Association Between Pretreatment Chest Imaging and Immune Checkpoint Inhibitor Pneumonitis Among Patients With Lung Cancer.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are a first-line and perioperative treatment for lung cancer. Pneumonitis is a potentially life-threatening complication of ICI treatment in 2% to 5% of patients; however, risk factors for developing ICI pneumonitis (ICI-p) remain undefined. METHODS: We conducted a retrospective cohort study of consecutive patients with lung cancer who received at least one dose of ICI from 2015 through 2020 at The Ohio State University. Pneumonitis cases were documented by the treating oncologist and retrospectively evaluated for agreement between an oncologist and a pulmonologist. Patient demographic and clinical characteristics were recorded and summarized between those with and without pneumonitis for the overall cohort. Univariate and multivariable survival analyses using the Fine-Gray competing risk model were used to examine the associations. RESULTS: A total of 471 patients with lung cancer were included, of which 402 had non-small cell lung cancer and 69 had small cell lung cancer; 39 (8%) patients in the overall cohort developed ICI-p. Preexisting interstitial abnormalities and prior chest radiation were both significantly associated with ICI-p on univariate analysis (hazard ratio [HR], 8.91; 95% CI, 4.69-16.92; P<.001; and HR, 2.81; 95% CI, 1.50-5.28; P=.001). On multivariable analyses, interstitial abnormalities remained a strong independent risk factor for ICI-p when controlling for chest radiation and type of immunotherapy (HR, 9.77; 95% CI, 5.17-18.46; P<.001). Among patients with ICI-p (n=39), those with severe (grade 3-5) pneumonitis had worse overall survival compared with those with mild (grade 1 or 2) pneumonitis (P=.001). Abnormal pulmonary function test results at both 12 and 18 months prior to ICI initiation were not significantly associated with ICI-p. CONCLUSIONS: Preexisting interstitial abnormalities on chest CT and prior chest radiation are independent risk factors that are strongly associated with ICI-p in patients with lung cancer. These findings highlight a potential need for closer observation for ICI-p among patients with these risk factors.

Mean platelet volume, thrombocytosis, and survival in non-small cell lung cancer patients treated with first-line pembrolizumab alone or with chemotherapy.

INTRODUCTION: Patients treated with immune checkpoint inhibitors (ICIs) may not response to treatment and are at risk for immune-related adverse events (irAEs). Platelet function has been linked to both oncogenesis and immune evasion. We studied the association between the change in mean platelet volume (MPV), platelet count, survival, and the risk of developing irAEs in patients with metastatic non-small cell lung cancer (NSCLC) who have received first-line ICI. METHODS: In this retrospective study, delta (∆) MPV was defined as the difference between cycle 2 and baseline MPV. Patient data were collected via chart review, and Cox proportional hazard and Kaplan-Meier method were used to assess the risk and estimate median overall survival. RESULTS: We identified 188 patients treated with first-line pembrolizumab, with or without concurrent chemotherapy. There were 80 (42.6%) patients received pembrolizumab monotherapy, and 108 (57.4%) received pembrolizumab in combination with platinum-based chemotherapy. Patients whose MPV (∆MPV ≤ 0) decreased had hazard ratio (HR) = 0.64 (95% CI 0.43-0.94) for death with p = 0.023. Patients with ∆MPV ≤ - 0.2 fL (median), there was a 58% increase in the risk of developing irAE (HR = 1.58, 95% CI 1.04-2.40, p = 0.031). Thrombocytosis at baseline and cycle 2 was associated with shorter OS with p = 0.014 and 0.039, respectively. CONCLUSION: Change in MPV after 1 cycle of pembrolizumab-based treatment was significantly associated with overall survival as well as the occurrence of irAEs in patients with metastatic NSCLC in the first-line setting. In addition, thrombocytosis was associated with poor survival.

Predictors of Response, Progression-Free Survival, and Overall Survival in Patients With Lung Cancer Treated With Immune Checkpoint Inhibitors.

