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Gene-Specific Primers and Flow Antibodies

Supplemental Figures S1-S6

LKB1 inactivating mutations are commonly observed in patients with KRAS-mutant non–small cell lung cancer (NSCLC). Although treatment of NSCLC with immune checkpoint inhibitors (ICI) has resulted in improved overall survival in a subset of patients, studies have revealed that co-occurring KRAS/LKB1 mutations drive primary resistance to ICIs in NSCLC. Effective therapeutic options that overcome ICI resistance in LKB1-mutant NSCLC are limited. Here, we report that loss of LKB1 results in increased secretion of the C–X–C motif (CXC) chemokines with an NH2-terminal Glu-Leu-Arg (ELR) motif in premalignant and cancerous cells, as well as in genetically engineered murine models (GEMM) of NSCLC. Heightened levels of ELR+ CXC chemokines in LKB1-deficient murine models of NSCLC positively correlated with increased abundance of granulocytic myeloid-derived suppressor cells (G-MDSC) locally within the tumor microenvironment and systemically in peripheral blood and spleen. Depletion of G-MDSCs with antibody or functional inhibition via all-trans-retinoic acid (ATRA) led to enhanced antitumor T-cell responses and sensitized LKB1-deficent murine tumors to PD-1 blockade. Combination therapy with anti–PD-1 and ATRA improved local and systemic T-cell proliferation and generated tumor-specific immunity. Our findings implicate ELR+ CXC chemokine-mediated enrichment of G-MDSCs as a potential mediator of immunosuppression in LKB1-deficient NSCLC and provide a rationale for using ATRA in combination with anti–PD-1 therapy in patients with LKB1-deficient NSCLC refractory to ICIs.Significance:These findings show that accumulation of myeloid-derived suppressor cells in LKB1-deficient non–small cell lung cancer can be overcome via treatment with all-trans-retinoic acid, sensitizing tumors to immunotherapy.

Conditional genetically engineered mouse models (GEMMs) of non-small cell lung cancer (NSCLC) harbor common oncogenic driver mutations of the disease, but in contrast to human NSCLC these models possess low tumor mutational burden (TMB). As a result, these models often lack tumor antigens that can elicit host adaptive immune responses, which limits their utility in immunotherapy studies. Here, we establish Kras-mutant murine models of NSCLC bearing the common driver mutations associated with the disease and increased TMB, by in vitro exposure of cell lines derived from GEMMs of NSCLC [KrasG12D (K), KrasG12DTp53−/−(KP), KrasG12DTp53+/−Lkb1−/− (KPL)] to the alkylating agent N-methyl-N-nitrosourea (MNU). Increasing the TMB enhanced host anti-tumor T cell responses and improved anti-PD-1 efficacy in syngeneic models across all genetic backgrounds. However, limited anti-PD-1 efficacy was observed in the KPL cell lines with increased TMB, which possessed a distinct immunosuppressed tumor microenvironment (TME) primarily composed of granulocytic myeloid-derived suppressor cells (G-MDSCs). This KPL phenotype is consistent with findings in human KRAS-mutant NSCLC where LKB1 loss is a driver of primary resistance to PD-1 blockade. In summary, these novel Kras-mutant NSCLC murine models with known driver mutations and increased TMB have distinct TMEs and recapitulate the therapeutic vulnerabilities of human NSCLC. We anticipate that these immunogenic models will facilitate the development of innovative immunotherapies in NSCLC.

