cell priming, lack of tumor recognition due to defective antigen presentation, suppression via other checkpoints, tumor cell resistance to interferon, and local immunosuppressive factors in the tumor-immune microenvironment (TiME).10

To date, there have been no established therapeutic breakthroughs for thwarting resistance, and there has been a remarkably unmet need to investigate the mechanisms of resistance to ICIs. Understanding the underlying mechanisms of resistance may allow the rational design of combinatorial therapies to overcome primary resistance. Our objective was to develop a potential therapeutic approach to overcome resistance to PD-1 blockade in MPM through comprehensive immunogenomic analysis.

MATERIAL and METHODS

Patients and Data Acquisition

This study was performed in accordance with the Institutional Review Board protocol at Baylor College of Medicine (H-43208). Informed consent was obtained for the collection of clinical data and biospecimens. We performed transcriptome mRNA arrays using the pre-immunotherapy tumor biopsies of eight MPM patients with advanced and unresectable MPM, whom we treated with nivolumab (anti–PD-1) therapy after they had progressed following treatment with a platinum-based agent and pemetrexed (Supplementary Table 1). Original mRNA expression data deposited in the National Center for Biotechnology Information’s (NCBI) Gene Expression Omnibus (GEO) database (GSE99070) were used to generate a transcriptome-based anti-PD-1-resistant signature.11 We used the GSE117358 dataset to evaluate the performance of mRNA signature to predict response and resistance to checkpoint immunotherapy in mouse mesothelioma tumor models.12 Furthermore, to identify the association of genetic alteration with this anti-PD-1-resistant signature, we utilized mRNA sequencing data from 73 samples in The Cancer Genome Atlas (TCGA) portal (https://portal.gdc.cancer.gov/).13

Briefly, anti-PD-1-resistant signature was used to generate a support vector machine (SVM) classifier for estimating the likelihood that a particular MPM tumor belonged to the subgroup in which the anti-PD-1-resistant signature is present (anti-PD-1-resistant subgroup) or the subgroup in which the signature is absent (anti-PD-1-responsive subgroup). The robustness of the classifier was estimated by the misclassification rate determined during leave-one-out cross-validation (LOOCV) of the training set. After LOOCV, the sensitivity and specificity of the prediction models were estimated by the fraction of samples correctly predicted.11

Mouse malignant mesothelioma cell line and syngeneic mouse tumor model

AB1 mouse malignant mesothelioma (MM) cell lines, purchased from Sigma-Aldrich, were thawed and cultured at 37°C with 5% CO2 in RPMI 1640 (with 2mM L-Glutamine+25mM HEPES) supplemented with 5% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Six-week-old male BALB/cJ mice (Jackson Laboratory) were used under the protocol approved by the Institutional Animal Care and Use Committee, in accordance with all relevant animal-use guidelines and ethical regulations. To develop the immunocompetent mouse model of MM, 5×105 tumor cells in 50 μL of the serum-free medium were inoculated subcutaneously on the right flank area, where was prepared with 70% alcohol. Tumor size was monitored three times per week. The greatest longitudinal diameter (length) and the greatest transverse diameter (width) were determined to determine by external calipers and used to calculate tumor volume. The tumor volume, based on these caliper measurements, was calculated using the modified ellipsoidal formula: tumor volume = ½(length×width2).

Before in vivo drug treatment, mice were randomly divided into 6 groups when the length of tumor of each mouse reached 5mm. For anti-PD-1 therapy, anti-mouse PD-1 Abs (10mg/kg, clone 29F.1A12, BioXCell, Cat# BE0273) or isotype control (Clone 2A3, BioXCell, Cat# BE0089) were injected intraperitoneally twice per week for 3 weeks. For cyclin-dependent kinase (CDK) 4/6 inhibition, abemaciclib (Cat# S5716) and palbociclib (Cat# S1116) was purchased from Selleckchem. Abemaciclib (75mg/kg)14 and palbociclib (100mg/kg)15 dissolved in normal saline were administered by oral gavage daily for 3 weeks. Mice were euthanized three weeks after the first treatment to measure tumor burdens. Methods for Imaging mass cytometry (IMC) and time-of-flight mass cytometry (CyTOF)11,16,17 were described in the supplementary section (Supplementary Table 2 and 3).

