STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment

The discovery of a series of oncogene driver mutations and the concept of oncogene addiction has changed the therapeutic approach for subsets of patients with non–small cell lung cancers (NSCLC; ref. 1). While this targeted approach for tumors with specific kinase alterations has been successful, KRAS mutation is the most common genetic alteration driving NSCLCs and remains refractory to targeted treatment strategies. KRAS-mutated NSCLCs are genomically more complex than those harboring mutated EGFR or EML4-ALK and the concurrent loss of key tumor suppressors such as TP53 or STK11 is common in KRAS-mutated lung adenocarcinomas.

STK11/LKB1 is inactivated in approximately one-third of the KRAS-mutated lung adenocarcinomas, a frequency comparable with TP53 loss in this background, though STK11 and TP53 mutations rarely overlap in KRAS-mutant lung tumors (2). Lkb1-deficient Kras-mutated (Kras/Lkb1) tumors show a more invasive and metastatic phenotype with significantly reduced survival (3), and differential drug sensitivities as compared with Kras-mutant Lkb1-wild-type tumors and Kras-mutated compound Tp53-deficient animals (4). A more metastatic phenotype in KRAS/LKB1 tumors has also been described in clinical studies (5, 6).

Recent clinical trials in NSCLC have demonstrated response to immune checkpoint blockade and nominated predictive markers for the efficacy of specific immunotherapies (7–9). Our previous work suggests that oncogenes impact immune evading mechanisms by directly activating immune checkpoints (10). Immune evasion can also be achieved by the release of proinflammatory cytokines into the tumor microenvironment that play an important role in promoting tumor growth, metastasis, and immune suppression (11, 12). Previous work has shown that Kras-mutated tumors display activation of the noncanonical IkappaB kinase TBK1 (13, 14), and the activation of this signaling pathway induces several proinflammatory cytokines, such as IL6 and CXC-chemokine ligands. Myeloid cells, especially tumor-associated macrophages (TAM) and neutrophils (TAN), support tumor cell proliferation and impede host immune surveillance through cytokine production and cell–cell interactions (15).

To elucidate how Stk11/Lkb1 (hereafter referred to as Lkb1 in the mouse model) loss affects the inflammatory phenotype in Kras-driven lung cancer, we compared immune cell populations and cytokine/chemokine profiles among Kras and Kras/Lkb1 mouse lung cancer models. We found that neutrophil-attracting soluble factors and neutrophil numbers were significantly increased and both T-cell numbers and function were significantly decreased in Lkb1-deficient tumors. Moreover, Lkb1 loss of function negatively impacted PD-L1 expression in lung tumor cells in mouse and human tumors and cell lines. By depleting the neutrophils in Kras/Lkb1–mutant mice, T-cell numbers and function were significantly improved affirming the immunosuppressive properties of this cell type. Finally, we functionally validated the therapeutic utility of blocking the cytokine feedback loop with a neutralizing anti-IL6 antibody, which resulted in an increase of T-cell numbers and function. Together, the results suggest that in the Lkb1-deficient tumors, immune evasion is achieved through suppressive myeloid cells and aberrant cytokine production and not the PD-1:PD-L1 interaction.

Mouse strains were described previously (3). Mice were dosed with 200 μg of IL6-neutralizing antibody (MP5-20F3, BioXcell), anti Ly-6G/Gr-1 antibody (RB6-8C5, BioXcell), PD-1–blocking antibody (clone 29F.1A12), and isotype controls (BioXcell) three times a week via intraperitoneal injections. MRI quantification was performed as described previously (10). Murine cell lines bearing mutated Kras and p53-loss (Kras/p53:KP) and mutated Kras and both p53 and Lkb1-loss (Kras/p53/Lkb1:KPL) were established and characterized as described previously (16). Recombinant mouse IL1α was from PeproTech.

