Julia Kargl

Mutant IDH1 regulates the tumor-associated immune system in gliomas

Gliomas harboring mutations in isocitrate dehydrogenase 1/2 (IDH1/2) have the CpG island methylator phenotype (CIMP) and significantly longer patient survival time than wild-type IDH1/2 (wtIDH1/2) tumors. Although there are many factors underlying the differences in survival between these two tumor types, immune-related differences in cell content are potentially important contributors. In order to investigate the role of IDH mutations in immune response, we created a syngeneic pair mouse model for mutant IDH1 (muIDH1) and wtIDH1 gliomas and demonstrated that muIDH1 mice showed many molecular and clinical similarities to muIDH1 human gliomas, including a 100-fold higher concentration of 2-hydroxygluratate (2-HG), longer survival time, and higher CpG methylation compared with wtIDH1. Also, we showed that IDH1 mutations caused down-regulation of leukocyte chemotaxis, resulting in repression of the tumor-associated immune system. Given that significant infiltration of immune cells such as macrophages, microglia, monocytes, and neutrophils is linked to poor prognosis in many cancer types, these reduced immune infiltrates in muIDH1 glioma tumors may contribute in part to the differences in aggressiveness of the two glioma types.

Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE−/− mice are protected from asbestos‐induced lung fibrosis. NE−/− mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF‐β‐independent fashion. Treatment of asbestos‐recipient mice with ONO‐5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE‐inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.

Lung Cancer Subtypes Generate Unique Immune Responses

Lung cancer, the leading cause of cancer-related deaths worldwide, is a heterogeneous disease comprising multiple histologic subtypes that harbor disparate mutational profiles. Immune-based therapies have shown initial promise in the treatment of lung cancer patients but are limited by low overall response rates. We sought to determine whether the host immune response to lung cancer is dictated, at least in part, by histologic and genetic differences, because such correlations would have important clinical ramifications. Using mouse models of lung cancer, we show that small cell lung cancer (SCLC) and lung adenocarcinoma (ADCA) exhibit unique immune cell composition of the tumor microenvironment. The total leukocyte content was markedly reduced in SCLC compared with lung ADCA, which was validated in human lung cancer specimens. We further identified key differences in immune cell content using three models of lung ADCA driven by mutations in Kras, p53, and Egfr. Although Egfr-mutant cancers displayed robust myeloid cell recruitment, they failed to mount a CD8+ immune response. In contrast, Kras-mutant tumors displayed significant expansion of multiple immune cell types, including CD8+ cells, regulatory T cells, IL-17A–producing lymphocytes, and myeloid cells. A human tissue microarray annotated for KRAS and EGFR mutations validated the finding of reduced CD8+ content in human lung ADCA. Taken together, these findings establish a strong foundational knowledge of the immune cell contexture of lung ADCA and SCLC and suggest that molecular and histological traits shape the host immune response to cancer.

Insulin receptor substrate-1 deficiency drives a proinflammatory phenotype in KRAS mutant lung adenocarcinoma

Significance Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant lung adenocarcinoma remains an intractable lung cancer subtype for which efficacious targeted therapies do not exist. This study identified a subpopulation of KRAS mutant lung adenocarcinoma with reduced insulin receptor substrate-1 (IRS-1) content and showed that this group is particularly amenable to Janus kinase/signal transducers and activators of transcription (JAK/STAT) inhibition. This finding is the first definitive report of a prohost role for IRS-1 in a solid tumor malignancy in humans. Furthermore, the data supports a novel role for IRS-1 in the sculpting of immune cell composition within the tumor microenvironment. Insulin receptor substrate-1 (IRS-1) is a signaling adaptor protein that interfaces with many pathways activated in lung cancer. It has been assumed that IRS-1 promotes tumor growth through its ability to activate PI3K signaling downstream of the insulin-like growth factor receptor. Surprisingly, tumors with reduced IRS-1 staining in a human lung adenocarcinoma tissue microarray displayed a significant survival disadvantage, especially within the Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant subgroup. Accordingly, adenoviral Cre recombinase (AdCre)-treated LSL-Kras/Irs-1fl/fl (Kras/Irs-1−/−) mice displayed increased tumor burden and mortality compared with controls. Mechanistically, IRS-1 deficiency promotes Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling via the IL-22 receptor, resulting in enhanced tumor-promoting inflammation. Treatment of Kras/Irs-1+/+ and Kras/Irs-1−/− mice with JAK inhibitors significantly reduced tumor burden, most notably in the IRS-1-deficient group.