Monoclonal antibodies that target immune checkpoint proteins, so-called immune checkpoint inhibitors, prevent tumor evasion of the immune system and are often effective in the treatment of lung cancer. Studies have revealed improved objective response rates, progression-free survival, and overall survival with immune checkpoint inhibitors when used in both first and subsequent-line settings. Unfortunately, only a subset of unselected patients with lung cancer responds to these therapies. An important area of ongoing research is to identify biomarkers that can predict which patients are most likely to derive clinical benefit. This review will discuss established and emerging biomarkers from some of the clinical trials that have demonstrated the efficacy of immune checkpoint inhibitors for the treatment of both NSCLC and SCLC.

A phase I/II study of pemetrexed with sirolimus in advanced, previously treated non-small cell lung cancer.

BACKGROUND: Single-agent pemetrexed is a treatment for recurrent non-squamous non-small cell lung cancer (NSCLC) that provides limited benefit. Preclinical studies showed promising synergistic effects when the mammalian target of rapamycin (mTOR) inhibitor sirolimus was added to pemetrexed. METHODS: This was a single-institution phase I/II study of pemetrexed in combination with sirolimus. The primary endpoint for the phase I was to determine the maximum tolerated dose (MTD) and safety of the combination. The primary endpoint for the phase II portion was to determine the overall response rate at the MTD. Key eligibility criteria included recurrent, metastatic NSCLC, ECOG performance status of 0-2, and adequate organ function. Sirolimus was administered orally daily after an initial loading dose, and pemetrexed was given intravenously on day 1 of every 21-day cycle. RESULTS: Forty-two patients with recurrent, metastatic NSCLC were enrolled, 22 in phase I and 20 in phase II. The MTD was pemetrexed 500 mg/m2 every 3 weeks, and sirolimus 10 mg on day 1, and 3 mg daily thereafter. Treatment-related adverse events (AEs) occurred in 38 (90.5%) patients. The most common grade 3-4 treatment-related AEs were lymphopenia (31%) and hypophosphatemia (19%). Two treatment-related deaths occurred due to febrile neutropenia and infection, respectively. Among 27 total patients treated at the MTD, 6 (22.2%) had a partial response (PR), 12 (44.4%) had stable disease (SD) and 5 (18.5%) had progressive disease. Median progression-free survival (PFS) was 18.4 weeks (95% CI: 7.0-29.4). CONCLUSIONS: The combination of pemetrexed and sirolimus is active in heavily-pretreated NSCLC (ClinicalTrials.gov Identifier: NCT00923273).

A Pilot Study of Sirolimus in Subjects with Cowden Syndrome or Other Syndromes Characterized by Germline Mutations in PTEN.

LESSONS LEARNED: This is the first human interventional study in patients with Cowden syndrome that is driven by inactivation of germline PTEN gene.Single-agent sirolimus, a mTOR inhibitor, suppressed mTOR signaling in surrogate human tissues without significant toxicity. BACKGROUND: Cowden syndrome is characterized by inactivating germline PTEN mutations, which can lead to activation of the PI3K-Akt-mTOR pathway. METHODS: Adult subjects with germline PTEN mutation who met international diagnostic criteria for Cowden syndrome and who had Eastern Cooperative Oncology Group (ECOG) performance status 0-2 and adequate organ function were enrolled. Subjects were treated with a 56-day course of daily oral sirolimus. In addition to symptom assessment and physical examination, dermatologic, endoscopic, neurologic (cerebellar), and radiographic assessments were conducted. Inhibition of the mTOR pathway in benign skin and gastrointestinal (GI) lesion was assessed by immunohistochemistry. RESULTS: A total of 18 patients and 16 families were enrolled. PTEN mutations were located at exons 1-8. Regression of skin and GI lesions was observed by dermoscopy or endoscopy. Neurological evaluation showed improvement in cerebellar function score at 1 month. Immunohistochemistry (IHC) analysis in skin and GI benign lesions showed a decrease in the ratio of phosphorylated (p)S6 to total S6 in response to sirolimus. Ratios of pS6K to total S6 at days 14 and 56 were significantly lower than at baseline (p = .0026, p = .00391, respectively). A 56-day course of sirolimus was well tolerated. CONCLUSION: A 56-day course of sirolimus was well tolerated in subjects with Cowden syndrome and was associated with some evidence of improvement in symptoms, skin and GI lesions, cerebellar function, and decreased mTOR signaling.