Lung cell isolation, mononuclear cell enrichment, and characterization of immune cell populations in murine tissue samples were described previously (10). Total cell count was divided by tumor-bearing lung weight utilized for each assay. Antibodies are listed in Supplementary Methods. Intracellular staining for Ki-67, IFNγ, CTLA-4, FOXP3, and LGALS9 was performed according to the manufacturer's protocol (eBioscience and BD Biosciences). Sorting of tumor cells (CD45⁻EpCAM⁺) and neutrophils (CD45⁺CD11b⁺Ly-6G⁺) was performed on a BD FACSAria II. Gating methods for immune analysis and sorting are mentioned in Supplementary Methods.

RNA isolation from sorted cells was performed using the PicoPure RNA Isolation Kit (Life Technologies) according to the manufacturer's protocol. Ten to 100 ng of total RNA was used as input for the generation libraries using the Nugen Ovation Kit. Libraries were quality controlled on an Agilent high sensitivity DNA chip and sequencing of pooled libraries was performed on the Illumina HiSeq platform to a minimum depth of 30 million reads.

For details, see Supplementary Methods.

Human lung cancer cell lines were obtained from ATCC and used prior to 6 months of passage in culture and not further authenticated. Short hairpin RNA constructs and stable isogenic cell lines were established as described previously (17). Recombinant human IL1α was from PeproTech (18).

IHC for TUNEL and Ki-67 was performed as previously described (19). For the PROSPECT samples, 4-μm thick tissue sections were stained using an automated staining system (Leica Bond Max, Leica Microsystems), according to the standard protocols. The38 Aperio Image Analysis Toolbox (Aperio, Leica Microsystems) was used for digital analysis of images obtained from scanned slides. PDL1 clone E1L3N from Cell Signaling Technology, CD3 A0452 from Dako, and CD8 C8/144B from Thermo Scientific were used.

Tumor nodules resected from Kras and Kras/Lkb1 mice were homogenized in RIPA buffer and proteinase inhibitor (Cell Signaling Technology). Western blotting was performed as described previously (18) with anti pSTAT3, STAT3, LKB1, and actin antibodies (Cell Signaling Technology).

For details, see Supplementary Methods.

All numerical data are presented as mean ± SD. Data were analyzed using two-tailed unpaired Student t test for comparisons of two groups and one-way ANOVA with Tukey post-test for three groups. P values for the survival curves have been calculated using a log-rank test. Mann–Whitney U tests were used to assess correlation of PD-L1 and T-cell markers in human tumor samples with LKB1 status. Multivariate testing of The Cancer Genome Atlas (TCGA) data with respect to genotype and clinical factors (sex, primary tumor stage, nodal stage, metastasis stage, overall stage, age, and smoking) was performed using one-way ANOVA.

We previously showed that oncogene activation contributes to escape from immune surveillance by modulating the tumor microenvironment (10). However, the loss of tumor suppressors has not been previously investigated in this context. To elucidate how Lkb1 deficiency impacts the immune microenvironment in lung tumors, we compared the immune cell populations and cytokine profiles of Kras and Kras/Lkb1 mouse models with similar degrees of tumor burden (Supplementary Fig. S1A). We found that Lkb1-deficient tumors showed a greater variation in the number of total hematopoietic (CD45⁺) cells (Supplementary Fig. S1B) and an increase in the total CD11b⁺ myeloid cell population among three major clusters (CD11c⁻CD11b⁻, CD11c⁺CD11b⁻, CD11b⁺) in the lung (Fig. 1A). Detailed analysis of these myeloid cell populations showed that total numbers of TAN (CD11b⁺Ly-6G⁺) are significantly elevated and tumor-associated alveolar macrophages (TAM: CD11c⁺CD11b⁻CD103⁻) are significantly decreased in Kras/Lkb1 tumors compared with Kras tumors (Fig. 1A). Minor myeloid cell populations including eosinophils, Ly-6C^hi inflammatory monocytes, and CD103⁺ dendritic cells did not show significant differences (Supplementary Fig. S1C). Interestingly, the increase of neutrophils was also observed in the spleen and peripheral blood of Kras/Lkb1 mice (Fig. 1B).