Abstract A12: Non-small cell lung tumor-derived autoantibodies can distinguish benign from malignant pulmonary nodules

High-risk smokers are currently being screened with low-dose CT imaging after the National Lung Screening Trial demonstrated a 20% reduction in lung cancer mortality. Unfortunately, small pulmonary nodules are a common finding in smokers (present in 25-50%), with very few ( Citation Format: Kristin J. Lastwika, Julia Kargl, Yuzheng Zhang, Xiaodong Zhu, David Shelley, Edward Lo, Wei Wu, Sudhakar N. Pipavath, Paul E. Kinahan, A. McGarry Houghton, Paul D. Lampe. Non-small cell lung tumor-derived autoantibodies can distinguish benign from malignant pulmonary nodules [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A12.

Abstract B26: Novel pro-host role for insulin receptor substrate-1 in lung adenocarcinoma

Lung cancer is the leading cause of cancer deaths worldwide. In the United States alone, lung cancer accounts for ~160,000 deaths per year while the five-year survival rate remains stagnant at ~15%. Lung adenocarcinoma (ADCA) accounts for over 50% of non-small lung cancer cases. Within this subset, K-ras is the most common activating mutation accounting for ~35% of cases. Kras mutant tumors have remained a largely un-targetable subtype and a better understanding of the signaling pathway involved in the different ADCA subtypes will be necessary for the development of therapeutics. Insulin Receptor Substrate-1 (IRS-1) is a signaling adaptor protein that interfaces with many of the pathways that are activated in lung cancer. We have undertaken a controlled study of this protein within the context of lung ADCA. Analysis of a well-annotated human lung ADCA TMA (n=136) revealed that positive IRS-1 staining provided a 75-month median survival advantage among all ADCA cases (p=0.01) and an 81-month median advantage within the K-ras subtype (p=0.006). EGFR and non-K-ras/non-EGFR cases did not display differences. To focus in on the K-ras subtype, we generated LSL-K-ras/IRS-1fl/fl mice and studied them over a time course. LSL-K-ras/IRS-1-/- mice displayed highly significant early mortality (p Citation Format: Heather E. Metz, Julia Kargl, Stephanie Busch, Kyoung-Hee Kim, McGarry Houghton. Novel pro-host role for insulin receptor substrate-1 in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B26.

Abstract PR04: Inflammatory responses within the lung tumor microenvironment correlate with oncogenic mutation and histologic subtype

Lung cancer is a heterogeneous disease comprised of multiple histologic subtypes that harbor disparate mutational landscapes. Small cell lung carcinoma (SCLC), in which RB1 and TP53 mutations are common, accounts for 10-20% of lung cancer diagnoses. Approximately half of non-small cell lung carcinoma cases are classified as lung adenocarcinomas (ADCA), in which KRAS , EGFR , and TP53 mutations are the predominant genetic drivers. Novel immunotherapeutic strategies have offered new hope for the management of ADCA and SCLC, but the clinical success of immunomodulatory agents will depend on a strong foundational knowledge of the cells that comprise the lung tumor microenvironment (TME) in these molecularly and histologically distinct diseases. We therefore sought to determine whether the host immune response to lung cancer is predicated, at least in part, by histologic and genetic differences, as such correlations would have important clinical ramifications. Using three mouse models of lung ADCA Kras LSL-G12D , Kras LSL-G12D ;Trp53 Fl/Fl , and Egfr L858R as well as the Rb1 Fl/Fl ;Trp53 Fl/Fl model of SCLC, we show that SCLC and ADCA tumors exhibit unique immune cell composition. Lung specimens from tumor-bearing and control animals ( n ≥ 5 per cohort) were harvested at multiple time points and subjected to histological assessment and comprehensive flow cytometric immunophenotyping. Tumor-associated inflammation was discernibly lower in SCLC compared to all ADCA models. Moreover, the leukocyte composition of SCLC was dominated by cells of the adaptive rather than innate arm of the host immune system, a finding subsequently validated in 2 human SCLC surgical specimens. We further identified key differences in leukocyte content of mouse ADCA that correlated with oncogenic mutation. Although Egfr -mutant cancers displayed robust myeloid cell recruitment, they failed to mount a CD8 + cellular immune response. In contrast, Kras -mutant tumors displayed significant expansion of multiple immune cell types, including CD8 + cells, regulatory T cells, IL17-producing lymphocytes, and myeloid cells. Although loss of Trp53 promoted malignancy, it had minimal effect on immune cell composition within the Kras TME. Pre-clinical in vivo investigations with immune checkpoint inhibitor compounds are currently being conducted to assess the importance of these distinct immune responses to tumor progression and survival. To determine whether the differential inflammatory responses observed in mice are clinically relevant, we conducted an immune profiling study of a large lung cancer patient cohort that included 55 ADCA specimens. Notably, patients with EGFR mutations ( n = 5) exhibited fewer total CD8 + T cells, as well as decreased exhausted CD8 + PD1 + and CD8 + TIM3 + content, compared to all other ADCA cases, thereby substantiating the murine phenotype. Taken together, these findings establish a strong foundational knowledge of the immune cell contexture of lung ADCA and SCLC and suggest that molecular and histological traits shape the host immune response to cancer. This abstract is also presented as Poster A01. Citation Format: Stephanie E. Busch, Julia Kargl, Mark L. Hanke, Heather E. Metz, Kyoung-Hee Kim, A McGarry Houghton. Inflammatory responses within the lung tumor microenvironment correlate with oncogenic mutation and histologic subtype. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR04.

