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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.

BACKGROUND The computed tomography angiography (CTA) “spot sign” is a well-recognized radiographic marker in primary intracerebral hemorrhage (ICH). Although it has been demonstrated to represent an area of active hemorrhage or contrast extravasation, the exact pathophysiology remains unclear. Vascular mimics of the spot sign have been identified; however, those representing pseudoaneurysm and small vessel aneurysm have rarely been reported. OBSERVATIONS A 57-year-old female with a past medical history of hypertension and diabetes mellitus presented with 2 weeks of acute-onset, worsening headache. Computed tomography scanning showed a right interior frontal lobe intraparenchymal hemorrhage. CTA demonstrated a punctate focus of hyperattenuation within the hematoma, consistent with a spot sign, which corresponded to a distal anterior cerebral artery pseudoaneurysm on a cerebral angiogram. The patient subsequently underwent emergent resection of the pseudoaneurysm and hematoma evacuation without complications. Her postoperative course was unremarkable without acute concerns or residual symptoms at the 4-month follow-up. LESSONS The authors present a unique case of a distal anterior cerebral artery pseudoaneurysm presenting as a spot sign in a relatively young patient without underlying vascular disease. Given the need for emergent intervention, intracranial pseudoaneurysm is an important diagnosis to consider in the presence of a spot sign in atypical clinical presentations of primary ICH.

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.

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.

A three-dimensional (3D) polycatenation supramolecular network with chemical formulas, [Zn(bpp)(BDC)]·1.5H2O (1), (bpp = 1,3-bis(4-pyridyl)propane, BDC2- = dianion of terephthalic acid), was synthesized and structurally determined. In compound 1, the coordination geometry of Zn(II) ion is distorted tetrahedral, where its 2D undulated layered framework is constructed via the bridges of Zn(II) ions with bpp and BDC2− ligands. Adjacent 2D layers are arranged in a combined parallel and interpenetrated manner to complete its 3D polycatenation supramolecular architecture. Compound 1 shows a one-step dehydration process with the weight losses of 6.1 %, approximately equal to the weight percentage of losing 1.5 guest water molecules. The cyclic thermogravimetric analysis reveals that compound 1 shows reversible, sponge-like water de-/adsorption behavior during de-/rehydration processes. Compound 1 also exhibits significant water vapor hysteresis isotherm.

Small fiber neuropathy (SFN) is a condition that affects small nerve fibers leading to a variety of symptoms, including pain, discomfort, tingling, and sensory loss. The development of new drugs is commonly complicated by variable medication pharmacokinetics and patients’ response to treatment due to comorbidities. In this review, we discuss currently used drugs, including the evidence about their efficacy. We also mention the medications with a questionable response in SFN treatment and those that are potential emerging therapies. SFN treatment is aimed towards addressing the cause of neuropathy and alleviating its symptoms. Determining the best treatment option may be challenging. Current drug treatments include antiseizure drugs, antidepressants, opioids, immunomodulating agents, topical medications, and NMDA antagonists. Each of these drugs has different molecular targets, efficacy, and adverse effect profiles. In some cases, the exact underlying mechanisms are not well understood. Current drug treatments provide patients with varying, often suboptimal, relief of symptoms. The need for new, effective, and safe therapies for SFN warrants intensive development and testing of new agents.

A two-dimensional (2D) coordination polymer, [Co(4-dpds)(bdc)(H2O)2]&middot, 4-dpds (1) (4-dpds = 4,4&prime, dipyridyldusulfide and bdc2&minus, = dianion of benzenedicarboxylic acid), has been synthesized and structurally determined by single-crystal X-ray diffractometer. In 1, the bdc2&minus, and 4-dpds both act as bridging ligands connecting the Co(II) ions to form a 2D wrinkled-like layered coordination polymer. Two wrinkled-like layered coordination polymers are mutually penetrated to generate a doubly interpenetrated framework, and then extended to its 3D architecture via the supramolecular forces between doubly interpenetrated 2D frameworks and free 4-dpds ligands by intermolecular O&ndash, H&sdot, &sdot, N hydrogen bonding interaction. Crystal packing arrangements were characterized by fingerprint plots, which were derived from the Hirshfeld Surfaces analysis, and showed that intermolecular hydrogen bonding interactions are the most important interactions on the construction of the crystal 1.