Chronic inflammation facilitates tumor progression. We discovered that a subset of non-small cell lung cancer cells underwent a gradually progressing epithelial-to-mesenchymal (EMT) phenotype following a 21-day exposure to IL-1β, an abundant proinflammatory cytokine in the at-risk for lung cancer pulmonary and the lung tumor microenvironments. Pathway analysis of the gene expression profile and in vitro functional studies revealed that the EMT and EMT-associated phenotypes, including enhanced cell invasion, PD-L1 upregulation, and chemoresistance, were sustained in the absence of continuous IL-1β exposure. We referred to this phenomenon as EMT memory. Utilizing a doxycycline-controlled SLUG expression system, we found that high expression of the transcription factor SLUG was indispensable for the establishment of EMT memory. High SLUG expression in tumors of lung cancer patients was associated with poor survival. Chemical or genetic inhibition of SLUG upregulation prevented EMT following the acute IL-1β exposure but did not reverse EMT memory. Chromatin immunoprecipitation and methylation-specific PCR further revealed a SLUG-mediated temporal regulation of epigenetic modifications, including accumulation of H3K27, H3K9, and DNA methylation, in the CDH1 (E-cadherin) promoter following the chronic IL-1β exposure. Chemical inhibition of DNA methylation not only restored E-cadherin expression in EMT memory, but also primed cells for chemotherapy-induced apoptosis.

Chronic inflammation facilitates tumor progression. We discovered that a subset of non-small cell lung cancer cells underwent a gradually progressing epithelial-to-mesenchymal (EMT) phenotype following a 21-day exposure to IL-1β, an abundant proinflammatory cytokine in the at-risk for lung cancer pulmonary and the lung tumor microenvironments. Pathway analysis of the gene expression profile and in vitro functional studies revealed that the EMT and EMT-associated phenotypes, including enhanced cell invasion, PD-L1 upregulation, and chemoresistance, were sustained in the absence of continuous IL-1β exposure. We referred to this phenomenon as EMT memory. Utilizing a doxycycline-controlled SLUG expression system, we found that high expression of the transcription factor SLUG was indispensable for the establishment of EMT memory. High SLUG expression in tumors of lung cancer patients was associated with poor survival. Chemical or genetic inhibition of SLUG upregulation prevented EMT following the acute IL-1β exposure but did not reverse EMT memory. Chromatin immunoprecipitation and methylation-specific PCR further revealed a SLUG-mediated temporal regulation of epigenetic modifications, including accumulation of H3K27, H3K9, and DNA methylation, in the CDH1 (E-cadherin) promoter following the chronic IL-1β exposure. Chemical inhibition of DNA methylation not only restored E-cadherin expression in EMT memory, but also primed cells for chemotherapy-induced apoptosis.

Despite recent advances in lung cancer immunotherapy, a major obstacle to the progress in the field is the lack of preclinical models that recapitulate the genetic and immunologic complexity of human disease. Conditional genetically engineered mouse models (GEMMs) of non-small cell lung cancer (NSCLC) harbor the common oncogenic mutations of the disease, but these models possess low tumor mutational burden (TMB), which limits their utility in immunotherapy studies. Here, we establish novel Kras-mutant murine models of NSCLC bearing common genetic alterations associated with the disease and increased TMB, by in vitro exposure of cell lines derived from GEMMs of NSCLC [KrasG12D (K), KrasG12DTp53−/− (KP), KrasG12DTp53+/−Lkb1−/− (KPL)] to the alkylating agent N-methyl-N-nitrosourea (MNU). Increased TMB was associated with enhanced anti-tumor T cell responses and improved anti-PD-1 efficacy in syngeneic models, across all genetic backgrounds. However, anti-PD-1 efficacy was comparatively modest in the KPL cell lines with increased TMB, which possessed a distinct immunosuppressed tumor microenvironment (TME) primarily composed of granulocytic myeloid-derived suppressor cells (G-MDSCs). This phenotype is consistent with findings in human NSCLC where LKB1 loss is a driver of primary resistance to PD-1 blockade. In summary, these novel Kras-mutant murine NSCLC models bearing common co-occurring mutations with increased TMB possess clinically relevant TMEs and recapitulate the genetic complexity and therapeutic vulnerabilities of human NSCLC. We anticipate that these immunogenic models will facilitate the development of novel immunotherapies in NSCLC.