To identify the cytokines and chemokines driving the immune phenotype of Lkb1-deficient tumors, we sorted CD45⁻EpCAM⁺ cells from Kras and Kras/Lkb1 lung tumors using FACS and performed mRNA sequencing. We discovered higher expression of a number of chemokines in the Kras/Lkb1 tumor cells: Ppbp [proplatelet basic protein: chemokine (C-X-C motif) ligand 7 (Cxcl7)], Cxcl3, and Cxcl5, all of which act through chemokine receptor CXCR2 on neutrophils (Supplementary Methods), and cytokines: Csf3 [colony stimulating factor 3: granulocyte colony stimulating factor (G-Csf)] and two of the IL1 family of proinflammatory cytokines, Il33 and Il1α (Fig. 1C). On the contrary, we identified a decrease in the expression of chemokine (C-C motif) ligand 5 (Ccl5) and Cxcl12 in Kras/Lkb1 tumor cells as compared with Kras. Both of these chemokines play an important role in recruiting lymphocytes and dendritic cells (20, 21) and these cell types are underrepresented in Kras/Lkb1 tumors (Supplementary Fig. S1C and S1D).

In addition to tumor cells, we sorted TAN from Kras/Lkb1 tumors and compared gene expression profiles with TAN from the uninduced normal lung from mice with the same genetic background. Analysis of mRNA sequencing revealed that TAN from the Kras/Lkb1 tumors produced elevated T-cell–suppressive factors (22) including Il10, Lgals9, Arginase 1 (Arg1), and Milk fat globulin EGF factor 8 protein (Mfge8) and the tumor-promoting cytokine, Il6, as compared with neutrophils from normal lung (Supplementary Fig. 2A).

To confirm these findings at the protein level, we analyzed CXCL7, G-CSF, and IL1α in culture supernatants from cell lines derived from mouse tumors (16). There was a significant increase in CXCL7 and G-CSF in Kras-mutated Tp53-deficient Lkb1-deficient cell lines (KPL) compared with Kras-mutated Tp53-deficient Lkb1-wild type cell lines (KP; Fig. 1D) but IL1α was under the detection limit in all cell lines (data not shown). In addition to the cytokines identified as differentially expressed in mRNA sequencing, we analyzed IL6 and IL17, two well-characterized cytokines that contribute to neutrophil accumulation and production (23, 24), and found that IL6 was significantly increased in KPL compared with KP (Fig. 1D), but IL17 was not detected (data not shown). Of the cytokines in bronchoalveolar lavage fluids (BALF), CXL7 showed significant difference in Kras/Lkb1 versus control and G-CSF, MFG-E8, and IL10 in Kras/Lkb1 versus control and Kras (Fig. 1E and Supplementary Fig. S2B). While Il6 upregulation was not apparent at the mRNA level in EpCAM⁺ Kras/Lkb1 tumors cells (data not shown), we detected the cytokine in BALFs from Kras/Lkb1 lungs and in cultured sorted CD45⁻EpCAM⁺ cells and TAN from Kras/Lkb1 tumors (Supplementary Fig. S2C). Considering the higher number of neutrophils in Kras/Lkb1 tumors as compared with Kras tumors, TANs likely play an important role in aberrant production of this cytokine in addition to tumor cells. Given that IL6 mediates its downstream effects through STAT3 (25), we measured the levels of phosphorylated STAT3. We found that Kras/Lkb1 tumor tissue had higher levels of phospho-STAT3 (pSTAT3) than the Kras tumors (Fig. 1F). These findings suggest that Lkb1 inactivation is associated with neutrophil accumulation to the immune microenvironment and overproduction of tumor-promoting cytokines.

