p53-Driven Lung Cancer

ORIGINAL ARTICLE

The Combination of MEK Inhibitor With Immunomodulatory Antibodies Targeting Programmed Death 1 and Programmed Death Ligand 1 Results in Prolonged Survival in Kras/p53Driven Lung Cancer Jong Woo Lee, PhD,a Yu Zhang, MD,b Kyung Jin Eoh, MD, PhD,a,c Roshan Sharma, MS,a Miguel F. Sanmamed, MD,b Jenny Wu, BS,a Justin Choi, MD,a Hee Sun Park, MD, PhD,d Akiko Iwasaki, PhD,e Edward Kaftan, PhD,a Lieping Chen, MD,b Vali Papadimitrakopoulou, MD,f Roy S. Herbst, MD, PhD,a Ja Seok Koo, PhDa,g,* a

Section of Medical Oncology, Department of Internal Medicine, Yale Comprehensive Cancer Center, Yale School of Medicine, New Haven, Connecticut b Department of Immunobiology, Yale Comprehensive Cancer Center, Yale School of Medicine, New Haven, Connecticut c Department of Obstetrics and Gynecology, Yonsei University College of Medicine, Seoul, South Korea d Department of Internal Medicine, Chungnam National University Hospital, Daejeon, South Korea e Department of Immunobiology and Molecular, Cellular and Developmental Biology, Yale School of Medicine, New Haven, Connecticut f Department of Thoracic, Head and Neck Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas g Developmental Therapeutics Translational Research Program, Yale Comprehensive Cancer Center, New Haven, Connecticut Received 3 October 2018; revised 19 January 2019; accepted 1 February 2019 Available online - 13 February 2019

ABSTRACT Introduction: This study aimed to characterize the tumorinfiltrating immune cells population in Kras/tumor protein 53 (Trp53)-driven lung tumors and to evaluate the combinatorial antitumor effect with MEK inhibitor (MEKi), trametinib, and immunomodulatory monoclonal antibodies (mAbs) targeting either programmed death -1 (PD-1) or programmed cell death ligand 1 (PD-L1) in vivo. Methods: Trp53FloxFlox;KrasG12D/þ;Rosa26LSL-Luciferase/LSL-Luciferase (PKL) genetically engineered mice were used to develop autochthonous lung tumors with intratracheal delivery of adenoviral Cre recombinase. Using these tumor-bearing lungs, tumor-infiltrating immune cells were characterized by both mass cytometry and flow cytometry. PKL-mediated immunocompetent syngeneic and transgenic lung cancer mouse models were treated with MEKi alone as well as in combination with either anti–PD-1 or anti–PD-L1 mAbs. Tumor growth and survival outcome were assessed. Finally, immune cell populations within spleens and tumors were evaluated by flow cytometry and immunohistochemistry. Results: Myeloid-derived suppressor cells (MDSCs) were significantly augmented in PKL-driven lung tumors compared to normal lungs of tumor-free mice. PD-L1

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expression appeared to be highly positive in both lung tumor cells and, particularly MDSCs. The combinatory administration of MEKi with either anti–PD-1 or anti–PD-L1 *Corresponding author. Disclosure: Dr. Chen has received personal fees from Pfizer, Vcanbio, GenomiCare; is the founder of NextCure and TAYU Biotech; and has received grants from NextCure. Dr. Papadimitrakopoulou has received grants from Eli Lilly and Company, Novartis, Astra Zeneca Pharmaceuticals, F. Hoffman-La Roche, Nektar Therapeutics, Janssen, BristolMyers Squibb, Checkmate, and Incyte; and has received personal fees from Nektar Therapeutics, Astra Zeneca Pharmaceuticals, Arrys Therapeutics, Merck and Company, Loxo Oncology, Araxes Pharma, F.Hoffman-La Roche Ltd., Janssen Research Foundation, Bristol-Myers Squibb, Clovis Oncology, Eli Lilly and Company, Novartis Pharmaceuticals Corp., Takeda Pharmaceuticals, Abbvie, TRM Oncology, Exelixis, and Tesaro. Dr. Herbst has received grants from Astra Zeneca, Eli Lilly and Company, and Merck and Company; and has received personal fees from Jun Shi Pharmaceuticals, Loxo Oncology, Merck and Company, Nektar, NextCure, Novartis, Pfizer, Sanofi, Seattle Genetics, Shire PLC, Spectrum Pharmaceuticals, Symphogen, TESARO, Infinity Pharmaceuticals, and Neon Therapeutics. The remaining authors declare no conflict of interest. Address for correspondence: Ja Seok Koo, PhD, The Section of Medical Oncology, Department of Internal Medicine, Yale Comprehensive Cancer Center, Yale School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520. E-mail: [email protected] ª 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. ISSN: 1556-0864 https://doi.org/10.1016/j.jtho.2019.02.004

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mAbs synergistically increased antitumor response and survival outcome compared with single-agent therapy in both the PKL-mediated syngeneic and transgenic lung cancer models. Theses combinational treatments resulted in significant increases of tumor-infiltrating CD8þ and CD4þ T cells, whereas attenuation of CD11bþ/Gr-1high MDSCs, in particular, Ly6Ghigh polymorphonuclear-MDSCs in the syngeneic model. Conclusions: These findings suggest a potential therapeutic approach for untargetable Kras/p53-driven lung cancers with synergy between targeted therapy using MEKi and immunotherapies.  2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved. Keywords: Trametinib; Programmed death 1; Programmed death ligand 1; Myeloid-derived suppressor cells; Kras/p53driven lung cancer

