Introduction: Although obesity is associated with adverse cancer outcomes in general, most retrospective clinical studies suggest a beneficial effect of obesity in NSCLC.Methods: Hypothesizing that this "obesity paradox" arises partly from the limitations of using body mass index (BMI) to measure obesity, we quantified adiposity using preoperative computed tomography images. This allowed the specific determination of central obesity as abdominal visceral fat area normalized to total fat area (visceral fat index [VFI]). In addition, owing to the previously reported salutary effect of metformin on high-BMI patients with lung cancer, metformin users were excluded. We then explored associations between visceral obesity and outcomes after surgical resection of stage I and II NSCLC. We also explored potential immunologic underpinnings of such association using complimentary analyses of tumor gene expression data from NSCLC tumors and the tumor transcriptome and immune microenvironment in an immunocompetent model of lung cancer with diet-induced obesity.Results: We found that in 513 patients with stage I and II NSCLC undergoing lobectomy, a high VFI is associated with decreased recurrence-free and overall survival. VFI was also inversely related to an inflammatory transcriptomic signature in NSCLC tumors, consistent with observations made in immunocompetent murine models wherein diet-induced obesity promoted cancer progression while exacerbating elements of immune suppression in the tumor niche.Conclusions: In all, this study uses multiple lines of evidence to reveal the adverse effects of visceral obesity in patients with NSCLC, which align with those found in animal models. Thus, the obesity paradox may, at least in part, be secondary to the use of BMI as a measure of obesity and the confounding effects of metformin use.

While immune checkpoint inhibitors (ICIs) have transformed the therapeutic landscape in oncology, they are effective in select subsets of patients. Efficacy may be limited by tumor-driven immune suppression, of which 1 key mechanism is the development of myeloid-derived suppressor cells (MDSCs). A fundamental gap in MDSC therapeutics is the lack of approaches that target MDSC biogenesis. We hypothesized that targeting MDSC biogenesis would mitigate MDSC burden and bolster tumor responses to ICIs. We tested a class of agents, dihydroorotate dehydrogenase (DHODH) inhibitors, that have been previously shown to restore the terminal differentiation of leukemic myeloid progenitors. DHODH inhibitors have demonstrated preclinical safety and are under clinical study for hematologic malignancies. Using mouse models of mammary cancer that elicit robust MDSC responses, we demonstrated that the DHODH inhibitor brequinar (a) suppressed MDSC production from early-stage myeloid progenitors, which was accompanied by enhanced myeloid maturation; (b) augmented the antitumor and antimetastatic activities of programmed cell death 1–based (PD-1–based) ICI therapy in ICI-resistant mammary cancer models; and (c) acted in concert with PD-1 blockade through modulation of MDSC and CD8 + T cell responses. Moreover, brequinar facilitated myeloid maturation and inhibited immune-suppressive features in human bone marrow culture systems. These findings advance the concept of MDSC differentiation therapy in immuno-oncology.

BackgroundBronchoalveolar lavage (BAL) is an underutilized tool in the search for pulmonary disease biomarkers. While leukocytes with effector and suppressor function play important roles in airway immunity and tumors, it remains unclear if frequencies and phenotypes of BAL leukocytes can be useful parameters in lung cancer studies and clinical trials. We therefore explored the utility of BAL leukocytes as a source of biomarkers interrogating the impact of smoking, a major lung cancer risk determinant, on pulmonary immunity.MethodsIn this ‘test case’ observational study, BAL samples from 119 donors undergoing lung cancer screening and biopsy procedures were evaluated by conventional and spectral flow cytometry to exemplify the comprehensive immune analyses possible with this biospecimen. Proportions of major leukocyte populations and phenotypic markers levels were found. Multivariate linear rank sum analysis considering age, sex, cancer diagnosis, and smoking status was performed.ResultsSignificantly increased frequencies of myeloid derived suppressor cells and PD-L1-expressing macrophages were found in current and former smokers compared to never smokers. While cytotoxic CD8T cells and conventional CD4 helper T cell frequencies were significantly reduced in current and former smokers, expression of immune checkpoints PD-1 and LAG-3 as well as Tregs proportions were increased. Lastly, the cellularity, viability and the stability of several immune readouts under cryostorage suggested BAL samples are useful for correlative endpoints in clinical trials.ConclusionsSmoking is associated with heightened markers of immune dysfunction, readily assayable in BAL, that may reflect a permissive environment for cancer development and progression in the airway.