Rapamycin downregulates thymidylate synthase and potentiates the activity of pemetrexed in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancer cases, and almost half of newly diagnosed patients have metastatic disease. Pemetrexed is a widely used drug for NSCLC and inhibits several folate-dependent enzymes including thymidylate synthase (TS). Increased expression of TS confers resistance to pemetrexed in vitro and predicts poor response to pemetrexed. Rapamycin is an mTOR inhibitor and suppresses cap-dependent synthesis of specific mRNA species. Here, we show that the combination of rapamycin and pemetrexed synergistically inhibits proliferation of NSCLC cells. Although pemetrexed as a single agent induced TS, pretreatment with rapamycin suppressed pemetrexed-induced TS expression. In vivo, the combination of rapamycin and pemetrexed inhibited growth of NSCLC xenografts, which correlated with decreased mTOR activity and suppression of pemetrexed-induced TS expression. The ability of rapamycin to enhance the efficacy of pemetrexed and prevent TS expression has implications for the design of Phase I and/or Phase II NSCLC clinical trials with mTOR inhibitors in combination with pemetrexed.

Inhibition of lung tumorigenesis by metformin is associated with decreased plasma IGF-I and diminished receptor tyrosine kinase signaling.

Metformin is the most commonly prescribed drug for type II diabetes and is associated with decreased cancer risk. Previously, we showed that metformin prevented tobacco carcinogen (NNK)-induced lung tumorigenesis in a non-diabetic mouse model, which was associated with decreased IGF-I/insulin receptor signaling but not activation of AMPK in lung tissues, as well as decreased circulating levels of IGF-I and insulin. Here, we used liver IGF-I-deficient (LID) mice to determine the importance of IGF-I in NNK-induced lung tumorigenesis and chemoprevention by metformin. LID mice had decreased lung tumor multiplicity and burden compared with wild-type (WT) mice. Metformin further decreased lung tumorigenesis in LID mice without affecting IGF-I levels, suggesting that metformin can act through IGF-I-independent mechanisms. In lung tissues, metformin decreased phosphorylation of multiple receptor tyrosine kinases (RTK) as well as levels of GTP-bound Ras independently of AMPK. Metformin also diminished plasma levels of several cognate ligands for these RTKs. Tissue distribution studies using [(14)C]-metformin showed that uptake of metformin was high in liver but four-fold lower in lungs, suggesting that the suppression of RTK activation by metformin occurs predominantly via systemic, indirect effects. Systemic inhibition of circulating growth factors and local RTK signaling are new AMPK-independent mechanisms of action of metformin that could underlie its ability to prevent tobacco carcinogen-induced lung tumorigenesis.

Repositioning metformin for cancer prevention and treatment.

Metformin is the most commonly prescribed drug for type 2 diabetes (T2DM). Retrospective studies show that metformin is associated with decreased cancer risk. This historical correlation has driven vigorous research campaigns to determine the anticancer mechanisms of metformin. Consolidating the preclinical data is a challenge because unanswered questions remain concerning relevant mechanisms, bioavailability, and genetic factors that confer metformin sensitivity. Perhaps the most important unanswered question is whether metformin has activity against cancer in non-diabetics. In this review we highlight the proposed mechanisms of metformin action in cancer and discuss ongoing clinical trials with metformin in cancer. Improved understanding of these issues will increase the chances for successful application of metformin as an inexpensive, well-tolerated, and effective anticancer agent.