Statistical Analysis

Student’s t-tests or Mann-Whitney U tests were used to compare continuous variables, and Chi-square or Fisher’s exact tests were performed to analyze categorical variables. Z values were calculated by subtracting the overall average gene expression from the raw data for each gene and dividing that result by the standard deviations of all of the measured expression. All statistical analyses were performed with SPSS 27.0 (IBM Corp, Released 2020. IBM SPSS Statistics for Windows, Version27.0. Armonk, NY), Prism 8.0 (GraphPad Software, Inc, San Diego, CA), and R language and software environment version 4.0.3 (http://www.r-project.org). Statistical significance was considered at P<0.05, and all tests were two-tailed.

RESULTS

Development of anti-PD-1-resistant mRNA signature.

This study scheme is illustrated in Figure 1. We first designed an anti-PD-1-resistant mRNA signature representative of resistance to PD-1 checkpoint immunotherapy by comparing mRNA expression in 4 responders (CR+PR) and 4 MPM patients with progressive disease after nivolumab treatment. Differential gene expression between responders and progressors was found in 158 differential mRNAs with a p-value less than 0.01 and above 2-fold changes, which was defined as our anti-PD-1-resistant signature (Fig.2A and Supplementary Table 4). Exploratory analysis of upstream regulators through the Ingenuity pathway analysis (http://www.ingenuity.com) revealed that the anti-PD-1-resistant gene signature was significantly associated with CDK4/6 and cyclin D1 activation in the cell cycle pathway (P<0.001) (Fig.2B).

Figure 1.

Figure 1.

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Study scheme. A transcriptome signature of resistance to PD-1 blockade was generated from malignant pleural mesothelioma (MPM) patients treated with nivolumab. To identify target candidates to overcome resistance, the upstream regulator analysis was performed. Next, the TCGA MPM cohort was used to determine what genomic alterations are associated with immune resistance signature. Finally, to test if targeting candidate molecules can increase the efficacy of PD-1 blockade, we performed in vivo experiment.

Figure 2.

Figure 2.

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Development of anti-PD-1 resistance mRNA signature. A. Development of transcriptome-based anti-PD-1 resistance signature. The data are presented in a matrix format in which rows represent individual gene and columns represent individual experiment. The red and blue colors in the cells reflect relative high and low expression levels, respectively, as indicated in the scale bar (log2 transformed scale). B. Upstream regulator analysis through the Ingenuity pathway analysis revealed that anti-PD-1-resistant gene signature was significantly associated with Cyclin-dependent kinase 4/6 and cyclin D1 activation in cell cycle pathway.

Resistance to PD-1 blockade is associated with CDKN2A deletion.

Our immune signature was applied to the GSE117358 dataset and TCGA MPM cohort (N=73) to predict the subgroups, utilizing a previously developed prediction model (Fig.3A).11,18 We validated that such a signature could predict response to checkpoint immunotherapy in an independent cohort. We examined publicly available mRNA expression data from a study in which immunocompetent mice were inoculated with bilateral subcutaneous murine AB1 mesothelioma tumors and treated with an anti-PD-L1 and anti-CTLA-4 antibodies. Tumors with an anti-PD-1-resistant signature had significantly decreased responses to ICIs (P<0.001)(Fig.3B). Furthermore, we determined the genomic alterations associated with the anti-PD-1 resistance signature (Fig.3C). No clear associations of clinical features were observed between two subgroups. BAP1 mutation was observed only in the anti-PD-1-responsive subgroup (P=0.016). Furthermore, anti-PD-1-responsive tumors had abundant plasma cells and M1 macrophages, which were calculated using CIBERSORT (http://cibersort.stanford.edu/), an analytic deconvolution method of “in silico flow cytometry” that accurately estimates the abundances of specific leukocyte subsets using gene expression data.19 In contrast, MPM tumors predicted as anti-PD-1-resistant tumors had a significant deep deletion of Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) located at 9p21.3 (P=0.003). CDKN2A loss was found in 47% (N=34) of all 73 MPM patients. Among 34 patients with CDKN2A loss, 74% (N=25) were classified into anti-PD-1-resistant tumors. Resting memory CD4 T cells were significantly increased in this subgroup (P=0.018)(Fig.3C). Representative multiplexed images of an MPM patient with intact CDKN2A who had a partial response to nivolumab, showed abundant infiltration of CD8 T cells and B cells. In contrast, the other representative images of an MPM patient with CDKN2A loss who had progressive disease after nivolumab treatment, showed abundant infiltration of tumor-associated macrophages (TAMs) in the absence of CD8 T cells (Fig.4).