In concordance with RNA sequencing data, IL1α showed a significant increase in BALFs in Kras/Lkb1 versus that from control (Fig. 1G). Mouse BALFs, similar to the supernatants from cultured cells, did not have detectable IL17 (data not shown). To assess whether IL1α might promote feed forward cytokine signaling in Lkb1-deficient tumors, we stimulated a Kras-mutated, p53-loss, Lkb1-loss mouse lung cancer cell line (KPL cell line) with IL1α and analyzed cytokine secretion. Among the cytokines, we detected an increase in IL6, CXCL7, and G-CSF production in a dose-dependent manner (Fig. 1H and Supplementary Fig. S3A). These results are consistent with Lkb1-loss increasing IL1α production, which then promotes the activation of IL6–STAT3 signaling.

Clinical studies have demonstrated that the density of tumor-infiltrating lymphocytes is associated with a favorable prognosis and response of immunotherapy in cancer (7–9). We found that total counts of both CD4 and CD8 T cells were significantly decreased in Kras/Lkb1 mouse tumors (Fig. 2A) as compared with Kras tumors. Infiltrating T cells showed a significantly higher expression of T-cell–inhibitory markers: Programmed cell death protein 1 (PD-1), T-cell immunoglobulin mucin-3 (TIM-3), Lymphocyte-activation gene 3 (LAG-3), and CTL-associated protein 4 (CTLA-4; Fig. 2B). We also confirmed expression of the ligand for TIM-3, LGALS9, in both tumor cells and TAN from Kras/Lkb1 tumors by flow cytometry (Supplementary Fig. S2D). The ratio of regulatory T cells (FOXP3⁺) to total CD4 T cells was also significantly increased in Kras/Lkb1 tumor as compared with Kras tumors (Fig. 2B). We evaluated T-cell function in Kras/Lkb1 and Kras tumors with similar levels of disease (Supplementary Fig. S3B) and found significantly less IFNγ and Ki-67 expression in total CD4 and CD8 T cells from the Kras/Lkb1 tumors than those from Kras tumors (Fig. 2C). Thus, Lkb1 inactivation is associated with reduced T-cell number and increased markers of T-cell exhaustion.

To understand the role of neutrophils in this model, we used a neutrophil-depleting (anti-Ly-6G/Gr-1:RB6-8C5) antibody in mice with established tumors (Supplementary Fig. S4A and S4B). Kras/Lkb1 mice treated with anti Ly-6G/Gr-1 antibody for 1 or 2 weeks showed a significant reduction of TAN and of IL6 and G-CSF in BALFs (Supplementary Fig. S4C and S4D), resulting in a significant increase in total CD8 T-cell numbers, proliferation (Ki-67⁺), and T-cell function represented by IFNγ production (Supplementary Fig. S4E).

PD-L1 expression on tumor cells is a biomarker associated with a response to PD-1 blockade treatment (7, 10). Lkb1-deficient tumor cells expressed significantly lower levels of PD-L1 in CD45⁻EpCAM⁺ cells as compared with Kras tumor cells (Fig. 2D). PD-L1 expression is influenced by a variety of factors that include non–cell-autonomous factors such as release of IFNγ from T cells (26) in the tumor microenvironment in vivo. To dissect the intrinsic role of Lkb1 inactivation on PD-L1 expression specifically in tumor cells, we analyzed PD-L1 expression in cultured cell lines derived from mouse tumors of the KP and KPL genotypes. PD-L1 levels were significantly lower in KPL as compared with KP (Fig. 2E). To confirm that our findings in mouse models and cell lines were applicable to humans, we studied human lung cancer cell lines with endogenous KRAS mutation and wild-type or inactivated LKB1. We either performed knockdown of LKB1 using shRNA or reconstituted LKB1 with wild-type (WT) or kinase dead (KD) LKB1 to develop isogenic cell lines (wild -type cells: H441, H1792, and LKB1-mutant cell: A549; Supplementary Fig. S5A). PD-L1 expression was lower in both of the LKB1 WT lines when expressing sh-LKB1 (Fig. 2F). Expression of LKB1 (WT and KD) in the LKB1-deficient A549 cell line resulted in a modest increase in PD-L1 levels (Supplementary Fig. S5B). These data suggest LKB1 inactivation decreased PD-L1 levels independent of IFNγ.