Introduction Lung cancer is the second most common cancer, and despite all the advances in cancer treatment, the overall 5-year survival rate remains dismal at 18%.1,2 NSCLC, which accounts for approximately 85% of all lung cancers, is further divided mainly in two groups: adenocarcinoma (LUAD) and squamous cell carcinoma. KRAS mutation, known as oncogenic driver mutation, has been detected in 20% w 40% of LUAD and in 3% w 6% of lung squamous cell carcinoma.3-5 We have focused on finding a new strategy for the KRAS-mutant lung cancer because there is no clinically effective targeted treatment for this subtype.6-10 Targeting KRAS signaling has been aimed at its downstream targets, one of which is MEK.11 However, monotherapy with MEK inhibitor (MEKi) for KRASmutated NSCLC has been reported to be largely ineffective.12,13 Moreover, MEKi in combination with conventional chemotherapy caused greater toxicity without improvement in clinical outcomes.14,15 This observation indicated the need for dose reduction to complete a full cycle of the combination therapy. Finding a novel strategy for KRAS-mutated NSCLC, it is noteworthy that antagonist monoclonal antibodies (mAbs) targeting programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1), which mediates immunosuppression, have been approved for the treatment of advanced NSCLC.16-20 Previous studies have shown that KRAS-mutated NSCLC is correlated with upregulated PD-L1, which indicates that PD-L1 could function as an immune escape mechanism of KRASmutated NSCLC.19,21,22 In the clinical studies, however,

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KRAS mutational status did not seem to be associated with a survival benefit of immune checkpoints inhibitors.20,23 Therefore, the combinations of PD-1/PD-L1 blockade with other agents would be required to produce higher efficacy. Here, we have studied the antitumor effect of reduced dose of MEKi in combination with immune checkpoints inhibitors against Kras/tumor protein 53 (p53)–driven lung tumors using a Kras G12D mutation and p53 deficiency-driven lung cancer mouse model; Trp53floxflox;KrasLSL-G12D/þ;Rosa26LSL-Luciferase/LSL-Luciferase (PKL). We found that combinatorial treatment significantly blocked tumor growth and extended survival outcome in the preclinical models.

Materials and Methods Transgenic Lung Cancer Mouse Models All experiments with animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Yale University (#2017-11464). All animals were housed in the Yale University Animal Facility under the guidelines held by the Yale IACUC. KrasLSL-G12D/þ (strain 01XJ6, B6/129Sv) and Trp53Flox/þ (strain 01XC2, FVB/129) strains were obtained from the National Cancer Institute of Frederick Mouse Repository, and FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J (005125, FVB/129) strain was purchased from the Jackson Laboratory (Bar Harbor, Maine). Based on the method of DuPage et al.,24 the PKL mice were intratracheally infected with adenoviral particles encoding for Cre recombinase (Ad-Cre) (5  106 plaque-forming units University of Iowa, Iowa City, Iowa). Tumor incidence and growth were quantified by bioluminescence live animal imaging (IVIS Spectrum; Perkin Elmer, Waltham, Massachusetts). For IVIS images, signal intensity was quantified as the radiance unit of photons/sec/cm2/sr which means the number of photons per second that level a square centimeter of tissue and radiate into a solid angle of 1 steradian (sr). For ex vivo imaging, mice were injected with Dluciferin and its organs were collected after euthanasia.

Murine Lung Tumor Cells PKL5-2 murine lung tumor cell line was established from lung tumors derived from our PKL model (FVB dominant). Cells were cultured with Roswell Park Memorial Institute –1640 medium supplemented with 10% fetal bovine serum and 1% Antibiotic-antimycotic (Invitrogen, Carlasbad, California) and incubated at 37  C and 5% CO2. PKL5-2 cells were regularly tested for mycoplasma negativity in the study.

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Treatment PKL mice received food and water ad libitum. Lung tumors were induced by Ad-Cre in the transgenic mouse model as described above. Syngeneic tumors were established by subcutaneously implanting 3  105 PKL52 cells into the right flank of mice. Tumor volume was calculated using the equation (l  w2)/2, where l and w refer to the larger and smaller dimensions collected at each measurement. Treatments began with tumor size 50 to 200 mm3. Trametinib (Selleckchem, Houston, Texas) was daily administered as 0.25, 0.5, and 1 mg/kg in 2% DMSO-5% polyethylene glycol (Millipore-Sigma, St. Louis, Missouri) by oral gavage. Anti–PD-1 (RMP1-14) and anti–PD-L1 (10F.9G2) mAbs were purchased from BioXCell (West Lebanon, New Hampshire). These antibodies were intraperitoneally administered as 200 mg/ mouse in phosphate-buffered saline twice a week. Tumors were monitored daily, and mice were euthanized when an endpoint was reached, defined as tumor volume greater than 1,000 mm3, tumor ulceration, or study end, whichever came first. Tumor regressions, median tumor volume, and treatment tolerability were also considered. Tumor weight was measured at the endpoint of study. The median time to endpoint and its corresponding 95% confidence interval were calculated.

Mass Cytometry and Fluorescence-Based Flow Cytometry All tissue preparations were performed simultaneously from each individual mouse. Spleen and either lung tumor or subcutaneous tumor were digested using Mouse Tumor Dissociation Kit (Miltenyi Biotec Inc., Auburn, California). For mass cytometry (CyTOF) analysis, cells were incubated with anti-CD16/CD32 mAbs (clone 2.4G2, BD Bioscience, San Jose, California) and subsequently incubated with a cocktail of metalconjugated antibodies (Abs) against surface markers on ice (Supplementary Table 1). Cells were incubated with IrDNA intercalator (Fluidigm, South San Francisco, California) at 4 C for overnight. For flow cytometry, lung single cell suspensions were prepared as previously described. Cells were stained with fluorochromeconjugated Abs on ice. Cells were washed and followed by acquisition using the standard protocol on LSRII flow cytometry instrument (BD Bioscience). Acquired data were analyzed with CyToBank (Cytobank Inc., Santa Clara, California) and FlowJo (FlowJo LLC, Ashland, Oregon) software, for CyTOF and flow cytometry, respectively.