Figure 3.

Figure 3.

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CDKN2A deletion is associated with αPD-1 resistance. A. The anti-PD-1-resistant mRNA signature was used to predict the subgroups in two independent cohorts. B. Murine malignant mesothelioma tumors predicted as anti-PD-1-resistant tumors had little response to dual immune checkpoint inhibitors. C. MPM tumors predicted as anti-PD-1-resistant tumors had a significant deletion of CDKN2A located at 9p21.3. CDKN2a loss was found in 47% of MPM patients. Among them, 74% were classified into anti-PD-1-resistant tumors.

Figure 4.

Figure 4.

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Representative images to show the association of CDKN2A status with the tumor-immune microenvironment. Representative images of an MPM patient with intact CDKN2A who had a partial response, showed abundant infiltration of CD8 T cells and B cells. In contrast, the other representative images of an MPM patient with CDKN2A loss who had progressive disease, showed abundant infiltration of TAMs with the absence of CD8 T cells (Ir, iridium; Pan-CK, pan-cytokeratin).

In addition, antigen presenting machinery including HLA-A, HLA-B, HLA-C, and B2M was significantly downregulated in the anti-PD-1-resistant subgroup (Fig.5A). The cytolytic activity20 derived from the geometric mean of GZMA and PRF1, and the IFN-γ signaling signature (CIITA, GBP4, GBP5, IRF1, IRF2, and JAK2)21 were significantly lower in the anti-PD-1-resistant subgroup, compared with those of the anti-PD-1-responsive subgroup (Fig.5B). On the contrary, CDK4 and CDK6 mRNA expression was significantly increased in the anti-PD-1-resistant subgroup (Fig.5C), implying cell cycle activation through the depletion of CDKN2A.

Figure 5.

Figure 5.

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Comparison of mRNA expression related to anti-PD-1 response and resistance. A. The expression of B2M and MHC I molecules was significantly reduced in the anti-PD-1 resistance group. B. Cytolytic activity and IFN-γ signature. Subset predicted as anti-PD-1 resistance demonstrated significantly decreased cytolytic activity and the IFN-γ signaling signature. C. Cell cycle-related CDK4/6 expression (RPKM, Reads Per Kilobase of transcript, per Million mapped reads).

CDK4/6 inhibitors overcome resistance to PD-1 checkpoint immunotherapy.

CDKN2A is a well-known tumor suppressor, and CDKN2A gene loss increases the activity of cell cycle regulators, CDK4 and CDK6, leading to cell proliferation.22,23 Given the immunologic role of CDK4/6 inhibition in the anti-tumor immunity by enhancing antigen presentation on tumor cells, stimulating effector T cell activation, and inhibiting regulatory T cell proliferation,14,15,24 we hypothesized that CDK4/6 inhibition could sensitize the tumor-immune microenvironment in the CDKN2A-depleted tumors resistant to PD-1 blockade. Whole exome sequencing of fifteen asbestos-induced murine MM tumor cell lines from BALB/c, CBA, and C57BL/6 mouse strains25 showed homozygous loss of Cdkn2a in 14 out of 15 tumors, including AB1 MM cells, which demonstrated resistance to PD-1 blockade in vivo (Fig.6A). To test our hypothesis, we used a subcutaneous mouse malignant mesothelioma (MM) model using the AB1 cell line. Like human MPM resistant to PD-1 blockade, this mouse MM tumor contained abundant TAMs with less lymphocyte infiltration (Fig.6B).

Figure 6.

Figure 6.