Metformin prevents tobacco carcinogen--induced lung tumorigenesis.

Activation of the mammalian target of rapamycin (mTOR) pathway is an important and early event in tobacco carcinogen-induced lung tumorigenesis, and therapies that target mTOR could be effective in the prevention or treatment of lung cancer. The biguanide metformin, which is widely prescribed for the treatment of type II diabetes, might be a good candidate for lung cancer chemoprevention because it activates AMP-activated protein kinase (AMPK), which can inhibit the mTOR pathway. To test this, A/J mice were treated with oral metformin after exposure to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Metformin reduced lung tumor burden by up to 53% at steady-state plasma concentrations that are achievable in humans. mTOR was inhibited in lung tumors but only modestly. To test whether intraperitoneal administration of metformin might improve mTOR inhibition, we injected mice and assessed biomarkers in liver and lung tissues. Plasma levels of metformin were significantly higher after injection than oral administration. In liver tissue, metformin activated AMPK and inhibited mTOR. In lung tissue, metformin did not activate AMPK but inhibited phosphorylation of insulin-like growth factor-I receptor/insulin receptor (IGF-1R/IR), Akt, extracellular signal-regulated kinase (ERK), and mTOR. This suggested that metformin indirectly inhibited mTOR in lung tissue by decreasing activation of insulin-like growth factor-I receptor/insulin receptor and Akt upstream of mTOR. Based on these data, we repeated the NNK-induced lung tumorigenesis study using intraperitoneal administration of metformin. Metformin decreased tumor burden by 72%, which correlated with decreased cellular proliferation and marked inhibition of mTOR in tumors. These studies show that metformin prevents tobacco carcinogen-induced lung tumorigenesis and support clinical testing of metformin as a chemopreventive agent.

The role of the Akt/mTOR pathway in tobacco carcinogen-induced lung tumorigenesis.

Lung cancer is the leading cause of cancer-related death in the United States, and 85 to 90% of lung cancer cases are associated with tobacco use. Tobacco components promote lung tumorigenesis through genotoxic effects, as well as through biochemical modulation of signaling pathways such as the Akt/mammalian target of rapamycin (mTOR) pathway that regulates cell proliferation and survival. This review will describe cell surface receptors and other upstream components required for tobacco carcinogen-induced activation of Akt and mTOR. Preclinical studies show that inhibitors of the Akt/mTOR pathway inhibit tumor formation in mouse models of carcinogen-induced lung tumorigenesis. Some of these inhibitors will be highlighted, and their clinical potential for the treatment and prevention of lung cancer will be discussed.

The chemopreventive agent myoinositol inhibits Akt and extracellular signal-regulated kinase in bronchial lesions from heavy smokers.

Myoinositol is an isomer of glucose that has chemopreventive activity in animal models of cancer. In a recent phase I clinical trial, myoinositol administration correlated with a statistically significant regression of preexisting bronchial dysplastic lesions in heavy smokers. To shed light on the potential mechanisms involved, activation of Akt and extracellular signal-regulated kinase (ERK), two kinases that control cellular proliferation and survival, was assessed in 206 paired bronchial biopsies from 21 patients who participated in this clinical trial. Before myoinositol treatment, strongly positive staining for activation of Akt was detected in 27% of hyperplastic/metaplastic lesions and 58% of dysplastic lesions (P = 0.05, chi(2) test). There was also a trend toward increased activation of ERK (28% in regions of hyperplasia/metaplasia to 42% of dysplastic lesions). Following myoinositol treatment, significant decreases in Akt and ERK phosphorylation were observed in dysplastic (P < 0.01 and 0.05, respectively) but not hyperplastic/metaplastic lesions (P > 0.05). In vitro, myoinositol decreased endogenous and tobacco carcinogen-induced activation of Akt and ERK in immortalized human bronchial epithelial cells, which decreased cell proliferation and induced a G(1)-S cell cycle arrest. These results show that the phenotypic progression of premalignant bronchial lesions from smokers correlates with increased activation of Akt and ERK and that these kinases are targets of myoinositol. Moreover, they suggest that myoinositol might cause regression of bronchial dysplastic lesions through inhibition of active Akt and ERK.