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mol/L citrate buffer, pH 6.0. Slides were incubated with Dual Endogenous Enzyme Block (Agilent, Santa Clara, California), and followed blocking with 1% bovine serum albumin in phosphate-buffered saline/0.1% Tween-20. Primary antibodies against anti-CD45 (ab10558) and anti-cleaved Caspase-3 (ab4051) purchased from Abcam (Cambridge, Massachusetts), CD3 (Biocare Medical, CP215) purchased from Biocare Medical (Pacheco, California), PD-L1(#13684), and PCNA (#2586) purchased from Cell Signaling Technology (Danvers, Massachusetts) were used for the staining following dilution in SignalStain Antibody Diluent. SignalStain Boost (HRP, rabbit or mouse, Cell Cignalling Technology) secondary antibodies were used and followed by DABþ Chromogen (Agilent) staining. For immunofluorescence, staining was followed the protocol as previously described.25 For western blotting, PKL5-2 cells were simultaneously exposed to the combinational treatment of trametinib (10 nmol/L) and anti–PD-L1 antibody (2 mg/mL) with or without interferon-r (IFN-r) (10 ng/mL) (Peprotech, Rocky Hill, New Jersey) or IFN-a (40 ng/mL) (Thermo Fisher Scientific, Waltham, Massachusetts) for 24 hours. Cell lysates were prepared with radioimmunoprecipitation assay lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L EGTA, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 1% NP-40) and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting with anti-cleaved PARP (#5625), anti-PARP (#9542), anti-Caspase 7 (#12827), anticleaved Caspase 3 (#9664), and anti-Caspase 3 (#9665) antibodies (Cell Signaling Technology, Danvers, Massachusetts). Anti-b-actin (#A2228; Sigma, St. Louis, Missouri) was used for loading control.

Statistical Analysis Plot and bar graphs show the mean and standard error of the mean (SEM) as calculated by Student’s t test. Differences between treatment groups were determined by one- or two-way analysis of variance followed by Bonferroni’s post-test. A Kaplan-Meier plot was generated to show survival by treatment group and significance was assessed by log-rank (Mantel-Cox) test. Analyses were conducted using GraphPad Prism 7 and differences were considered to be significant at p less than 0.05.

Results

Immunohistochemistry, Immunofluorescence Staining, and Western Blotting

Immunogenic Autochthonous Lung Adenocarcinomas Driven by PKL Harbor Abundant Polymorphonuclear Myeloid-Derived Suppressor Cells

Tissue sections were deparaffinized, rehydrated, and subjected to high-temperature antigen retrieval in 0.01

We independently generated a PKL model by intercrossing of Trp53flox/flox;KrasLSL-G12D/þ mouse

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Figure 1. Prominent infiltration of immune cells in lung tumor driven by KrasG12D and p53 knockout lung cancer mouse model. (A) Representative lungs from Krasþ/þ;Trp53flox/flox (nontumor, n ¼ 6) and KrasLSL-G12D/þ;Trp53flox/flox (tumor) mice at 10 (n ¼ 6) and 12 weeks (n ¼ 6) after adenoviral particles encoding for Cre recombinase (Ad-Cre) infection. Scale bar ¼ 1 cm (left). Grossly enlarged tumor bearing lungs relative to tumor-free lungs as shown an average percentage of whole lung weight of body weight (right). (B) A significant increase in the infiltration of immune cells as shown by immunohistochemistry for CD45 in lung tumors (n ¼ 3 each timepoint) compared with tumor-free lung (n ¼ 3). (C) SPADE tree derived from CyTOF (15 markers) analysis of whole tumor cell population from either Kras/p53 mutants (right) or control (left) mice at 12 weeks after Ad-Cre induction (n ¼ 3). (D) Total number of the indicated immune cells in lung. Results are presented as mean ± SEM. M-MDSC, monocytic myeloid-derived suppressor cells; PMN, polymorphonuclear; SPADE, Spanning-tree Progression Analysis of Density Normalized Events; CyTOF, mass cytometry; ns, not significant. p values were determined by two-tailed Student’s t test. *p < 0.05, **p < 0.005

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Figure 2. Programmed death ligand 1 (PD-L1) expression is significantly elevated not only in non-immune cells but also in myeloid-derived suppressor cells (MDSCs) of tumor-bearing lungs as compared with tumor-free lungs. (A) A prominent expression of PD-L1 is seen in tumors derived from Kras/p53-mutated mice in a time course manner as shown by immunohistochemistry for PD-L1 (n ¼ 3). Scale bar ¼ 100 mm. (B) Highly overexpressed PD-L1 population in CD45- (non-immune) cells of tumor-bearing lungs compared with tumor-free lung. Isotype control antibody for PD-L1 was applied in this flow cytometry analysis (n ¼ 6). (C) Elevated PD-L1 expression in CD11bþ/Gr-1þ MDSCs of lung tumors compared with control lungs as shown by flow cytometry analysis. Results are presented as mean ± SEM. p values were determined by two-tailed Student’s t test. *p < 0.05.

with a mouse harboring loxP-stop-loxP followed by firefly luciferase in Rosa 26 locus (Supplementary Fig. 1A). Using the mice, intratracheal delivery of Ad-Cre was performed for the development of autochthonous lung tumors. We confirmed that luciferase-mediated bioluminescence is specific to the PKL-driven tumors but not visualized in other organs by live animal imaging using IVIS system (Supplementary Fig. 1B).