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Anti-PD-1-resistant mouse malignant mesothelioma model using Cdkn2a(−) AB1 cell line. A. AB1 MM tumors were resistant to PD-1 blockade in vivo. B. Like human MPM resistance to PD-1 blockade, this mouse mesothelioma tumor contained abundant tumor-associated macrophages (TAMs) with less lymphocyte infiltration.

FDA-approved oral CDK4/6 inhibitors, palbociclib (100mg/kg) and abemaciclib (75mg/kg), were administered daily for 3 weeks, and anti-mouse PD-1 antibody (10mg/kg) was administered intraperitoneally twice per week for 3 weeks when tumors were established (Fig.7A). CDK4/6 inhibition slowed down tumor growth compared with untreated and anti-PD-1 treated groups. Single-agent treatment with abemaciclib or palbociclib showed modest effects for inhibiting tumor growth. A combination of CDK4/6 inhibitor and PD-1 blockade markedly suppressed tumor growth (both P<0.001) (Fig.7B–7C).

Figure 7.

Figure 7.

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CDK4/6 inhibitors overcome resistance to αPD-1 mAb. A. Treatment scheme. FDA-approved oral CDK4/6 inhibitors, palbociclib and abemaciclib, were daily administered 7 days after tumor inoculation for 3 weeks, and anti-mouse PD-1 antibody was administered intraperitoneally twice per week (each group n=5). B, C. Both abemaciclib and palbociclib showed modest effects for controlling tumor growth. A combination of CDK4/6 inhibitor and PD-1 blockade markedly suppressed tumor growth.

CyTOF displayed that the majority of immune compositions in untreated AB1 tumors were TAMs (Fig.8). Treatment of anti-PD-1 antibody led to a significant increase of T cells (P=0.003) and a slight decrease of TAMs (P=0.006). Palbociclib treatment led to a significant increase of both B cells (P<0.001) and CD4 T cells (P=0.014), and a significant decrease of TAMs (P<0.001). Combination therapy resulted in abundant infiltration of B cells (P<0.001) and CD4 T cells (P=0.017) and a dramatic decrease of TAMs (P<0.001). Taken together, a combination of CDK4/6 inhibitors (palbociclib or abemaciclib) and a PD-1 inhibitor eradicated anti-PD-1-resistant Cdkn2a(−) AB1 MM tumors with tumor cells through the reprogramming of the tumor-immune microenvironment.

Figure 8.

Figure 8.

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CDK4/6 inhibitors alter the tumor-immune microenvironment. Time of flight mass cytometry showed that most immune compositions in untreated AB1 tumors were tumor-associated macrophages (TAMs). A single treatment of anti-PD-1 led to a modest increase of T cells and a slight decrease of TAMs. Palbociclib treatment led to a significant increase of B cells and CD4 T cells, and a significant decrease of TAMs. Combination therapy resulted in abundant infiltration of B cells and CD4 T cells and a dramatic decrease of TAMs.

COMMENT

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have changed the standard-of-care treatment for a variety of advanced cancers. In MPM, PD-1 inhibitors have recently shown encouraging clinical activity with good tolerability in patients previously treated with chemotherapy.3–6,26,27 However, the mechanisms of response and resistance to immune checkpoint blockade in human tumors are only beginning to be understood and are still unknown in MPM. Through comprehensive immunogenomic analysis, we uncovered that CDKN2A loss is associated with primary resistance to PD-1 blockade. Our in vivo experiment using an immunocompetent mouse MM model with primary resistance to anti-PD-1 treatment supported that suppressing CDK4/6 activated by CDKN2A loss overcomes primary resistance to checkpoint immunotherapy.