The ability of CD8+ T cells to mount an anti-tumor immune response is compromised by immune suppression in the tumor microenvironment (TME). Tumor Associated Macrophages (TAMs) and Myeloid Derived Suppressor Cells (MDSCs) are a major part of this immune suppressive network. Targeting these populations remains challenging. Previously, we have reported that pharmacological and genetic blockade of p38 MAPK impeded the expansion and mobilization of monocytic and granulocytic MDSCs in mouse mammary carcinoma models. We also found that blockade of p38 or depletion of MDSCs reduced tumor growth and metastasis while enhancing the levels of CD8+ T cells in the primary tumors. In the present study, we asked whether CD8+ T cells contribute to the anti-metastatic activity of p38 inhibitor (p38i) and how p38 blockade affects the functional status of T cells and MDSCs. By using the mouse mammary carcinoma 4T1 model, we found that depletion of CD8+ T cells negated the effects of p38i on tumor growth and metastasis, indicating that CD8+ T cells contribute to the anti-tumor and anti-metastatic effects of p38 blockade. Next, we examined whether p38i exhibits a direct effect on T cells. The results of the T cell proliferation in vitro assays revealed that p38 blockade did not have a direct impact on T cell proliferation in response to αCD3/αCD28 stimulation. To determine the effect of p38 blockade on T cells in vivo, we performed single cell RNA-seq on the 4T1 tumor models treated with p38i and the 4T1 model with p38α (Mapk14) knockout (p38ko). This study revealed that p38 blockade by p38i or by inactivation of p38 in tumor cells decreased the amount of exhausted T cells and increased Th1 cells in the TME, indicating a positive effect on T cell functions. Furthermore, we observed a significant decrease in inflammatory signaling in granulocytes and monocytes upon p38 blockade. Our previous study showed that p38i did not affect generation of MDSCs in vitro in response to G-CSF & GM-CSF. To determine whether p38i alters MDSCs in vivo, we assessed MDSC gene signature in monocytic and granulocytic MDSCs isolated from spleens of tumor-bearing mice subjected to p38 blockade. This work revealed that the MDSC gene signature was reduced in both p38i and p38ko groups compared to tumor bearing mice treated with vehicle-control. These results indicated a reduction in the MDSC generation in the in vivo model. Our study revealed that blockade of p38 reduces tumor induced immune suppression and may enhance anti-tumor immune response in metastatic breast cancer. Citation Format: Priyanka Rajan, Justin Zonneville, Robert Zollo, Mackenzie Honikel, Sofija Raudins, Sean Colligan, Brian Morreale, Mohammed Alruwaili, Mohammed Alqarni, Scott Olejniczak, Joseph Barbi, Scott Abrams, Andrei Bakin. Blockade of p38 MAPK reduces the tumor-induced immune suppressive microenvironment in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 73.

Dysregulated protein synthesis is seen in many aggressive cancers, including metastatic breast cancer. However, the specific contributions of certain translation initiation factors to in vivo disease remain undefined. This is particularly true of eIF4B, an RNA-binding protein and cofactor of the RNA helicase eIF4A and associated eIF4F cap-binding complex. While eIF4A, eIF4G, and eIF4E are well-known to contribute to the progression of many cancer types including metastatic breast cancers, the role played by eIF4B in breast cancer remains relatively unclear. We therefore explored how naturally divergent and experimentally modulated eIF4B levels impact tumor growth and progression in well- characterized murine triple negative breast cancer (TNBC) models. Surprisingly, we found that higher eIF4B levels in mouse and human breast cancers were associated with less aggressive phenotypes. shRNA-mediated eIF4B knockdown in TNBC lines failed to markedly alter proliferation and global translation in the cells in vitro and only modestly hindered their growth as primary mammary tumors growth in mice. However, eIF4B knockdown significantly enhanced invasive growth in vitro and exacerbated both tumor burden and mortality relative to nontargeting shRNA controls in a model of metastatic disease. Analysis of eIF4B levels and breast cancer patient survival reinforced a link to better outcomes. Interestingly, low eIF4B expression was also associated with more formidable immune evasion in vitro and in vivo, implicating a novel immunomodulatory role for this factor in the malignant setting that suggests a mode of action beyond its historical role as a co-activator of eIF4A/F.