The tumor initiation by Ad-Cre yielded a large number of lung tumors at week 10, as indicated by 2.43-fold higher of the increased percentage of whole lung weight per body weight ratio in tumor-bearing mice compared to tumor-free mice (Fig. 1A) (p ¼ 0.0262). Furthermore, these tumors constituted the majority of lung composition at 12 weeks after Ad-Cre (Fig. 1A). We next examined the status of immune cells in these lung tumor specimens by immunohistochemical staining. CD45þ

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cells were dramatically accumulated mainly at the periphery, but not inside, of tumor nodules (peritumoral regions) in PKL-driven lung tumors in comparison with normal lungs from tumor-free Kras wild-type (Krasþ/þ) mice at week 10 and 12 (Fig. 1B). Both Trp53flox/flox; Rosa26LSL-Luc/LSL-Luc concomitant with either Krasþ/þ or KrasLSL-G12D/þ mice were identically infected by Ad-Cre to rule out the effect, if any, of the immune response to viral infection and luciferase-mediated immunogenicity. To characterize the tumor-infiltrated immune cells in this model, we conducted a multivariate single-cell analysis using CyTOF. Using CyTOF analysis, we first observed drastic increases of both CD45þ cells (p ¼ 0.0217) and CD45- cells in tumor-bearing lungs compared to tumor-free lungs (Figs. 1C and D; and Supplementary Fig. 2). We also identified that neutrophils/PMN-MDSCs (CD45þ/CD11bþ/Ly6Ghigh/ low Ly6C ) were significantly more abundant in tumorbearing lungs (p ¼ 0.0042) whereas CD3þ/CD8þ T cells showed a tendency to decrease in tumor-bearing lungs compared to tumor-free lungs. To get further insight into subpopulation of MDSCs, we used major histocompatibility complex class II (MHC-II) marker on these cells and we observed that the PMN-MDSC (CD45þ/CD11bþ/Ly6Ghigh/Ly6Clow/MHC-IIlow) cell population tended to be increased in tumor-bearing lungs, compared with tumor-free lungs (Figs. 1C and D). The PMN-MDSC population in tumor-bearing lungs was accounted for in approximately 17% of the CD45þ immune cells in the whole lung. We did not observe a significant difference in the percentage of regulatory T cells (FoxP3þ) in this tumor model in comparison with tumor-free lung (Supplementary Fig. 3).

PD-L1 Is Highly Expressed in Both Tumor Cells and MDSCs in Murine Lung Tumors Driven by PKL Given that the PD-L1 is an immunosuppressive pathway that is found to be active in the tumor microenvironment, we conducted immunohistochemistry with a mouse-specific antibody against PD-L1 using the formalin-fixed paraffin-embedded lung tumor tissues to see the status of PD-L1 expression. Elevated PD-L1 expression in tumor-bearing lungs was exhibited in a time-course manner upon Ad-Cre induction in the mice (Fig. 2A). Moreover, PD-L1 expression was also higher in CD45- cells isolated from tumor-bearing lungs compared to CD45- cells from tumor-free lungs as determined by flow cytometry, as we expected (Fig. 2B). Neutrophils/ MDSCs isolated from the tumor-bearing lungs also showed substantially higher PD-L1 expression than those found in the tumor-free lungs (Fig. 2C). Taken together, these results indicate that PD-L1 expressed

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MDSCs; in particular, neutrophils/PMN-MDSCs were accumulated in our Kras/p53-driven lung tumors, suggesting that this accumulation may precede tumor progression.

Combination of MEKi and Anti–PD-1/PD-L1 mAbs Attenuated Tumor Growth Leading to Prolonged Survival in PKL-Transgenic Lung Cancer Mouse Model Upon our findings of the elevated PD-L1 expression in the Kras/p53-driven lung tumors as favorable immunotherapy marker, we first decided to explore a potential combinational treatment with anti–PD-1/ PD-L1 immunotherapies and MEKi using our PKL transgenic lung cancer mouse model since MEK has been considered as a direct downstream effector of KRAS oncogenic signaling. Lung tumor-bearing PKL mice induced by intratracheal administration of AdCre were treated with MEKi, trametinib, for 3 days before initiation of combination treatment and followed by further administration of MEKi as a single or combination treatment with either anti–PD-1 or anti–PD-L1 mAbs for the following 3 weeks (Fig. 3A). We observed that any single-agent therapies using either anti–PD-1 or anti–PD-L1 mAbs failed to control tumor progression (Figs. 3B and C). In contrast, MEKi alone and in combination with either anti–PD-1 or anti–PD-L1 mAbs significantly decreased both number and size of tumor nodules as shown grossly visualized and on hematoxylin and eosin–stained specimens resulting in an attenuation of lung weight/body weight ratio at the endpoint (Figs. 3B and C). Moreover, we confirmed that these combinational treatments of MEKi with either anti–PD-1 or anti–PD-L1 mAbs were associated with significant suppression of tumor progression driven by Kras/p53 mutations by IVIS imaging (Figs. 3D and E). The combinational therapies, but not single-agent therapies, resulted in significant improvement of survival compared to vehicle-treated control mice, in particular, the combination with MEKi and anti–PD-L1 mAb (Fig. 3F).

