Stephanie E. Busch

CTCF Haploinsufficiency Destabilizes DNA Methylation and Predisposes to Cancer

Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf(+/-) tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf(+/-) tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.

Neutrophils dominate the immune cell composition in non-small cell lung cancer.

The response rate to immune checkpoint inhibitor therapy for non-small-cell lung cancer (NSCLC) is just 20%. To improve this figure, several early phase clinical trials combining novel immunotherapeutics with immune checkpoint blockade have been initiated. Unfortunately, these trials have been designed without a strong foundational knowledge of the immune landscape present in NSCLC. Here, we use a flow cytometry panel capable of measuring 51 immune cell populations to comprehensively identify the immune cell composition and function in NSCLC. The results show that the immune cell composition is fundamentally different in lung adenocarcinoma as compared with lung squamous cell carcinoma, and that neutrophils are the most prevalent immune cell type. Using T-cell receptor-β sequencing and tumour reactivity assays, we predict that tumour reactive T cells are frequently present in NSCLC. These results should help to guide the design of clinical trials and the direction of future research in this area.

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.

COPD Alters Immune Cell Composition and Immune Checkpoint Inhibitor Efficacy in NSCLC

Rationale: Chronic obstructive pulmonary disease (COPD) and non‐small cell lung cancer (NSCLC) are interrelated diseases with substantial mortality, and the pathogenesis of both involves aberrant immune functioning. Objectives: To profile immune cell composition and function in patients with NSCLC and describe the effects of COPD on lung and tumor microenvironments. Methods: We profiled resected lung and tumor tissue using flow cytometry and T‐cell receptor sequencing in patients with and without COPD from a prospective cohort of patients undergoing resection of NSCLC. A murine cigarette smoke exposure model was used to evaluate the effect on pulmonary immune populations. A separate retrospective cohort of patients who received immune checkpoint inhibitors (ICIs) was analyzed, and their survival was quantified. Measurements and Main Results: We observed an increased number of IFN‐&ggr;‐producing CD8+ and CD4+ (T‐helper cell type 1 [Th1]) lymphocytes in the lungs of patients with COPD. In both humans and mice, increased Th17 content was seen with smoke exposure, but was not associated with the development or severity of COPD. COPD‐affected lung tissue displayed increased Th1 differentiation that was recapitulated in the matching tumor sample. PD‐1 (programmed cell death protein 1) expression was increased in tumors of patients with COPD, and the presence of COPD was associated with progression‐free survival in patients treated with ICIs. Conclusions: In patients with COPD, Th1 cell populations were expanded in both lung and tumor microenvironments, and the presence of COPD was associated with longer progression‐free intervals in patients treated with ICIs. This has implications for understanding the immune mediators of COPD and developing novel therapies for NSCLC.

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 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 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

Abstract A34: Arf suppresses the growth and progression of Kras-driven lung tumors

Non-small cell lung carcinoma (NSCLC) is among the deadliest of human cancers, with an overall five-year survival rate of 10–20%. Activating mutations in the Kras proto-oncogene occur in approximately one-third of NSCLC patients, but tumor suppression in the lung remains poorly elucidated. The Cdkn2a locus, which houses both the p16 lnk4a and Arf tumor suppressor genes, is frequently altered in NSCLC; however, the specific role of Arf in lung tumorigenesis has not been well characterized. Several lines of evidence indicate that Kras and other oncogenes induce the expression of Art, which in turn stabilizes p53 activity and arrests the growth of transformed cells. To determine the extent to which Arf suppresses NSCLC development in vivo, we employed the urethane chemical carcinogenesis model of Kras -driven NSCLC. We treated both wild-type and Arf -deficient mice with urethane and found that early-stage lung adenomas in wild-type mice robustly expressed Arf, In contrast to adenomas, malignant lung adenocarcinomas exhibited little induction of Art. Moreover, Arf -deficient mice exposed to urethane displayed accelerated lung tumor-associated morbidity and significantly increased lung tumor size compared to Arf wild-type and heterozygous littermates. Malignant progression of adenomas to adenocarcinomas was also markedly elevated in Arf -deficient mice. These data demonstrate that Arf functions as a barrier to tumor promotion and progression in a carcinogen model of Kras -driven NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A34.