David J. Seward

Serine Threonine Kinase 11 (STK11) loss of function (LoF) correlates with anti-PD-1 therapy resistance in patients with KRAS-driven lung adenocarcinoma (LUAD). The molecular mechanisms governing this observation remain unclear and represent a critical outstanding question in the field of lung oncology. As an initial approach to understand this phenomenon, we knocked-out (KO) STK11 in multiple KRAS-driven, STK11-competent human LUAD cell lines and performed whole transcriptome analyses to identify STK11-loss-dependent differential gene expression. Subsequent pathway enrichment studies highlighted activation of the HIPPO/YAP1 signaling axis, along with the induction of numerous tumor-intrinsic cytokines. To validate that YAP1-mediated transcriptional activation occurs in response to STK11 loss, we pursued YAP1 perturbation as a strategy to restore an STK11-competent gene expression profile in STK11-KO LUAD cell lines. Together, our data link STK11 loss with YAP1-mediated transcriptional activation, including the upregulation of immune-evasion promoting cytokines IL-6, CXCL8 and CXCL2. Further, our results raise the intriguing possibility that YAP1 antagonism may represent a therapeutic approach to counter anti-PD-1 therapy resistance in STK11-null, KRAS-driven LUADs by modulating tumor-intrinsic gene expression to promote a “hot” tumor immune microenvironment.

Glutathione (GSH) is a critical component of the cellular redox system that combats oxidative stress. The glutamate-cystine antiporter, system x_C⁻, is a key player in GSH synthesis that allows for the uptake of cystine, the rate-limiting building block of GSH. It is unclear whether GSH or GSH-dependent protein oxidation [proteinS-glutathionylation (PSSG)] regulates the activity of system x_C⁻. We demonstrate that an environment of enhanced PSSG promotes GSH increases via a system x_C⁻–dependent mechanism. Absence of the deglutathionylase, glutaredoxin (GLRX), augmented SLC7A11 protein and led to significant increases of GSH content.S-glutathionylation of C23 or C204 of the deubiquitinase OTUB1 promoted interaction with the E2-conjugating enzyme UBCH5A, leading to diminished ubiquitination and proteasomal degradation of SLC7A11 and augmentation of GSH, effects that were reversed by GLRX. These findings demonstrate an intricate link between GLRX and GSH viaS-glutathionylation of OTUB1 and system x_C⁻and illuminate a previously unknown feed-forward regulatory mechanism whereby enhanced GSH protein oxidation augments cellular GSH.

Serine Threonine Kinase 11 (STK11) loss of function (LoF) correlates with anti-PD-1 therapy resistance in patients with KRAS-driven lung adenocarcinoma (LUAD). The molecular mechanisms governing this observation remain unclear and represent a critical outstanding question in the field of lung oncology. As an initial approach to understand this phenomenon, we knocked out STK11 in multiple KRAS-driven, STK11-competent human LUAD cell lines and performed whole transcriptome analyses to identify STK11-loss-dependent differential gene expression. Subsequent pathway enrichment studies highlighted activation of the HIPPO/YAP1 signaling axis, along with the induction of numerous tumor-intrinsic cytokines. To validate that YAP1-mediated transcriptional activation occurs in response to STK11 loss, we pursued YAP1 perturbation as a strategy to restore an STK11-competent gene expression profile in STK11-KO LUAD cell lines. Together, our data link STK11loss with YAP1-mediated transcriptional activation, including the upregulation of immune-evasion promoting cytokines IL-6, CXCL8 and CXCL2. Further, our results raise the intriguing possibility that YAP1 antagonism may represent a therapeutic approach to counter anti-PD-1 therapy resistance in STK11-null, KRAS-driven LUADs by modulating tumor-intrinsic gene expression to promote a “hot” tumor immune microenvironment.

γδ T cells have been demonstrated to both promote tumor growth/metastasis and to efficiently kill tumor cells. This dual role of γδ T cells has been attributed to their ability to produce either IL-17 (γδT17) or IFNγ (γδT1), the former being associated with tumor progression and the latter being associated with anti-tumor activity. γδ T cell function is influenced by multiple factors, including the recognition of stress-induced ligands, as well as cytokines and chemokines. However, the specific signals that lead to the selective activation and function of γδT17 or γδT1 subsets in different cancers remains unclear. Here, we explored these questions using a novel inducible mouse model of Kras-driven lung adenocarcinoma with or without a co-mutation in Stk11, a master regulator of metabolic pathways. Preliminary characterization of this model revealed that KrasG12D/Stk11fl/fl mice exhibited significantly greater morbidity than KrasG12D mice. While lung leukocyte numbers did not differ significantly between KrasG12D and KrasG12D/Stk11fl/fl mice, we noted a significantly increased frequency of γδT17, but not γδT1, subsets in KrasG12D/Stk11fl/fl lungs. Interestingly, the increased frequency of γδT17 was due to increased Vγ1 and Vγ6, but not Vγ4, γδT17 suggesting a selective expansion of these subsets. To examine a potential role for butyrophilins in these models, we assessed lung butyrophilin gene expression using qPCR. This analysis revealed increased expression of Btnl1, Btnl2, and Btnl9 in KrasG12D lungs mice as compared to controls. Together, these results suggest that this model has the potential to shed new light on how the interaction of the tumor microenvironment with specific γδ T cell subsets affects disease pathogenesis.