CDKN2A encodes the ADP-ribosylation factor (p14ARF) and p16INK4A through the alternative splicing, which play as cell cycle inhibitors.28 The p16INK4A binds to CDK4/6 to inhibit the binding of cyclin D1 (CCND1) to CDK4/6, leading to arresting cell cycle in the G1 phase, and the p14ARF protects p53 and prevents tumor formation.29 Deletions in the 9p21 area containing CDKN2A and CDKN2B genes lead to disruption of the CDKN2A-CCND1-CDK4/6-Rb axis, one of the cell cycle regulators promoting G1-S cell-cycle transition.30,31 In MPM, about 40–50% of patients had CDKN2A loss, which is associated with worse prognosis.13,32–35 However, the baseline and sustained expressions of p16INK4A during chemotherapy were associated with prolonged survival.35

Selective CDK4/6 inhibitors, including FDA-approved palbociclib, abemaciclib, and ribociclib, have been shown effective anti-tumor activity and manageable toxicity.36 Recent studies have indicated that CDK4/6 inhibitors enhance antigen processing and presentation as well as T cell immunity in the TiME of mouse breast cancer.24,37 A potential link between CDK4/6 activity and PD-L1 protein stability was recently reported.38 CDK4/6 inhibition also had a radiosensitization effect in MPM.39 Based on promising preclinical and clinical results in other cancers, phase II clinical trials with single-agent CDK4/6 inhibitors have been designed in patients with MPM [NCT03654833 and NCT02187783].

To face the challenge of primary resistance to immunotherapy, constant efforts to develop combination strategies have been made to broaden the responders. Furthermore, there has been an extensive search for predictive biomarkers for initial ICI response. PD-L1 expression, tumor mutational burden, tumor-infiltrating lymphocytes, or related gene expression signatures have been explored as potential predictors, and various other markers are currently under investigation. Our study also provides the potential role of homozygous deletion of CDKN2A, which can be detected by fluorescence in situ hybridization as a mechanistic predictive marker to recommend the combination treatment of CDK4/6 inhibitors with ICIs. Therefore, the combination treatment approach of CDK4/6 inhibitors with ICIs can be a rational strategy to overcome the treatment resistance of tumors.

Our study limitations include the small number of patients with more advanced disease receiving ICIs to generate the immunologic profile that was applied to the TCGA cohort with earlier disease, requiring further mechanistic investigation with multiple strains to determine the effect of CDK4/6 inhibitors on various cellular compositions as well as cancer cells, and uncovering the cause and effect between CDKN2A and the alteration of tumor milieu. To implement our research findings to clinical practice, we are investigating the association of CDKN2A status with resistance to immune checkpoint inhibitors in the additional independent MPM cohort in our institute. Furthermore, we will test the efficacy of the combination of CDK4/6 inhibitors and anti-PD-1 treatment on multiple mesothelioma strains with or without CDKN2A overexpression. A preclinical study using patient-derived xenograft (PDX) models may provide a rationale to immediately initiate a clinical trial of the combination of CDK4/6 inhibitors with immune checkpoints in MPM patients.

Conclusions

Through comprehensive immunogenomic approaches, we have identified CDK4/6 as a therapeutic target for overcoming primary resistance to PD-1 blockade. These data provide the rationale for undertaking clinical trials of CDK4/6 inhibitors combined with PD-1 blockade in more than 40% of patients with malignant pleural mesothelioma who demonstrate loss of CDKN2A.

Supplementary Material

1

ACKNOWLEDGEMENT:

This project was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the Cancer Prevention and Research Institute of Texas Core Facility Support Award (RP180672), the National Institutes of Health (NIH) (CA125123 and RR024574) and the assistance of Joel M. Sederstrom.

GLOSSARY OF ABBREVIATIONS

B2M

beta-2-microglobulin

CDKN2A

Cyclin Dependent Kinase Inhibitor 2A

CDK4/6

Cyclin Dependent Kinase 4/6

CR

complete response

CyTOF

time-of-flight mass cytometry

PDX

Patient-derived xenograft

FFPE

formalin-fixed paraffin-embedded

GEO

gene expression omnibus database

ICIs

immune checkpoint inhibitors

IMC

imaging mass cytometry

LOOCV

leave-one-out cross validation

MM

malignant mesothelioma

MPM

malignant pleural mesothelioma

NCBI

the National Center for Biotechnology

PD

progressive disease

PD-1

Programmed Cell Death 1

PD-L1

Programmed Cell Death 1 Ligand 1

PR

partial response

RPKM

reads per kilobase of transcript, per million mapped reads

SD

stable disease

SVM

support vector machine

TCGA

The Cancer Genome Atlas

TAMs

tumor-associated macrophages

Footnotes

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