We next assessed the cytokine and chemokine profiles in human isogenic cell lines to determine whether similar patterns would be observed. We analyzed culture supernatants from these cell lines and found that IL6 and G-CSF were significantly increased in LKB1-inactivated cells as compared with LKB1-intact cells (Fig. 3A and Supplementary Fig. S5C). There was also a significant increase of CXCL7 that was only detected in A549 cells (Supplementary Fig. S5C). IL1α stimulation of these cell lines led to an increase in IL6, G-CSF, and CXCL7 in a dose-dependent manner (Fig. 3B and Supplementary Fig. S5D), which was consistent with the Lkb1-deficient mouse cell line data (Fig. 1D). We also found that IL6 induction by IL1α stimulation was more pronounced in LKB1-deficient cell lines as compared with LKB1-intact cell lines (Supplementary Fig. S5E) except for H1792, which showed modest stimulation. This line has high baseline IL1α production (Fig. 3A) and the shRNA only partially knocked down LKB1 (Supplementary Fig. S5A), which makes this result difficult to interpret.

Analysis of lung cancer cell lines from The Cancer Cell Line Encyclopedia (CCLE; ref. 27) confirmed that PD-L1 expression is significantly lower in cell lines with LKB1 mutation (32 LKB1 WT and 4 LKB1-mutant cell lines; Fig. 3C), although the small number of LKB1-mutant lines precluded any significant associations among the immune-related genes displayed in Fig. 1C and Supplementary Fig. S2A and LKB1 status when corrected for multiple hypotheses. In addition, analysis of KRAS-mutated lung adenocarcinomas from The Cancer Genome Atlas (TCGA-52 LKB1 WT, 15 LKB1 mutant; ref. 2) showed that PD-L1 expression was significantly reduced in LKB1-mutated NSCLCs (Fig. 3C). In a multivariate analysis of the TCGA dataset with respect to LKB1 status and clinical factors, PD-L1 and LKB1 status were also significantly associated (P = 0.005). To validate these findings in an independent dataset, PD-L1 mRNA was assessed in the MD Anderson PROSPECT (Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax and Therapeutic Target Identification) cohort (MDACC-108 LKB1 WT, 44 LKB1-MUTANT cases). We again observed an association among PD-L1 expression and LKB1 status. We also validated and quantitated the expression of PD-L1 at the protein level by reverse-phase protein arrays (RPPA) in 106 cases from the MDACC cohort and detected significantly lower levels of PD-L1 in LKB1-mutant tumors (Fig. 3D). As the current clinical standard detection method for the PD-L1 expression is IHC, we confirmed the difference in PD-L1 expression by IHC (Fig. 3E).

Next, to evaluate the functional effect of LKB1-loss in the tumor microenvironment in patient tumors, we analyzed the T-cell infiltrate in the tumors by IHC. In 19 LKB1 WT and 11 LKB1-mutant tumors, total T-cell (CD3⁺) and CD8 T-cell counts and densities were significantly lower in LKB1-inactivated tumors as compared with LKB1-intact tumors (Fig. 3F), In sum, observations in patient cell lines and tumor samples are consistent with our findings in Kras/Lkb1 mice, suggesting that LKB1 mutation negatively regulates PD-L1 expression and reduces CD8 T-cell infiltration.