A central role for Foxp3+ regulatory T cells in K-Ras-driven lung tumorigenesis.

BACKGROUND: K-Ras mutations are characteristic of human lung adenocarcinomas and occur almost exclusively in smokers. In preclinical models, K-Ras mutations are necessary for tobacco carcinogen-driven lung tumorigenesis and are sufficient to cause lung adenocarcinomas in transgenic mice. Because these mutations confer resistance to commonly used cytotoxic chemotherapies and targeted agents, effective therapies that target K-Ras are needed. Inhibitors of mTOR such as rapamycin can prevent K-Ras-driven lung tumorigenesis and alter the proportion of cytotoxic and Foxp3+ regulatory T cells, suggesting that lung-associated T cells might be important for tumorigenesis. METHODS: Lung tumorigenesis was studied in three murine models that depend on mutant K-Ras; a tobacco carcinogen-driven model, a syngeneic inoculation model, and a transgenic model. Splenic and lung-associated T cells were studied using flow cytometry and immunohistochemistry. Foxp3+ cells were depleted using rapamycin, an antibody, or genetic ablation. RESULTS: Exposure of A/J mice to a tobacco carcinogen tripled lung-associated Foxp3+ cells prior to tumor development. At clinically relevant concentrations, rapamycin prevented this induction and reduced lung tumors by 90%. In A/J mice inoculated with lung adenocarcinoma cells resistant to rapamycin, antibody-mediated depletion of Foxp3+ cells reduced lung tumorigenesis by 80%. Likewise, mutant K-Ras transgenic mice lacking Foxp3+ cells developed 75% fewer lung tumors than littermates with Foxp3+ cells. CONCLUSIONS: Foxp3+ regulatory T cells are required for K-Ras-mediated lung tumorigenesis in mice. These studies support clinical testing of rapamycin or other agents that target Treg in K-Ras driven human lung cancer.

Akt-dependent and -independent mechanisms of mTOR regulation in cancer.

The protein kinase mTOR (mammalian target of rapamycin) is a critical regulator of cellular metabolism, growth, and proliferation. These processes contribute to tumor formation, and many cancers are characterized by aberrant activation of mTOR. Although activating mutations in mTOR itself have not been identified, deregulation of upstream components that regulate mTOR are prevalent in cancer. The prototypic mechanism of mTOR regulation in cells is through activation of the PI3K/Akt pathway, but mTOR receives input from multiple signaling pathways. This review will discuss Akt-dependent and -independent mechanisms of mTOR regulation in response to mitogenic signals, as well as its regulation in response to energy and nutrient-sensing pathways. Preclinical and clinical studies have demonstrated that tumors bearing genetic alterations that activate mTOR are sensitive to pharmacologic inhibition of mTOR. Elucidation of novel pathways that regulate mTOR may help identify predictive factors for sensitivity to mTOR inhibitors, and could provide new therapeutic targets for inhibiting the mTOR pathway in cancer. This review will also highlight pharmacologic approaches that inhibit mTOR via activation of the AMP-activated protein kinase (AMPK), an important inhibitor of the mTOR pathway and an emerging target in cancer.

A novel G-quadruplex-forming GGA repeat region in the c-myb promoter is a critical regulator of promoter activity.