Abstract 463: Successful generation of tumor infiltrating lymphocytes from human non-small cell lung cancer specimens

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Tumor infiltrating lymphocytes (TILs) are naturally occurring cancer-fighting T cells found within malignant tumors. After decades of research, patient-derived TIL populations have been successfully re-infused into melanoma subjects, and have produced impressive treatment effects in a subset of cases. To date, the successful generation and usage of TIL populations for non-small cell lung cancer (NSCLC) patients has not been reported. Generating TILs from NSCLC tissue presents several challenges, as immune cell populations residing within the lungs are constantly exposed to numerous inhaled antigens and other particulate matter. Here, we report the successful generation of TILs from three NSCLC specimens from subjects undergoing surgical resection for curative intent. Six distinct lung cancer tissue fragments were cultured in the presence of IL-2 for each of the three cases. Each fragment yielded a TIL population, although their proliferative rates differed substantially. Each TIL population was subjected to flow cytometry to identify the cellular subtypes, and these results were compared to the lymphocyte subset content measured in the original in vivo tumor specimen. Preliminary findings suggest that the ability to generate CD8+ enriched TILs is dependent upon the CD8+ content present in vivo. Low CD8+ content was associated with an abundance of either Tregs or NK cells. Greater than 70% of the CD8+ populations generated displayed an effector phenotype (CCR7-CD45RA+), regardless of the overall cellular composition of the specimen. However, these cells displayed heterogeneous levels of exhaustion markers (PD1, Tim3), which inversely correlated with proliferative rates. Our results show that TIL populations can be successfully generated from primary human NSCLC specimens. Although the relative cellular content differed for each tissue fragment, we were able to generate at least one CD8+ effector population with low levels of exhaustion for each case. In vitro and in vivo studies to determine the tumor killing capacity of these TIL populations are currently underway. Note: This abstract was not presented at the meeting. Citation Format: Kyoung-Hee Kim, Julia Kargl, Sylvia Lee, McGarry Houghton. Successful generation of tumor infiltrating lymphocytes from human non-small cell lung cancer specimens. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 463. doi:10.1158/1538-7445.AM2015-463

Abstract 1290: Distinct immune cell composition for lung squamous cell carcinoma (SCCA) and adenocarcinoma (ADCA)