OBJECTIVES:Adequate assessment of fluid responsiveness in shock necessitates correct interpretation of hemodynamic changes induced by preload challenge. This study evaluates the accuracy of point-of-care Doppler ultrasound assessment of the change in carotid corrected flow time induced by a passive leg raise maneuver as a predictor of fluid responsiveness. Noninvasive cardiac output monitoring (NICOM, Cheetah Medical, Newton Center, MA) system based on a bioreactance method was used. DESIGN:Prospective, noninterventional study. SETTING:ICU at a large academic center. PATIENTS:Patients with new, undifferentiated shock, and vasopressor requirements despite fluid resuscitation were included. Patients with significant cardiac disease and conditions that precluded adequate passive leg raising were excluded. INTERVENTIONS:Carotid corrected flow time was measured via ultrasound before and after a passive leg raise maneuver. Predicted fluid responsiveness was defined as greater than 10% increase in stroke volume on noninvasive cardiac output monitoring following passive leg raise. Images and measurements were reanalyzed by a second, blinded physician. The accuracy of change in carotid corrected flow time to predict fluid responsiveness was evaluated using receiver operating characteristic analysis. MEASUREMENTS AND MAIN RESULTS:Seventy-seven subjects were enrolled with 54 (70.1%) classified as fluid responders by noninvasive cardiac output monitoring. The average change in carotid corrected flow time after passive leg raise for fluid responders was 14.1 ± 18.7 ms versus -4.0 ± 8 ms for nonresponders (p < 0.001). Receiver operating characteristic analysis demonstrated that change in carotid corrected flow time is an accurate predictor of fluid responsiveness status (area under the curve, 0.88; 95% CI, 0.80-0.96) and a 7 ms increase in carotid corrected flow time post passive leg raise was shown to have a 97% positive predictive value and 82% accuracy in detecting fluid responsiveness using noninvasive cardiac output monitoring as a reference standard. Mechanical ventilation, respiratory rate, and high positive end-expiratory pressure had no significant impact on test performance. Post hoc blinded evaluation of bedside acquired measurements demonstrated agreement between evaluators. CONCLUSIONS:Change in carotid corrected flow time can predict fluid responsiveness status after a passive leg raise maneuver. Using point-of-care ultrasound to assess change in carotid corrected flow time is an acceptable and reproducible method for noninvasive identification of fluid responsiveness in critically ill patients with undifferentiated shock.