The eukaryotic translation initiation factor 4A (eIF4A) resolves mRNA structures to support protein synthesis, yet little is known about its regulation. Here we analyzed eIF4A phosphorylation during alternate stages of the cell cycle, and found three residues near the DEAD box motif (T73, T146, and S177) underwent substantial phosphorylation changes. Phosphomimetic mutations T73D and T146D led to G2/M phase arrest, and abolished eIF4A interaction with RNA, suggesting eIF4A activity is needed for completion of cell division. In addition to these repressive events, we found that S177, a site immediately adjacent to the DEAD-box, showed diametrically opposed phosphorylation, with only phosphorylated S177 present during G1/S arrest and dephosphorylated S177 peptides during G2/M arrest. Phosphomimetic S177D eIF4A increased polysome levels and enhanced normally reduced eIF4A-eIF4G-interaction during G2/M, while phosphodeficient S177A decreased polysome levels and reduced growth, suggesting phosphorylation of S177 enhances eIF4A-mediated translation during G1/S. Together these results suggest that dynamic phosphorylation of eIF4A S177 serves to stimulate translation during G1/S, while inhibitory phosphorylation of additional sites holds the potential to rapidly transition eIF4A to an inactive state and turn off translation. These results also suggest an important role for eIF4A in coupling translation to cell cycle stages.

Metformin, the widely used and studied diabetes drug, has been reported to have anti-cancer effects. However, in retrospective clinical studies, the survival benefit associated with metformin use in cancer patients is often modest. Consequently, the design and conduct of sufficiently powered prospective clinical trials aimed at exploring this drug’s anti-tumor potential are hampered by the absence of predictive biomarkers. Testing the notion that specific patient characteristics such as obesity may impact the relative potency of metformin’s effects on lung cancer survival, we analyzed the patient outcomes of 756 non-small cell lung cancer (NSCLC) cases with early- (stage I/II, n = 490) or late-stage disease (stage III/IV, n = 266) that either did or did not use metformin (n = 121 and 635, respectively) while not receiving neoadjuvant therapy. We found that metformin use is associated with improved overall and recurrence free survival only in overweight and obese patients defined by a body mass index (BMI) greater than 25 kg/m2. Corroborative findings obtained from immunocompetent mouse lung cancer models aligned with these observations. In these studies, despite supporting accelerated growth of both primary and metastatic tumors compared to their normal weight counterparts, animals subjected to diet induced obesity (DIO) experienced significantly reduced tumor development upon metformin treatment. In contrast, the drug had little-to-no effect on tumor burden in normal weight controls. Furthermore, while flow cytometric evaluation of obese mouse-derived tumors identified several previously described mediators of immune dysfunction previously described in animals with DIO (e.g., widespread up-regulation of the immune checkpoint factors PD-1 and Lag3, elevated suppressor cell proportions and activity), metformin treatment was associated with their reversal specifically in the tumors of obese mice. Interestingly, the drug had little effect on the tumor immune contexture of normal weight mice, and gene expression analysis failed to reveal considerable alterations in canonical cancer pathways. Thus, our preclinical and clinical studies suggest that the anti-diabetic drug metformin has an anti-cancer effect in NSCLC that is restricted to overweight individuals and reflects its potential to direct favorable, context-specific immune reprogramming. This work also provides the rationale for using high BMI as a predictive biomarker of the anti-cancer effect of metformin in lung cancer. Citation Format: Joseph Barbi, Randall J. Smith, Robert Zollo, Stephanie N. Sass, Deschana Washington, Cara Petrucci, Aravind Srinivasan, Eric Kannisto, Santosh Patnaik, Sai Sai Yendamuri. Obesity dependent benefits of metformin for improved anti-tumor immunity and outcomes in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2082.

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