Lung cancer is the leading cause of cancer deaths worldwide, with ∼15% of lung cancer patients surviving for five years. Recent approaches in cancer therapy target immunosuppressive factors in the tumor microenvironment (TME) capable of derailing an effective immune response, using checkpoint inhibitors (e.g. anti-PD-1/PDL-1). Unfortunately, only ∼20% of patients benefit from these treatments and appropriate studies are of importance to determine the underlying molecular mechanisms. Lung cancer is a heterogeneous disease classified by multiple histologic subtypes, with ADCA and SCCA representing the majority of non-small cell lung cancer (NSCLC). To evaluate the complexity of the immune cell composition of the TME of human NSCLC we examined tumor and non-adjacent lung tissue from the same patients. Tissue specimen (n = 43) with corresponding clinicopathologic information were analyzed by flow cytometry and immunohistochemistry. Next generation sequencing for TCRs was performed to assess the TCR repertoire. Surprisingly we found that immune cells comprise ∼80% of the tumor mass in NSCLC. Many immune cell types are significantly increased in tumor tissue, when compared to normal lung, such as CD3+ and CD4+ T-cells, as well as B-cells. Notably, CD4+ cell subtypes including Th17 and regulatory T cells (Treg), and IL-17-expressing γδ T cells are increased. In contrast, decreased presence of Th1 cells was observed in tumor tissue. Remarkably, immune cell compositions are unique for lung ADCA and SCCA. In SCCA we observed a Treg (p In contrast, ADCA represents a Th17 signature, revealing elevated levels of IL17-expressing CD4+ T-cells and γδ T cells. While SCCA immune cell content clusters well, immune cell composition in ADCA is very heterogeneous. Preliminary data suggest that driver-mutations in ADCA such as EGFR predict distinct immune cell composition. These data provide evidence that the immune cell composition present within different NSCLC subtypes displays unique phenotypes and identifying the immunosuppressive factors in different subsets will be important for successful immune-based therapy. Citation Format: Julia Kargl, Kyoung-Hee Kim, Stephanie E. Busch, Mark L. Hanke, Heather E. Metz, Martin W. McIntosh, A McGarry Houghton. Distinct immune cell composition for lung squamous cell carcinoma (SCCA) and adenocarcinoma (ADCA). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1290. doi:10.1158/1538-7445.AM2015-1290

Abstract 4860: Insulin receptor substrate-1 regulates immune cell content in lung adenocarcinoma

Lung cancer is the leading cause of cancer deaths worldwide. In the United States, lung cancer accounts for ∼160,000 deaths per year while the five-year survival rate remains stagnant at ∼15%. A better understanding of the pathobiology for this disease is imperative for development of novel therapeutics to improve mortality rates. One of the most common activating mutations in lung cancer is K-ras. Our group previously showed that K-ras activation generates an inflammatory response in lung tumors, mediated largely through the increased production of neutrophil chemokines by tumor cells. The corresponding increase in neutrophil recruitment in this model increased tumor growth, which was associated with degradation of the intracellular protein insulin receptor substrate-1 (IRS-1). A study of non-small cell lung cancer demonstrated that IRS-1 was low or absent in 46% of cases. To further dissect the role of IRS-1 in tumor growth we generated LSL-K-ras/IRS-1 fl/fl mice and subjected them to adenoviral cre. LSL-K-ras/IRS-1-/- mice displayed early mortality and increased tumor burden when compared to LSL-K-ras/IRS-1+/+ controls. Surprisingly, IRS-1 loss in tumor cells generated a robust immune response. The bronchial alveolar lavage fluid of LSL-K-ras/IRS-1-/- mice showed increased infiltration of macrophages, neutrophils and lymphocytes. Further examination of the chemokine profiles of LSL-K-ras/IRS-1-/- and LSL-K-ras/IRS-1+/+ mice showed a significant increase in many immune cell-recruiting chemokines in the LSL-K-ras/IRS-1-/- mice including CCL2, CCL3, CCL4, CXCL1, CXCL2 and CXCl12. Many cancers are able to manipulate the host immune response through signaling pathways that interface with IRS-1, including: phosphoinositol 3-kinase (PI3K), extracellular signal regulated kinase (MEK/ERK) and Janus kinase/signal transducer and activator of transcription (JAK/STAT). As some cytokines can activate IRS-1 via JAK/STAT signaling, we suspected that IL-17 and IL-22 might function in this regard. Loss of IRS-1 increases pSTAT3 production, which triggers chemokine release and the generation of a pro-tumor immune response. Preliminary data to support this hypothesis include: 1) presence of IL-17 and IL-22 producing cells (e.g. Th17) at sites of tumor in LSL-K-ras mice, 2) induction of pTyr-IRS-1 upon IL-17 and IL-22 stimulation in A549 cells, and 3) increased CC and CXC chemokines in IRS-1 deficient cells upon IL-17 and IL-22 stimulation. Experimentation is ongoing to further elucidate the mechanism behind this phenotype. Citation Format: Heather E. Metz, Stephanie E. Busch, Mark L. Hanke, Julia Kargl, Kyoung-Hee Kim, A McGarry Houghton. Insulin receptor substrate-1 regulates immune cell content in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4860. doi:10.1158/1538-7445.AM2014-4860