Combination of MEKi and Anti–PD-1/PD-L1 mAbs Synergistically Abolished Tumor Growth in Syngeneic Kras/P53 Mouse Model The antitumor effect of the combinational treatment was evaluated in the immunocompetent syngeneic model. We initially determined whether one of our murine lung tumor cell lines, called PKL5-2 cells, derived from lung tumors in our PKL-transgenic mice could respond to the immunotherapies by measuring the expression of PD-L1 in the cells. PD-L1 expression

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was low in resting PKL5-2 tumor cells, whereas its expression on these cells was dramatically induced by IFN-g addition, as determined by flow cytometry, indicating the possibility of response to immunotherapy (Supplementary Fig. 4). Thus, PKL mice were subcutaneously implanted with PKL5-2 lung tumor cells followed by administration of MEKi, anti–PD-1 or anti–PD-L1 as a single or a combination (Fig. 4A). Inconsistent with our finding in the transgenic model, single-agent anti–PD-1 or anti–PD-L1 treatments were ineffective or even worse than the control group in tumor growth (Fig. 4B). However, MEKi alone and in combination with either anti–PD-1 or anti–PD-L1 mAbs substantially reduced tumor growth as determined by bioluminescence imaging and tumor weight at the endpoints (Fig. 4B). Consistent with the result from our transgenic model, MEKi in combination with either anti–PD-1 or anti–PD-L1 mAbs also dramatically resulted in prolonged survival of mice, but not MEKi alone (Fig. 4C). Although the combination with MEKi and immunecheckpoint inhibitor mAbs showed substantially synergism in our in vivo models, a single treatment with 1 mg/ kg of MEKi also exhibited a significant suppressive effect on tumor growth. We thus questioned whether lower doses of MEKi in combination with anti–PD-L1 mAb also show a synergism of antitumor effect. MEKi 1 mg/kg is likely a similar dose as that of patients who showed no therapeutic benefit. In this regard, the validation of lowerdose MEKi, which would not show any antitumor effect as a single-agent in our model, was required to overcome this limitation. Thus, we first tested with various doses of MEKi less than 1 mg/kg as a single treatment using our syngeneic model. Tumor-bearing syngeneic mice were administered 0.25 or 0.5 mg/kg MEKi for 10 days followed by modification of this treatment regime to test the combinational effect with anti–PD-L1 mAb (Fig. 4D). We simultaneously monitored tumor growth and body weight every other day (Figs. 4E and F). When MEKi was used on its own, a lower dose of MEKi showed modest suppressive effects on tumor growth, but it was not statistically

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significant, whereas 1 mg/kg MEKi displayed a significant suppressive effect on tumor growth in consistent with our previous finding (Fig. 4E). Remarkably, combinational treatment of anti–PD-L1 mAb with the lowest dose of MEKi (0.25 mg/kg) exhibited a sufficient antitumor effect which was more than 1 mg/kg of MEKi single treatment (Fig. 4B and Supplementary Fig. 5). In this study, we did not see any abnormalities in both body weight and behavior of the mice (Fig. 4F).

PMN-MDSC Population in the Tumor is Decreased by the Combination of MEKi With Anti–PD-1/PDL1 mAbs To further determine the effect of PD-1/PD-L1 pathway blockade in combination with MEKi in the immune cell composition of the Kras/p53-driven lung tumors, we isolated the infiltrated tumor leukocytes and studied them by flow cytometry. A combination of anti–PD-1 or anti–PD-L1 mAbs with MEKi significantly augmented the percentage of CD4þ (MEKi þ anti–PD-1: 7.29 ± 2.4%, MEKi þ anti–PD-L1: 9.04 ± 0.6%; as mean ± SEM) and, more profoundly, CD8þ T cells (MEKi þ anti–PD-1: 2.35 ± 0.28%, MEKi þ anti–PD-L1: 1.32 ± 0.2%) in the tumor in comparison with vehicle-treated control (4.27. ± 0.34% and 0.15 ± 0.02%, respectively), whereas MEKi alone did not increase these populations of CD4þ (3.72 ± 0.81%) and CD8þ (0.17 ± 0.03%) T cells in the tumor (Figs. 5A and B). The combination of either anti–PD-1 or anti–PD-L1 mAbs with MEKi significantly ablated the MDSC population (MEKi þ anti–PD-1: 21.77 ± 2.00%, MEKi þ anti– PD-L1: 20.6 ± 1.36%) in the tumor, in particular, the PMN-MDSCs, CD45þ/CD11bþ/Ly6Ghigh/Ly6Clow (MEKi þ anti–PD-1: 2.68 ± 0.19% and MEKi þ anti– PD-L1: 2.89 ± 0.18%) (Figs. 5C and E). We did not observe any differences on monocytic-MDSCs as indicated by CD45þ/CD11bþ/Ly6G-/Ly6Chigh (Fig. 5D). There is a dramatic decline in the PD-L1þ MDSCs compartment post-treatment with MEKi and either anti–PD-1 or anti–PD-L1 mAbs (Fig. 5F).

