- Email: [email protected]
Lineage Specifiers in Lung Cancer Are Ahead of Their TIME
Immunity
Previews underscore the importance of elucidating the detailed mechanism by which 20 30 cGAMP mounts the antitumor immune response. The data from Marcus et al. (2018) suggest that targeting cGAMP to effector populations that facilitate a robust type I IFN response capable of activating NK cells would be most effective in priming tumor clearance. As opposed to direct tumor injection, it is possible that more sophisticated approaches to retaining cGAMP in the tumor microenvironment and targeting it to certain cell populations will be more effective. In addition, this work also suggests that therapeutic strategies that naturally engage cGAMP-STING signaling, such as treatment with certain DNA damaging agents, are likely to prime innate immune responses, which could be tailored in combination therapies to maximize NK cell activation. Regardless, these findings provide a rationale to further our understanding of the mechanisms of 20 30 -cGAMP transport and modulation of cGAS activity in order to maximally augment tumor immunogenicity and broaden the therapeutic utility of cancer immunotherapy.
DECLARATION OF INTERESTS D.A.B. is a consultant for N-of-One and has received honoraria from Loxo Oncology and Madalon Consulting and research grants from Bristol-Myers Squibb and Novartis. REFERENCES Bridgeman, A., Maelfait, J., Davenne, T., Partridge, T., Peng, Y., Mayer, A., Dong, T., Kaever, V., Borrow, P., and Rehwinkel, J. (2015). Viruses transfer the antiviral second messenger cGAMP between cells. Science 349, 1228–1232. Chen, Q., Boire, A., Jin, X., Valiente, M., Er, E.E., Lopez-Soto, A., Jacob, L., Patwa, R., Shah, H., Xu, K., et al. (2016). Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer. Nature 533, 493–498. €ck, S., Guey, B., Gulen, M.F., Wolter, K., Kang, Glu T.W., Schmacke, N.A., Bridgeman, A., Rehwinkel, J., Zender, L., and Ablasser, A. (2017). Innate immune sensing of cytosolic chromatin fragments through cGAS promotes senescence. Nat. Cell Biol. 19, 1061–1070. Lam, A.R., Bert, N.L., Ho, S.S., Shen, Y.J., Tang, L.F., Xiong, G.M., Croxford, J.L., Koo, C.X., Ishii, K.J., Akira, S., et al. (2014). RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma. Cancer Res. 74, 2193–2203. Marcus, A., Mao, A.J., Lensink-Vasan, M., Wang, L., Vance, R.E., and Raulet, D.H. (2018). Tumor-
derived cGAMP triggers a STING-mediated interferon response in non-tumor cells to activate the NK cell response. Immunity 49, this issue, 754–763. Ng, K.W., Marshall, E.A., Bell, J.C., and Lam, W.L. (2018). cGAS-STING and cancer: Dichotomous roles in tumor immunity and development. Trends Immunol. 39, 44–54. Sade-Feldman, M., Jiao, Y.J., Chen, J.H., Rooney, M.S., Barzily-Rokni, M., Eliane, J.P., Bjorgaard, S.L., Hammond, M.R., Vitzthum, H., Blackmon, S.M., et al. (2017). Resistance to checkpoint blockade therapy through inactivation of antigen presentation. Nat. Commun. 8, 1136. Woo, S.R., Fuertes, M.B., Corrales, L., Spranger, S., Furdyna, M.J., Leung, M.Y., Duggan, R., Wang, Y., Barber, G.N., Fitzgerald, K.A., et al. (2014). STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity 41, 830–842. Xia, T., Konno, H., and Barber, G.N. (2016). Recurrent loss of STING signaling in melanoma correlates with susceptibility to viral oncolysis. Cancer Res. 76, 6747–6759. Zaretsky, J.M., Garcia-Diaz, A., Shin, D.S., EscuinOrdinas, H., Hugo, W., Hu-Lieskovan, S., Torrejon, D.Y., Abril-Rodriguez, G., Sandoval, S., Barthly, L., et al. (2016). Mutations associated with acquired resistance to PD-1 blockade in melanoma. N. Engl. J. Med. 375, 819–829.
Lineage Specifiers in Lung Cancer Are Ahead of Their TIME Kelli A. Connolly,1 Brittany Fitzgerald,1 and Nikhil S. Joshi1,* 1Yale University School of Medicine, Department of Immunobiology, New Haven, CT 06519, USA *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2018.10.004
The factors that shape the distinctive tumor-immune landscapes of various types and subtypes of cancer remain poorly understood. In this issue of Immunity, Mollaoglu et al. (2018) reveal a mechanistic link between the function of lineage specifiers SOX2 and NKX2-1 and the presence of neutrophils in the tumor-immune microenvironment of lung cancer. Non-small-cell lung cancer (NSCLC) is a complex disease that accounts for approximately 85% of lung cancer. NSCLC is composed of two primary subtypes, lung adenocarcinoma (LADC) and lung squamous-cell carcinoma (LSCC). NSCLC patient tumors contain a variety of driver mutations that contribute to the heterogeneity between subtypes.