Serine/Threonine Kinase 11 (STK11) encodes an important tumor suppressor that is frequently mutated in lung adenocarcinoma. Clinical studies have shown that mutations in STK11 resulting in loss of function correlate with resistance to anti-PD-1 monoclonal antibody therapy in KRAS-driven non-small cell lung cancer (NSCLC), but the molecular mechanisms responsible remain unclear. Despite this uncertainty, STK11 functional status is emerging as a reliable biomarker for predicting non-response to anti-PD-1 therapy in NSCLC patients. The clinical utility of this biomarker ultimately depends upon accurate classification of STK11 variants. For nonsense variants occurring early in the STK11 coding region, this assessment is straightforward. However, rigorously demonstrating the functional impact of missense variants remains an unmet challenge. Here we present data characterizing four STK11 splice-site variants by analyzing tumor mRNA, and 28 STK11 missense variants using an in vitro kinase assay combined with a cell-based p53-dependent luciferase reporter assay. The variants we report were identified in primary human NSCLC biopsies in collaboration with the University of Vermont Genomic Medicine group. Additionally, we compare our experimental results with data from 22 in silico predictive algorithms. Our work highlights the power, utility and necessity of functional variant assessment and will aid STK11 variant curation, provide a platform to assess novel STK11 variants and help guide anti-PD-1 therapy utilization in KRAS-driven NSCLCs.

Glycolysis is a well‐known process by which metabolically active cells, such as tumor or immune cells meet their high metabolic demands. Previously, our laboratory has demonstrated that in airway epithelial cells, the pleiotropic cytokine, interleukin‐1 beta (IL1B) induces glycolysis and that this contributes to allergic airway inflammation and remodeling. Activation of glycolysis is known to increase NADPH reducing equivalents generated from the pentose phosphate pathway, linking metabolic reprogramming with redox homeostasis. In addition, numerous glycolytic enzymes are known to be redox regulated. However, whether and how redox chemistry regulates metabolic reprogramming more generally remains unclear. In this study, we employed a multi‐omics approach in primary mouse airway basal cells to evaluate the role of protein redox biochemistry, specifically protein glutathionylation, in mediating metabolic reprogramming. Our findings demonstrate that IL1B induces glutathionylation of multiple proteins involved in metabolic regulation, notably in the glycolysis pathway. Cells lacking Glutaredoxin‐1 ( Glrx) , the enzyme responsible for reversing glutathionylation, show modulation of multiple metabolic pathways including an enhanced IL1B‐induced glycolytic response. This was accompanied by increased secretion of thymic stromal lymphopoietin (TSLP), a cytokine important in asthma pathogenesis. Targeted inhibition of glycolysis prevented TSLP release, confirming the functional relevance of enhanced glycolysis in cells stimulated with IL1B. Collectively, data herein point to an intriguing link between glutathionylation chemistry and glycolytic reprogramming in epithelial cells and suggest that glutathionylation chemistry may represent a therapeutic target in pulmonary pathologies with perturbations in the glycolysis pathway.

Treatment for stage IA lung cancer may be too aggressive an approach in elderly patients with competing co-morbidities. We report outcomes for those electing active surveillance (AS) and investigate factors that may predict indolent disease. Retrospective review was performed for 12 consecutive patients, ≥70 years old, with medically inoperable stage IA, T1N0M0 lung cancer and significant co-morbidities, who chose AS with radiation therapy (RT) reserved for clear disease progression. Collected data included Charlson-Deyo Comorbidity Index (CDCI) grades, histology, and tumor size changes. Volume doubling time (VDT) calculations used a modified Schwartz equation. Fifteen nodules underwent AS in 12 patients; three patients had more than one nodule. Median age of all patients was 78 (range, 71–85). All patients’ CDCI grades were ≥1, 7 were ≥2. Eleven of 12 patients were deemed to be at high-risk for falls. Twelve nodules in 12 patients were biopsied; adenocarcinoma the prevailing common (47%) histology. The median, one, two and three year patient freedom-from-RT values were 21.4 months (95% CI: 11.6-not reached), 81%, 43%, and 29%, respectively. Median VDT of treated vs. untreated nodules was 189 days (range, 62-infinite) vs. 1153 days (range, 504-infinite), respectively. No patient progressed regionally or distantly, and there have been no cancer-related deaths. Due to cardiovascular events, two patients died and one remains on hospice. Median duration of AS for those still continuing computed tomography (CT) surveillance is 35.1 months. Selected elderly patients with stage IA lung cancer and significant co-morbidities may undergo AS without detriment in outcome. Prospective AS studies are warranted.