Supporting the notion that PD-L1 expression in tumor cells is critical in the response to PD-1 blockade, treatment of the Kras/Lkb1 mouse model with a PD-1 blocking antibody did not show a significant treatment response (Fig. 4A). Given our observation of elevated IL1α and IL6 in BALFs (Fig. 1E and G) and aberrant activation of pSTAT3 in tumor nodules (Fig. 1F) from Kras/Lkb1 mice as compared with Kras mice, we hypothesized that targeting aberrant cytokine production could be a rational therapeutic strategy in Kras/Lkb1–mutant tumors. To evaluate the in vivo efficacy of IL6 blockade, we treated Kras/Lkb1 mice with a neutralizing IL6 antibody (MP5-20F3). The therapeutic anti-IL6 antibody significantly inhibited tumor progression as compared with anti PD-1 antibody (Fig. 4A). In addition, IL6 antibody–treated mice showed significantly improved survival as compared with control mice (Fig. 4B). However, treatment of Kras/Lkb1 tumors with other checkpoint-blocking antibodies against CTLA-4 or a combination of PD-1 and TIM-3 also did not demonstrate any efficacy (data not shown). Taken together, these findings suggest that Lkb1-loss results in a T-cell–suppressed environment as a consequence of autocrine and neutrophil-induced cytokine production and not engagement of the PD-L1:PD-1 immune checkpoint.

To further investigate the effect of IL6–neutralizing antibody on the immune profile of Kras/Lkb1 tumors, we treated the mice for 2 weeks and then performed immune and histologic analyses (Supplementary Fig. S6A). There was a significant reduction in detectable IL6 as expected as well as G-CSF in BALFs from the treated mice (Fig. 4C). In keeping with the neutrophil-attracting cytokines and chemokines observed in BALFs, there was a significant reduction in the counts of total TAN with IL6 neutralization (Fig. 4D), resulting in functional recovery of T cells (Fig. 4E). Treated tumors also exhibited elevation of CD4 T cells, CD8 T cells, and TAM to levels that are comparable with the Lkb1 wild-type tumors (Supplementary Fig. S6B). In addition to the immune-related effects, IL6 antibody–treated tumor cells exhibited significantly less proliferation and increased apoptosis (Fig. 4F). Although therapeutic IL6 blockade improved T-cell function, concurrent therapy combining anti-IL6 and PD-1 treatment did not demonstrate additional benefit as compared with IL6 blockade alone in terms of survival (Supplementary Fig. S6C). This suggests a need to further define contexts in which cytokine suppression and immune checkpoint blockade might be utilized together to enhance therapeutic benefit as compared with either treatment alone.

Oncogenes and tumor suppressors promote self-sufficient signaling for autonomous proliferation of tumor cells. Recent work has shown that oncogenic mutations alter the tumor microenvironment, cause immune suppression, and can impact the response to immune-modulating treatment strategies (10). Somatic mutations also produce neoantigens, which are recognized by the immune system and mediate sensitivity of the tumors to immunotherapies (28–30). Here, we have shown that inactivation of the tumor suppressor gene STK11/LKB1 causes dramatic changes in the tumor microenvironment in addition to the previously reported effects on cell cycle, metabolism, differentiation, polarity, and other cellular pathways (16, 31).

We have shown that LKB1 inactivation promoted the production of proinflammatory cytokines CXCL7, G-CSF, and IL6 in both mouse tumors and cell lines, which contributes to neutrophil accumulation. Elevation of the proinflammatory cytokine IL1α was confirmed in vivo but not robustly in cell culture supernatants from Lkb1-deficient cells. Previous studies have shown that IL1α is released only under specific conditions including necrotic cell death and inflammasome activation (32, 33), suggesting that the release of IL1α could be caused by necrotic cell death in Kras/Lkb1 tumor microenvironment and that this facilitates the activation of IL6–STAT3 signaling pathway in Kras/Lkb1 tumors together with IL6, producing neutrophil accumulation.

The tumor microenvironment in Kras/Lkb1 tumors displayed characteristics of T-cell suppression with fewer lymphocytes, higher levels of checkpoint receptor expression in those, and an increase in TANs with suppressive properties compared with Kras tumors. TANs expressing high levels of Il10, Arginase1, and Mfge8 that have been implicated in T-cell suppression and Treg induction (15, 22, 34). Depleting TAN using an anti Ly-6G/Gr-1 antibody improved T-cell function. Although the role of TAN in suppressing T-cell function is controversial (35), TAN appear to act in an immunosuppressive fashion in this context, suggesting that therapies suppressing TAN should be further explored as immunomodulatory therapies.