LADC commonly contains alterations in KRAS and EGFR and in tumor suppressors TP53, LKB1, KEAP1, and NF1. In contrast, LSCC frequently harbors mutations in TP53 and CDKN2A and has some EGFR mutations that do not respond to small-molecule therapy (Herbst et al., 2018). The cell-lineagespecifying transcription factors NKX2-1
and SOX2 are often overexpressed in LADC (Tanaka et al., 2007) and LSCC (Bass et al., 2009), respectively. The growing understanding of the molecular heterogeneity of patient NSCLC over the past two decades has led to a shift from general chemotherapy treatments to first-line precision medicine, which ranges from administration of
Immunity 49, October 16, 2018 ª 2018 Elsevier Inc. 587
Immunity
Previews
Figure 1. The Mechanisms of Genetic Alterations in Cancer Could Inform More Personalized Therapy SOX2 recruits tumor-associated neutrophils via regulation of chemoattractants CXCL5 (in mice) and CXCL6 (in humans), and high TAN infiltration correlates with squamous subtype and poorer prognosis. NKX2-1 has an inverse effect and leads to lower TAN recruitment. The SOX2/NKX2-1 axis described by Mollaoglu et al. in this issue of Immunity reveals the therapeutic potential of using high SOX2 and low NKX2-1 gene expression as a marker for identifying patients who might benefit from combining checkpoint therapy.
small-molecule inhibitors in tyrosine-kinase-receptor-driven disease to the use of checkpoint-inhibitor immunotherapy in PDL1-expressing cancers. The lungcancer field has been a clinical leader for matching treatment to specific markers of disease. Accordingly, the search for molecular markers useful for therapeutic prediction remains fervent. In this issue of Immunity, Mollaoglu et al. (2018) provide insight into the molecular mechanisms driving LSCC and LADC and the corresponding impacts on the tumor immune microenvironment (TIME). The success of immunotherapies in producing long-term regression in cancer patients has made clear the immune system’s impact on cancer progression and treatment outcomes, and recent studies have shown effectiveness of checkpointblockade therapies in NSCLC patients. Nevertheless, efficacy of immunotherapies has been restricted to only a portion of NSCLC patients. Investigations into the mechanism(s) underlying responsiveness to immunotherapy have highlighted how much remains unknown about the TIME and its determinants. In NSCLC, these studies have led to the discovery that the immune landscapes of LSCC and LADC are distinct, suggesting a link between histologic subtypes and the quality and makeup of the TIME. Still, a direct relationship between molecular drivers 588 Immunity 49, October 16, 2018
of lung-cancer subtypes and the TIME has remained elusive. One reason for this is the level of difficulty required in experimentally uncoupling the functions of lineage-determining transcription factors from their associated histologic cancer subtypes. Oliver and colleagues overcome this challenge through the manipulation and development of various genetically engineered mouse models (GEMMs) that recapitulate the molecular characteristics of histologically distinct lung-cancer subtypes; such characteristics include the expression of lineage specifiers SOX2 and NKX2-1. Previous work by the Oliver group and others demonstrated that tumor-cell genetics can impact the differentiation of lung cancers into distinct histologic subtypes, for example by affecting Myc expression in a neuroendocrine-low subtype of small-cell lung cancer (Mollaoglu et al., 2017). More specifically, the combination of SOX2 overexpression and LKB1 deletion promotes LSCC histopathology (Mukhopadhyay et al., 2014). Following up on these findings, Mollaoglu et al. (2018) developed a novel GEMM of LSCC-like disease in which the overexpression of SOX2 and deletion of LKB1 were achieved by the expression of Cre recombinase in cells. LSCC-like disease from these Rosa26LSL-Sox2-IRES-GFP; Lkb1fl/fl (SL) mice share molecular drivers
with other previously established GEMMs of LSCC, such as Lkb1fl/fl;Ptenfl/fl (LP) and Sox2LSL/LSL;Ptenfl/fl;Cdkn2abfl/fl (Sox2PC) mice, but also with adenosquamous tumors from KrasLSL-G12D/+;Lkb1fl/fl (KL) mice. Tumors across all four of these GEMMs are abundant in neutrophils, suggesting a role for the shared molecular drivers in determining aspects of the TIME. Direct comparison of tumors from these models facilitated a deeper understanding of the molecular mechanism of LSCC tumorigenesis. Furthermore, the authors interrogated the precise functions of the lineage specifiers SOX2 and NKX2-1 in both tumor-cell fate and the overall TIME by temporally deleting NKX2-1 (highly associated with the adenocarcinoma subtype) and overexpressing SOX2 (highly associated with the squamous-cell subtype) in a mouse model of LADC. Although the inhibition of neutrophil infiltration in lung cancer has shown promise in preclinical studies, little is known of the mechanism(s) of recruitment or precise functions in various malignant settings. By combining their advanced genetic models with ChIP-seq and gene expression data, the authors outline a mechanism linking the expression of the transcription factors SOX2 and NKX2-1 in tumor cells and the chemotaxis of tumor-associated neutrophils (TAN). Using an unbiased comprehensive