The c-myb promoter contains multiple GGA repeats beginning 17 bp downstream of the transcription initiation site. GGA repeats have been previously shown to form unusual DNA structures in solution. Results from chemical footprinting, circular dichroism and RNA and DNA polymerase arrest assays on oligonucleotides representing the GGA repeat region of the c-myb promoter demonstrate that the element is able to form tetrad:heptad:heptad:tetrad (T:H:H:T) G-quadruplex structures by stacking two tetrad:heptad G-quadruplexes formed by two of the three (GGA)(4) repeats. Deletion of one or two (GGA)(4) motifs destabilizes this secondary structure and increases c-myb promoter activity, indicating that the G-quadruplexes formed in the c-myb GGA repeat region may act as a negative regulator of the c-myb promoter. Complete deletion of the c-myb GGA repeat region abolishes c-myb promoter activity, indicating dual roles of the c-myb GGA repeat element as both a transcriptional repressor and an activator. Furthermore, we demonstrated that Myc-associated zinc finger protein (MAZ) represses c-myb promoter activity and binds to the c-myb T:H:H:T G-quadruplexes. Our findings show that the T:H:H:T G-quadruplex-forming region in the c-myb promoter is a critical cis-acting element and may repress c-myb promoter activity through MAZ interaction with G-quadruplexes in the c-myb promoter.

Phosphatidylinositol ether lipid analogues induce AMP-activated protein kinase-dependent death in LKB1-mutant non small cell lung cancer cells.

Loss of function of the tumor suppressor LKB1 occurs in 30% to 50% of lung adenocarcinomas. Because LKB1 activates AMP-activated protein kinase (AMPK), which can negatively regulate mTOR, AMPK activation might be desirable for cancer therapy. However, no known compounds activate AMPK independently of LKB1 in vivo, and the usefulness of activating AMPK in LKB1-mutant cancers is unknown. Here, we show that lipid-based Akt inhibitors, phosphatidylinositol ether lipid analogues (PIA), activate AMPK independently of LKB1. PIAs activated AMPK in LKB1-mutant non-small cell lung cancer (NSCLC) cell lines with similar concentration dependence as that required to inhibit Akt. However, AMPK activation was independent of Akt inhibition. AMPK activation was a major mechanism of mTOR inhibition. To assess whether another kinase capable of activating AMPK, CaMKK beta, contributed to PIA-induced AMPK activation, we used an inhibitor of CaMKK, STO-609. STO-609 inhibited PIA-induced AMPK activation in LKB1-mutant NSCLC cells, and delayed AMPK activation in wild-type LKB1 NSCLC cells. In addition, AMPK activation was not observed in NSCLC cells with mutant CaMKK beta, suggesting that CaMKK beta contributes to PIA-induced AMPK activation in cells. AMPK activation promoted PIA-induced cytotoxicity because PIAs were less cytotoxic in AMPKalpha-/- murine embryonic fibroblasts or LKB1-mutant NSCLC cells transfected with mutant AMPK. This mechanism was also relevant in vivo. Treatment of LKB1-mutant NSCLC xenografts with PIA decreased tumor volume by approximately 50% and activated AMPK. These studies show that PIAs recapitulate the activity of two tumor suppressors (PTEN and LKB1) that converge on mTOR. Moreover, they suggest that PIAs might have utility in the treatment of LKB1-mutant lung adenocarcinomas.

Phosphatidylinositol ether lipid analogues that inhibit AKT also independently activate the stress kinase, p38alpha, through MKK3/6-independent and -dependent mechanisms.

Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.

Handicapping the race to develop inhibitors of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway controls many cellular processes that are important for the formation and progression of cancer, including apoptosis, transcription, translation, metabolism, angiogenesis, and cell cycle progression. Genetic alterations and biochemical activation of the pathway are frequent events in preneoplastic lesions and advanced cancers and often portend a poor prognosis. Thus, inhibition of the PI3K/Akt/mTOR pathway is an attractive concept for cancer prevention and/or therapy. Inhibitors of individual components, such as PI3K, PDK-1, Akt, and mTOR, are being developed at a rapid pace and have promise for improving the care of cancer patients. Here, we review the published data on inhibitors of the pathway and discuss relevant issues, such as the complex regulation of the pathway, the design of clinical trials, and the likelihood of finding a therapeutic index when targeting such a critical signaling pathway.