Figure 3. MEKi combined with anti–programmed death ligand 1 (PD-L1) yields antitumor efficacy and prolonged survival in the bioluminescent Trp53FloxFlox;KrasG12D/þ;Rosa26LSL-Luciferase/LSL-Luciferase (PKL) transgenic lung cancer mouse model. (A) Schematic of dosing schedule. 6- to 8-week–old PKL mice were infected by adenoviral particles encoding for Cre recombinase (Ad-Cre) through intratracheal intubation. Treatments began on day 54 after Ad-Cre induction, indicated as days 54–57 of drug treatment for MEKi alone and further days 57–80 of drug treatments for the combination of MEKi with anti–programmed death 1 (PD-1) or anti–PD-L1 antibodies (n ¼ 8 each group). (B) Representative images of gross lung tumors and hematoxylin and eosin (HE)–stained lung lobe in each treatment group. Scale bar ¼ 1 cm (lung pictures) or 1 mm (HE-stained lobe). (C) Percentages of whole lung weight per body weight in each group. (mean ± SEM; n ¼ 8; *p < 0.05; vehicle versus combination treatment groups). (D,E) Representative images of the bioluminescent in vivo (D) and its bioluminescence intensity monitoring (E) in a time-course manner (mean ± SEM; n ¼ 8; *p < 0.05). (F) Kaplan-Meier survival curve of PKL mice treated with MEKi and either anti–PD-1 or anti–PD-L1 antibodies compared to vehicle treated mice (vehicle ¼ 16, MEKi ¼ 8, MEKi þ anti– PD-L1 ¼ 9, MEKi þ anti–PD-L1 ¼ 8). Tx, treatment; IVIS, IVIS imaging software.

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These results suggest that the decreases in MDSCs and PD-L1–positive MDSCs likely lead to the abolishment of immune inhibition, as indicating the increased composition of tumor-infiltrating T cells by the combinational treatment with MEKi plus either anti–PD-1 or anti–PD-L1 mAbs groups.

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Antitumor Effect of the Combination of MEKi and Anti–PD-1/PD-L1 mAbs in PKL-Transgenic Mice is Mediated by MEKi-Induced Apoptosis and Augmentation of Tumor-Filtrating T Cells We next tested expressional status of pERK1/2 in lung tumors, which is a direct downstream of MEK signaling, and

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a proliferation marker, PCNA, to determine MEKi-mediated inhibitions of MEK signaling and tumor cell proliferation, respectively. In all the treatment groups with MEKi both single and combination, pERK1/2 expression was almost completely ablated in lung tumors when PCNA was also profoundly decreased, indicating the suppressive effect on tumor proliferation in the setting of MEK inhibition (Fig. 6A). The majority of the tumors were shrunk by the combination treatment groups. Moreover, in vehicletreated control tumors, CD3þ T cells were limited to the outer rim of the tumor, whereas MEKi-treated tumorbearing lungs exhibited infiltration of T cells into the tumor compartment (Fig. 6B). In addition, we observed that MEKi treatment not only suppressed tumor proliferation (Fig. 6A), but it also induced tumor cell apoptosis, as shown by the augmented expression of cleaved-caspase 3 in tumor bed (Fig. 6C). These results indicate that combinatorial treatment significantly ablated tumor growth and increased tumor-infiltration of T cells. Given the protective role of PD-1/PD-L1 on cancer cells from IFN-mediated cytotoxicity, we examined with PKL5-2 cells to see whether the combinational treatment of MEKi with anti–PD-L1 mAb sensitizes cancer cells to IFN-mediated apoptosis.26,27 PKL5-2 cells were exposed to MEKi and anti–PD-L1 mAb with or without either IFNg or IFN-a for 24 hours and followed by western blotting with antibodies against apoptotic markers. As expected, IFN-mediated apoptosis was dramatically induced in cells exposed to the combination as shown by increases of cleaved PARP, cleaved caspase 7, and cleaved caspase 3 (Fig. 6D). Overall, these results indicate that the combinational treatment of MEKi with antibodymediated PD-1/PD-L1 blockages synergistically suppresses KRAS-mutated lung tumor growth and also sensitize cancer cells to IFN-mediated cytotoxicity.

Discussion In this study, we have shown that a combinatorial treatment with a low dose of MEKi, trametinib, and anti–

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PD-L1 in a KRAS-mutated NSCLC could: (1) enhance Tcell infiltration in both tumor microenvironment and inside tumor bed; (2) decrease the number of Ly6Ghigh PMN-MDSCs in the tumor; and (3) suppress tumor cell proliferation and lead to apoptosis of tumor cells. Therefore, MEKi functioned as a sensitizer of previously unresponsive KRAS-mutated tumors to immunotherapy. There are two ongoing clinical trials relevant to the therapeutic regimen of trametinib and pembrolizumab (NCT 03299088, NCT03225664). To the best of our knowledge, our research is the first preclinical study that could provide rationale for the use of the combination treatment for KRAS-mutated NSCLC. Many clinical trials have evaluated the possible role of MEKi in KRAS-mutated NSCLC (Supplementary Tables 2 and 3). MEKi as single-agent treatment has revealed a limited efficacy in KRAS-mutated NSCLC.12,28 Also, the efficacy of MEKi in combination with conventional chemotherapy has been tested in randomized clinical trials.15,29 However, more than two-thirds of patients treated with the combination treatment were reported to experience grade 3 or higher adverse events. Accordingly, 46%, 28%, and 23% of the group had to receive hospitalization, dose reduction, and discontinuation, respectively, suggesting the need for dose reduction to complete a full cycle of the combination therapy.15 We observed that a combination of low-dose MEKi with anti–PD-L1 mAb showed a significant synergism of both antitumor effect and favorable survival in our mouse model. According to The U.S. Food and Drug Administration’s guideline on calculating human equivalent dose, the reduced dose of trametinib used in our research, for example, 0.25mg/kg/day for the mouse, is equivalent to 1.22 mg/day for a human who is assumed to weigh 60 kg, which is approximately half of the conventionally used dose.30 Some previous studies have shown that MEKi provoked adaptive drug resistance in KRAS-mutant lung cancer through fibroblast growth factor receptor signaling, indicating that a combination of MEKi and