Moreover, we found that Lkb1 inactivation caused a decrease in PD-L1 levels on tumor cells from Kras/Lkb1 tumors and in cultured cells from mice and patients. Conforming to the previous observations proposing an association of tumor cell PD-L1 expression, the magnitude of T-cell accumulation in the tumors and response to PD-1 blockade (7, 8, 36), treatment with a PD-1–blocking antibody did not show efficacy in the treatment of the Kras/Lkb1 mouse model. A recent study (30) as well as our own institutional experience suggest that while KRAS-mutated patients respond favorably to PD-1 blockade (PFS of 15 months on pembrolizumab) this is not seen in patients with LKB1 mutations although large cohorts will be needed to define genotype–response associations in detail. In addition, contributing to low PD-1:PD-L1 levels in Kras/Lkb1 tumors is the greater proportion of TAN that express low levels of PD-L1 as compared with TAM. The coordinated role of PD-L1 expression in the tumor cells and the cells constituting the immune microenvironment in this model requires further study to define improved immunotherapeutic strategies.

Treatment of Kras/Lkb1 tumors with an IL6–blocking antibody decreased tumor cell proliferation and increased T-cell function, resulting in a therapeutic effect in Kras/Lkb1 mouse model while immune checkpoint blockade was not efficacious. These data suggest that mouse models can be used to model the tumor microenvironment and predict response to novel immune-modulating treatment strategies based on rational predictions from studies of the tumor microenvironment. Future studies will examine whether cytokines other than IL6 also contribute to STAT3 activation (37), and whether combined cytokine blockade (e.g., with IL1 neutralization) will lead to more durable therapeutic effects. Although we found that neutralizing IL6 antibodies improved T-cell number and function in Lkb1-deficient tumors, the combination of anti PD-1 plus anti IL6 antibodies did not improve outcome when given concurrently. There may be technical limitations to this considering both of the antibodies are Rat IgG isotype and combination treatment can lead to antibody neutralization. Furthermore, dosing schedules of cytokine suppression and combinations with other therapeutic agents such as immune checkpoint blockade will need to be studied.

In summary, we have presented a novel set of findings that suggest that not only oncogene driver mutations but also tumor suppressor gene mutations can modify the immune microenvironment in lung cancer. In this example, focusing on Lkb1 loss, we observed a marked increase in inflammatory cytokines that recruited neutrophils and inhibited the function of T cells. We also showed that PD-1 checkpoint blockade was ineffective in Lkb1-mutant cancers, whereas targeting IL6 displayed a significant albeit short-lived treatment response in the Kras/Lkb1 model. These findings suggest that IL6-dependent signaling activation can be a therapeutic target in Lkb1-deficient Kras-driven lung tumors and potentially other tumors with high levels of IL6, and also suggest targeting aberrant inflammation by inhibiting cytokine signaling may represent a promising immunotherapeutic strategy in selected patients.

M. Hellmann reports receiving a commercial research grant from Bristol-Myers Squibb and is a consultant/advisory board member for Bristol-Myers Squibb, Genentech, Third Rock Ventures, and Inovio Pharmaceuticals. J.V. Heymach reports receiving other commercial research support from AstraZeneca, GlaxoSmithKline, Bayer and is a consultant/advisory board member for Genentech, AstraZeneca, Novartis, GlaxoSmithKline, Lilly, Boerhinger Ingelheim, Synta, and Exelixis. F.S. Hodi is a consultant in Merck and reports receiving a commercial research grant from Bristol Myers Squibb and Merck (to institution). G.J. Freeman has ownership interest (including patents) in Merck, Bristol Myers Squibb, Roche (immediate family member), EMD Serono, Amplimmune, Boehringer Ingelheim, Novartis (immediate family member) and is a consultant/advisory board member for Novartis (immediate family member), Roche, Bristol Myers Squibb, Eli Lilly, and Surface Oncology (immediate family member). D.A. Barbie is a consultant/advisory board member for N-of-One. G. Dranoff is a global head, exploratory immune-oncology in Novartis. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S. Koyama, E.A. Akbay, G.S. Herter-Sprie, P.E. Fecci, J.V. Heymach, G. Dranoff, P.S. Hammerman, K.-K. Wong