Dysregulated inflammation dictated by oncogene/tumor suppressor mutations can often lead to an immune suppressive tumor microenvironment (TME). Lung tumors with LKB1 mutations account for 30% of KRAS-mutant non-small cell lung cancer (NSCLC). These tumors are particularly aggressive and resistant to immunotherapy despite a high mutational burden (TMB). The mechanisms of this impaired immunogenicity remain obscure. Here, we report that LKB1 loss leads to a heightened secretion of the ELR(+) CXC chemokines, including CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8, in premalignant and cancerous cells in vitro. Among multiple NSCLC cell lines, cancer cells with both KRAS and LKB1 mutations tend to express higher levels of these ELR(+) CXC chemokines. Consistently, ectopic expression of LKB1 in LKB1-null cancer cells decreases the chemokine production. In a genetically-engineered mouse model, an elevation of these chemokines is also observed in KrasG12D;Lkb1-/- (KL) tumors compared to their KrasG12D (K) or KrasG12D;Tp53-/- (KP) counterparts. Mechanistic studies reveal that the LKB1-MARK axis regulates these chemokines in the premalignant cells. Immune phenotyping of KL or KrasG12D;Tp53+/-;Lkb1-/- (KPL) tumors in vivo demonstrates significantly increased granulocytic myeloid-derived suppressor cells (G-MDSCs), which harbor high levels of reactive oxygen species (ROS). Utilizing a syngeneic murine lung cancer model with a high TMB, we demonstrate that KPL tumors have a minimal response to anti-PD-1 therapy compared to KP tumors. Therefore, we hypothesize that G-MDSCs may cause resistance to anti-PD-1 monotherapy in KPL tumors. We find that inhibition of the G-MDSCs either by depletion or by reduction of ROS can potentiate the anti-PD-1 response and subsequently eliminate the KPL tumors. Investigation of the TME reveals that G-MDSC depletion primes tumor-infiltrating lymphocytes (TILs) for activation: there is an increased percentage of TILs with high Ki67 and PD-1 expression and an increased percentage of antigen presenting cells (macrophages and dendritic cells) with high PD-L1 expression. Combination with anti-PD-1 therapy enhances the function of these TILs, evidenced by IFN-γ and TNF-α secretion. Re-challenge of these mice three months after the initial combination therapy leads to a rapid tumor rejection, suggesting a durable systemic anti-tumor immune response. In conclusion, we find that LKB1 deficiency leads to an increased ELR(+) CXC chemokine production and tumor infiltration of G-MDSCs. Inhibition of G-MDSC enhances the efficacy of anti-PD-1 blockade in LKB1-deficient tumors bearing a high TMB. Citation Format: Rui Li, Ramin Salehi-Rad, Milica Momcilovic, Stephanie Ong, Raymond Lim, Zi Ling Huang, Linh Tran, Zhe Jing, Manash Paul, Michael Teitell, John Minna, David Shackelford, Krysan Kostyantyn, Bin Liu, Steven Dubinett. Inhibition of chemokine-induced myeloid cells potentiates the anti-PD-1 response in KRAS/LKB1 mutant non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1016.

Chronic inflammation facilitates tumor progression. Here, we found that chronic exposure of non-small cell lung cancer (NSCLC) cells to IL-1β, a critical cytokine in lung tumorigenesis, induces epithelialto-mesenchymal (EMT) memory, a prolonged EMT program in the absence of the original inflammatory stimulus. Induction of high SLUG expression is indispensable for the establishment of the memory phenotypes. However, SLUG is not required to maintain EMT memory once it is established. A temporal regulation of epigenetic modifications, including histone modifications and DNA methylation, drives the downregulation of Ecadherin in IL-1β-induced EMT. Inhibition of DNA methylation not only reverses E-cadherin suppression in EMT memory, but also primes these mesenchymal cells for chemotherapy-induced apoptosis. These findings enhance our understanding of the distinct molecular events between acute and chronic inflammation and suggest epigenetic modifications as a potential mechanism for temporal-spatial regulation of EMT during metastasis. Funding Statement: Funding sources for S.M.D.: NCI 1U01CA196408 Integrated Molecular, Cellular, and Imaging Characterization of Screen-detected Lung Cancer; NIH/NCI EDRN 1U01CA214182; DOD W81XWH-17-1-0399; Stand Up to Cancer AACR-DT23-17; Medical Research Funds from the Department of Veteran Affairs and NIH/NCATS UL1TR001881. Declaration of Interests: S.M.D. serves on the scientific advisory boards for Cynvenio Biosystems, T-Cure Bioscience, EarlyDx, Inc and the Johnson and Johnson Lung Cancer Initiative. Ethics Approval Statement: Not required.