Figure 4. Antitumor effect and prolonged survival benefit by the combination of MEKi with immunomodulators targeting programmed death 1 (PD-1) or programmed death ligand 1 (PD-L1) in the PKL5-2 murine lung tumor syngeneic model. (A) Schematic of dosing schedule. MEKi began on day 6 after tumor cell implantation, indicated as days 6–12 of drug treatment for MEKi alone and further days 12–33 of drug treatments for the combination of MEKi with anti–PD-1 or anti–PD-L1 antibodies. Mice (n ¼ 8 per group) were treated with MEKi at 1 mg/kg oral gavage daily, or with antibodies, a-mouse PD-1 (RMP1-14 clone) or a-mouse PD-L1 (10F.9G2 clone) monoclonal antibodies (mAbs) at 200 mg/mouse intraperitoneally twice weekly until tumors reached the endpoint of 1,000 mm3 or by study endpoint. (B) Significant decreased tumor masses by the combination of MEKi with either anti–PD-1 or anti–PD-L1 antibodies and representative bioluminescence imaging of treated mice. Tumor masses were harvested and weighted as shown by an average tumor weight per group. Results are presented as mean ± SEM, *p < 0.05. (C) Kaplan-Meier survival curves of different treatment groups (n ¼ 8). (D) Schematic of dosing schedule for the testing of lower doses MEKi in syngeneic model. Mice were initiated treatment with 0.25, 0.5, or 1 mg/kg of MEKi at 9 days after cell implantation and followed by either addition of anti–PD-L1 mAb or changing of regime at 20 days after cell implantation (n ¼ 3 to 4 per group). (E) Synergism of antitumor effect by the combinational treatment of lowest MEKi dose with anti–PD-L1 mAbs. (F) Body weight changes during the study.

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Figure 5. The combinatory therapies of MEKi with either anti–programmed death 1 (PD-1) or programmed death ligand 1 (PDL1) monoclonal antibodies (mAbs) augment tumor infiltrating T cells and ablate polymorphonuclear (PMN)–myeloid-derived suppressor cells (MDSCs) in Trp53FloxFlox;KrasG12D/þ;Rosa26LSL-Luciferase/LSL-Luciferase (PKL) syngeneic tumor beds. (A,B) Flow cytometry quantification of tumor-infiltrating immune cells including CD4þ (A) and CD8þ (B) T cells in the allografted tumors. (C–E) Flow cytometry quantification of tumor-infiltrating immune cells including CD11bþ/Gr-1high total MDSCs (C), CD11bþ/ Ly6Chigh monocytic MDSCs (D), and CD11bþ/Ly6Ghigh PMN-MDSCs (E) (mean ± SEM; n ¼ 8; *p < 0.05 versus vehicle and singletreatment groups). (F) Representative histogram of PD-L1–positive CD11bþ;Gr-1þ MDSCs after treatment with combination of MEKi with anti–PD-1 or anti–PD-L1. ns, not significant.

fibroblast growth factor receptor inhibitor would be effective.31,32 Also, there is a clinical trial that evaluated the efficacy of atezolizumab combined with cobimetinib in colorectal cancer (NCT02788279). However, they did not consider the need for dose adjustment to reduce drug toxicity–related complication when used in cancer patients. As a matter of fact, treatment-related grade 3-4 adverse events were reported in 45% of patients who received atezolizumab with cobimetinib.33 Our study

focused on the potential role of low-dose MEKi in modifying the immune-suppressive tumor microenvironment by attenuating PMN-MDSCs in the tumor bed, so that checkpoint inhibitors could be more effective. We observed that when we added either IFN-g or IFN-a to the combination treatment, for example, MEKi and anti– PD-L1 mAb, the cytotoxic effect was drastically increased in the cells compared to without IFN exposure. Consistent with other studies, the combinational treatment of

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Figure 6. The combination of MEKi with either anti–programmed death 1 (PD-1) or programmed death ligand 1 (PD-L1) antibodies shows a significant decrease in P-ERK whereas increases of highly infiltrated CD3þ T cells and apoptosis in Trp53FloxFlox;KrasG12D/þ;Rosa26LSL-Luciferase/LSL-Luciferase (PKL) lung tumors. (A) Reduction of P-ERK and PCNA in combinatory treated lung tumors as shown double immunofluorescence staining for P-ERK and PCNA. Tumor nodules were marked. (B) A significant increase of infiltrated CD3þ T cells in lung tumor treated with MEKi and anti–PD-L1 antibody compared to other groups as shown immunofluorescence staining for CD3. Hematoxylin and eosin stained image showed tumor nodules. (C) Immunohistochemistry staining for cleaved-caspase 3 in PKL lung tumor treated with the indicated agents. Eight weeks after induction lung tumor by adenoviral particles encoding for Cre recombinase, mice were administered with MEKi and either anti–PD-1 or anti–PD-L1 antibodies for 7 days and followed harvesting lung tumors. The number of cleaved-Caspase 3-positive cells were counted in tumor bed with at least 25 tumor nodules in three individual each group (mean ± SEM; *p < 0.05, **p < 0.005; Scale bar ¼ 100 mm). (D) Western blot for cleaved PARP, cleaved caspase 7, and cleaved caspase 3 in PKL 5-2 cells exposed to MEKi and anti–PD-L1 monoclonal antibodies with or without either interferon (IFN-g or IFN-a). PCNA, Proliferating cell nuclear antigen; DAPI, 40 ,6-diamidino-2-phenylindole; PARP, Poly (ADP-ribose) polymerase; P-ERK, Phospho-Extracellular signal-regulated kinase.