Development of methodology: S. Koyama, E.A. Akbay, A.R. Aref, F. Skoulidis, G.J. Freeman

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Koyama, E.A. Akbay, F. Skoulidis, G.S. Herter-Sprie, K.A. Buczkowski, Y. Liu, M.M. Awad, E.R. Parra Cuentas, I.I. Wistuba, M. Soucheray, H. Asahina, S. Kitajima, A. Altabef, J.D. Cavanaugh, K. Rhee, P.E. Fecci, T. Shimamura, M. Hellmann, J.V. Heymach, F.S. Hodi, D.A. Barbie, K.-K. Wong

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Koyama, E.A. Akbay, Y.Y. Li, F. Skoulidis, G.S. Herter-Sprie, K.A. Buczkowski, Y. Liu, M.M. Awad, W.L. Denning, L. Diao, J. Wang, E.R. Parra Cuentas, I.I. Wistuba, S. Kitajima, F.S. Hodi, D.A. Barbie, G. Dranoff, P.S. Hammerman

Writing, review, and/or revision of the manuscript: S. Koyama, E.A. Akbay, Y.Y. Li, F. Skoulidis, G.S. Herter-Sprie, K.A. Buczkowski, H. Zhang, M. Hellmann, J.V. Heymach, F.S. Hodi, G.J. Freeman, G. Dranoff, P.S. Hammerman, K.-K. Wong

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G.S. Herter-Sprie, Y. Liu, K. Rhee, P. Gao, H. Zhang, J.V. Heymach, G.J. Freeman, P.S. Hammerman

Study supervision: D.A. Barbie, G. Dranoff, P.S. Hammerman, K.-K. Wong

Other (pathology involving in the data analysis): E.R. Parra Cuentas

Other (Western blot analysis): T. Thai

Other (construction of cell line models): T. Shimamura

The authors thank Suzan Lazo-Kallanian, John Daley, Kristen Cowens, and Steven Paul for help with flow cytometry anlaysis, Christine Lam for tissue processing, Mei Zhang for IHC, Xiaoen Wang for helping with mouse studies, and the Dana Farber Center for Cancer Genome Discovery for RNA sequencing.

P.S. Hammerman is supported by a Clinical Investigator Award from the Damon Runyon Cancer Research Foundation, a Developmental Project Grant from NCI P50 CA090578 and the Starr Consortium for Cancer Research. K.-K. Wong is supported by NIH/NCI P01 CA120964, 5R01CA163896-04, 1R01CA195740-01, 5R01CA140594-07, 5R01CA122794-10, and 5R01CA166480-04 grants and support from Gross-Loh Family Fund for Lung Cancer Research and Susan Spooner Family Lung Cancer Research Fund at Dana-Farber Cancer Institute. P.S. Hammerman, K.K. Wong, J.V. Heymach, and M.D. Hellman are supported by a Stand Up To Cancer - American Cancer Society Lung Cancer Dream Team Translational Research Grant (Grant Number: SU2C-AACR-DT17-15). Stand Up To Cancer is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. S. Koyama is supported by Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship Award and The Kanae Foundation for the Promotion of Medical Science Fellowship Award. G.S. Herter-Sprie was supported by the Deutsche Forschungsgemeinschaft (HE 6897/1-1) and the Claudia Adams Barr Program for Innovative Cancer Research. T. Shimamura is supported by Uniting Against Lung Cancer Legacy Program and American Cancer Society Research Scholar Award. G.J. Freeman is supported by NIH R01AI08995.