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.

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.

In this study, a series of β-Ga2O3 films are prepared by using triethylgallium (TEGa) and nitrous oxide (N2O) as precursors to explore the effect of N2O/TEGa ratio on the characteristics of β-Ga2O3 films. A metal/semiconductor/metal (MSM)-type solar blind ultraviolet (UV) photodetector (PD) is fabricated using as-prepared β-Ga2O3 film. It is found that an increment of N2O/TEGa ratio tends to suppress the oxygen vacancies in β-Ga2O3 film so the device performance can be significantly improved. This work gives a deep insight into the impact of TEGa/N2O ratio for depositing β-Ga2O3 on the film quality, the surface morphology, the chemical composition and the device performance for UV PDs.

β-Ga2O3 films are deposited on (0001) sapphire substrates using triethylgallium (TEGa) and nitrous oxide (N2O) under high N2O/TEGa ratios by atomic layer deposition (ALD). Au-β-Ga2O3-Au metal/semiconductor/metal (MSM) solar-blind deep ultraviolet (DUV) photodetectors (PDs) are prepared using Au interdigitated electrodes deposited by thermal evaporation. The ALD-grown β-Ga2O3 films and Au-β-Ga2O3-Au DUV MSM PDs are irradiated with gamma ray to explore the response of gamma irradiation on the β-Ga2O3 films and β-Ga2O3 DUV MSM PDs. It is found that gamma irradiation tends to deteriorate the structural properties of the β-Ga2O3 films and dark current of the β-Ga2O3 DUV MSM PDs. Nevertheless, it also results in an increase in the 254 nm illuminated photocurrent of the Au-β-Ga2O3-Au DUV MSM PD. Energy band diagram schematics of the biased Au-β-Ga2O3-Au DUV MSM PDs are presented to interpret the influence of gamma irradiation-induced defects on the performances of the Au-β-Ga2O3-Au DUV MSM PDs.

Two coordination polymers (CPs), with chemical formulas {[Ni2(bpp)2(BT)(H2O)6] 1.5(EtOH) 1.5H2O}n (1) and [Zn(bpp)(BT)0.5]&middot, 5H2O (2) (bpp = 1,3-bis(4-pyridyl)propane, and BT4&minus, = tetraanion of 1,2,4,5-Benzenetetracarboxylic acid), have been synthesized and structurally characterized by single-crystal X-ray diffraction methods. In compound 1, the coordination environments of two crystallographically independent Ni(II) ions are both distorted octahedral bonded to two nitrogen donors from two bpp ligands and four oxygen donors from one BT4- ligand and three water molecules. Both bpp and BT4&minus, act as bridging ligands with bis-monodentate and 1,4-bis-monodentate coordination modes, respectively, connecting the Ni(II) ions to form a 2D layered metal-organic framework (MOF). Adjacent 2D layers are then arranged orderly in an ABAB manner to complete their 3D supramolecular architecture. In 2, the coordination environment of Zn(II) ion is distorted tetrahedral bonded to two nitrogen donors from two bpp ligands and two oxygen donors from two BT4&minus, ligands. Both bpp and BT4- act as bridging ligands with bis-monodentate and 1,2,4,5-tetrakis-monodentate coordination modes, respectively, connecting the Zn(II) ions to form a 3D MOF. The reversible water de-/adsorption behavior of 1 between dehydrated and rehydrated forms has been verified by cyclic Thermogravimetric (TG) analyses through de-/rehydration processes. Compound 1 also exhibits significant water vapor hysteresis isotherms.

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.