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MEKi and antibody-mediated PD-1/PD-L1 blockage sensitizes cancer cells to IFN-mediated apoptosis, suggesting that the combination could inhibit immune escape of cancer cells, at least in part, by synergistic enhancement of sensitivity to IFNs and cytotoxic T cell– mediated killing with suppression of MDSC.26,27 The efficacy of immunotherapy in KRAS-mutated NSCLC has not been consistently observed in previous clinical studies (Supplementary Table 4). Subgroup analysis of the CheckMate 057 trial showed that NSCLC patients harboring the KRAS mutation were more likely to benefit from nivolumab as shown by an improved overall survival compared with patients with wild-type KRAS.34 In contrast, two other clinical studies with immune checkpoint inhibitors showed that KRAS mutational status was not associated with survival benefit.20,23 A recent study corroborated that STK11/ LKB1 alterations function as a major driver of primary resistance to PD-1 blockade in KRAS-mutant NSCLC.35 Our results implicate that low-dose trametinib may degrade tumor burden by apoptosis and be followed by an increase of tumor antigens available which are recognized by T cells to mount antitumor T cell response. A recent study showed that MEKi G-38963 in combination with anti–PD-L1 mAb enhanced T cell activity against KRAS-mutated colon cancer cells in CT-26– mediated syngeneic mouse model.36,37 In melanoma cell lines, MEK inhibition with trametinib was also shown to increase the expression of apoptosis markers along with expression of human leukocyte antigen molecules.38 Simultaneously, trametinib could modify the suppressive tumor microenvironment by both attenuating MDSCs and enhancing T cell infiltration into the tumor bed. Also, a dose reduction of trametinib may prevent the possible adverse events which were observed in previous clinical trials using MEKi. There is an ongoing clinical trial, of which one of the objectives is to determine the highest tolerable dose of trametinib when given with pembrolizumab for NSCLC (NCT03225664). Together with our study, it is worthwhile to anticipate outcomes from this ongoing trial to determine the optimal dose of trametinib for future clinical trial that will enable us to evaluate the therapeutic efficacy of trametinib in combination with pembrolizumab for NSCLC. Inhibition of cytotoxic T lymphocytes in the tumor microenvironment seems to occur through the conversion of effector cells into potent immunosuppressive cell populations such as regulatory T cells and MDSCs.39,40 In mice, MDSCs have been classified as CD11bþ and Gr1þ and are made up of a heterogeneous mixture of immature myeloid cells and myeloid progenitor cells.41-44 Mouse tumor-infiltrating MDSCs have been shown to impair CD8þ T cell response and antitumor immune

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response.41 We found that MEK inhibition by trametinib reduced the accumulation of Ly6Gþ MDSCs in the tumor and modified the suppressive tumor microenvironment allowing immunotherapy with PD-1/PD-L1 blockade to properly function. KRAS-mutated cancer may especially have the propensity to recruit MDSCs. In a mouse pancreatic model, overexpression of constitutive active KRAS led to the expression of MIP-2 and MCP-1, which promoted recruitment of MDSC into the tumor stroma.45 MEK inhibition has been shown to reduce chemokines involved in the recruitment of MDSCs into the tumor and block MDSC differentiation. MEK inhibition with trametinib decreased tumor production of osteopontin, an MDSC chemotactic factor and reduced the recruitment of monocytic Ly6Cþ MDSCs into a KRAS-driven breast cancer mouse model and in a Lewis lung carcinoma model.46 We, however, did not find any differences in the monocytic MDSC population in our MEKitreated groups but rather found that MEKi reduced the PMN-MDSCs in our models. This may be due to differences in the tumor model. In addition, trametinib was also able to inhibit MDSC expansion from myeloid progenitors in the bone marrow.47 Similarly, MEK inhibitor U0126 was able to block the interleukin-11– induced MDSC differentiation.48 We investigated LUADs carrying common KRAS and/ or TP53 mutations because we identified these tumors to be inadequately infiltrated by CD8þ T cells in both humans and mice. As previously described, this model was found to be poorly immunogenic with no CD3þ T cell tumor infiltration.49 In contrast, we found that there was T cell tumor infiltration, but it was limited to the tumor edge in our mouse model. We evaluated whether the difference between our models was due to the incorporation of luciferase into our tumor model, which could act as a xenoantigen. However, in our model without luciferase, there was also T cell surrounding the tumor (Supplementary Fig. 6A). We postulate that the difference in T cell infiltration into the tumor may be due to the differences in the genetic background of the mouse models used. Pfirschke et al.49 used a mouse model on B6 or 129 backgrounds, which are of MHC haplotype b, whereas our mice were on FVB background, which is of MHC haplotype q (Supplementary Fig. 6B). Despite the difference in the level of T cell infiltration, our model remained poorly immunogenic and did not respond to single-agent immunotherapy treatment with either PD-1 or PD-L1 blockade. In conclusion, this study expands upon the emerging targeted therapy of MEK inhibition in KRAS-mutated NSCLC with trametinib by showing synergistic antitumor effect in combination with anti–PD-1 or anti–PD-L1 mAbs immunotherapies. This should prompt further

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investigation into this potential breakthrough in the treatment of the largest genotype subset of NSCLC, a leading cause of cancer mortality in the United States and worldwide.

Acknowledgments This work was supported by Beatrice Kleinberg Neuwirth Fund, National Cancer Institute grant P50CA196530 (Yale SPORE in Lung Cancer to Drs. Herbst, Chen, and Koo), R01-CA126801 (to Dr. Koo), and National Cancer Institute Cancer Center Support Grant CA-16359 (to the Yale Comprehensive Cancer Center).

Supplementary Data Note: To access the supplementary material accompanying this article, visit the online version of the Journal of Thoracic Oncology at www.jto.org and at https://doi. org/10.1016/j.jtho.2019.02.004.

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