Purpose: Passive leg raise (PLR), in combination with technologies capable of capturing stroke volume changes, has been widely adopted in the management of shock. However, dedicated evaluation of safety, feasibility, and receptiveness of patients and nursing staff to PLR maneuver is missing. Methods: A noninterventional, prospective trial recruited adult patients with onset of undifferentiated shock within 24 hours with persistent vasopressor requirements despite fluid resuscitation. A standardized PLR maneuver was used to compare two noninvasive hemodynamic monitoring systems, each without significant impact on the performance of the maneuver. Safety and efficacy of the PLR were evaluated via subjective and objective measures. Objective measures of patient comfort and tolerance were evaluated through changes in vital signs, sedation, and analgesia requirements. Nurses and awake patients completed surveys on their experience. Results: Seventy-nine patients were enrolled. Testing was aborted in 2 cases for medical reasons (one patient developed rapid atrial fibrillation, second had profound desaturation). Of all, 5.4% of patients required additional vasopressor support after completion of the PLR maneuver due to persistent hypotension and 4.1% of patients required additional sedation. Among awake patients (N = 35), 6% reported pain and 29% reported discomfort. A total of 11% of nurses reported minor technical difficulties with the maneuver. Conclusion: Passive leg raise maneuver leads to a few serious but reversible complications in a selected population of hemodynamically unstable patients. Although it provides relevant diagnostic information, it may impact patient care. Treating physician should be aware of infrequent but possible complications and appreciate the impact of the maneuver on patients’ comfort and nursing workload.

Checkpoint inhibitors such as PD-L1/PD-1 blockade have rapidly integrated into the paradigm of NSCLC treatment. However, a majority of patients do not benefit from monotherapy with checkpoint inhibitors. High tumor mutational burden (TMB), along with pre-existing intratumoral T cell infiltration and baseline high PD-L1 expression, predicts response to checkpoint blockade. Furthermore, a recent retrospective study identified LKB1 alterations as the most prevalent genomic driver of resistance to PD-1 axis inhibitors in KRAS-mutant lung adenocarcinoma. In this study, we investigate the mechanisms underlying LKB1-mediated immunosuppression in NSCLC. We show that loss of LKB1 in human bronchial epithelial cells (HBECs) and NSCLC cells leads to increased secretion of CXCR2 ligands, including CXCL1, CXCL2, CXCL3, CXCL5 and CXCL8. These CXCR2 ligands are also elevated in LKB1-deficient tumors from patient-derived xenografts and genetically-engineered murine models. We find abundant tumor infiltrating MDSCs in murine Lkb1-deficient NSCLC, consistent with the capacity for CXCR2 ligands to recruit MDSCs. MDSCs mediate potent immune suppressive activities at multiple levels including release of immunosuppressive cytokines, recruitment of regulatory T cells (Tregs), inhibition of CD8 T cell tumor infiltration and upregulation of PD-L1 expression. Although MDSC depletion activates interferon gamma signaling and decreases systemic Tregs in murine KrasK12D;Tp53-/-;Lkb1-/- (KPL) tumors, it does not sensitize KPL tumors to anti-PD-1 therapy. One of the major challenges in the preclinical assessment of lung cancer immunotherapy is that the commonly utilized murine models lack the mutational burden of human NSCLC. To assess this combination therapy in the context of a mutational burden that more accurately reflects the clinical disease, we generated tumors with high TMB by exposing KPL cells in vitroto the tobacco carcinogen N-methyl-N-nitrosourea. In the context of high TMB, MDSC depletion demonstrates remarkable anti-tumor effects in combination with anti-PD-1 therapy. Finally, we delineate the regulation of CXCR2 ligands by LKB1 which is dependent on the MARK-mediated NF-κB pathway. In conclusion, we find that LKB1 deficiency leads to increased CXCR2 ligand production and tumor infiltrating MDSCs. MDSC depletion enhances the efficacy of anti-PD-1 blockade in LKB1-deficient tumors bearing high TMB. Citation Format: Rui Li, Ramin Salehi-Rad, Stephanie Ong, Milica Momcilovic, Bin Liu, Raymond Lim, Linh Tran, Ziling Huang, Zhe Jing, Manash Paul, Kostyantyn Krysan, Stacy Park, John Minna, Michael Teitell, David Shackelford, Steven Dubinett. Depletion of CXCR2-dependent myeloid-derived suppressor cells (MDSCs) overcomes anti-PD-1 resistance in a murine model of LKB1-deficient non-small cell lung cancer (NSCLC) with high mutational load [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2710.