approach, Mollaoglu et al. (2018) uncovered direct transcriptional regulation of the neutrophil chemokine CXCL5, by both SOX2 and NKX2-1. They also found binding sites for both SOX2 and NKX2-1 in the promoter region and first exon of Cxcl5 and showed that CXCL5 expression is inversely regulated by these two lineage specifiers. Although specific genetic or pharmacologic targeting of CXCL5 is not explored in this report, these studies raise the possibility that neutrophil chemoattractants might mediate the histological determination of malignancies and therefore present an attractive therapeutic option for patients with LSCC. Furthermore, analysis of TCGA data of patients across all lungcancer subtypes revealed that CXCL6, the human ortholog of mouse CXCL5, correlated with squamous subtype and high SOX2 and low NKX2-1 expression in human NSCLC samples, highlighting the translational applicability of the findings. Thus, this elegant investigation takes
Immunity
Previews great strides toward answering the question of how changes in tumor-cell-intrinsic factors impact cancer histopathology and quality of the TIME. The role of neutrophils in cancer remains a contentious topic and, accordingly, the mechanisms of intratumoral neutrophil recruitment and function are active areas of research. Although neutrophils are often a prevalent immune cell type in tumors of NSCLC patients, they have been found in greater quantities in LSCC than in LADC patients (Kargl et al., 2017). In a mouse model of NSCLC, neutrophils contributed directly and indirectly to cancer progression by establishing immune exclusion and modifying blood vessels, resulting in increased hypoxia and stabilization of the transcription factor HIF1a (Faget et al., 2017). Additionally, data suggest that a rising neutrophil-tolymphocyte ratio in patients during nivolumab treatment is potentially predictive of a shorter time to treatment failure (Kiriu et al., 2018). Conversely, intratumoral neutrophils are often heterogeneous, and evidence for neutrophils with antitumor functions have been observed. For example, Singhal et. al. have revealed the existence of a sub-population of intratumoral neutrophils capable of presenting antigen and augmenting anti-tumor T cell responses (Singhal et al., 2016). In the present study, through a combination of TAN-depletion studies and single-cell RNAseq, Mollaoglu et al. (2018) demonstrated largely pro-tumor effects of TANs and cooperation with genetic alterations, which promoted the development of tumors of the LSCC histologic subtype. Notably, neutrophil depletion not only resulted in the development of fewer LSCC tumors but also seemed to promote a LADC phenotype. These data suggest that the TIME, and the prevalence of neutrophils in particular, could promote the growth of histologically distinct tumor cells. These findings have interesting clinical implications because transdifferentiation of LADC to LSCC has been
documented in patients with relapsing disease after therapy. Taken together, these findings open a number of questions regarding the potential impacts of current therapies on intratumoral neutrophils and whether combination with neutrophiltargeted therapies could inhibit LADC to LSCC transdifferentiation and improve responses of LADC patients to therapy. The mechanisms for how tumor genetics regulate the quality of the TIME are understudied and have implications for improving patient selection for more personalized therapies. For example, understanding how specific genetic alterations impact TAN infiltration could help identify cancer patients who would benefit from combining checkpoint-blockade therapy with CXCL6 inhibitors, as illustrated in Figure 1. The push in cancer genetics by way of large-consortium-style analysis of human patient samples opens important questions about what these data mean biologically for the treatment of particular patient subgroups. Efforts made by the International Cancer Genomic Consortium (https://icgc.org) and The Cancer Genome Atlas (TCGA; https://cancergenome.nih.gov/) have resulted in an abundance of genomic and transcriptomic data with potential relevance to precision cancer treatment. By revealing a mechanistic link between lineage specifiers and the shaping of the TIME, the findings by Oliver and colleagues highlight the importance of a deeper understanding of the relationship between tumor-cell-intrinsic factors and the determinants of the anti-tumor immune response. Complimentary studies that examine the immunological context associated with genetic alterations present in human cancers should ultimately improve our ability to design effective therapies for difficult-to-treat malignancies. REFERENCES Bass, A.J., Watanabe, H., Mermel, C.H., Yu, S., Perner, S., Verhaak, R.G., Kim, S.Y., Wardwell,
L., Tamayo, P., Gat-Viks, I., et al. (2009). SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas. Nat. Genet. 41, 1238–1242. Faget, J., Groeneveld, S., Boivin, G., Sankar, M., Zangger, N., Garcia, M., Guex, N., Zlobec, I., Steiner, L., Piersigilli, A., et al. (2017). Neutrophils and Snail Orchestrate the Establishment of a Pro-tumor Microenvironment in Lung Cancer. Cell Rep. 21, 3190–3204. Herbst, R.S., Morgensztern, D., and Boshoff, C. (2018). The biology and management of non-small cell lung cancer. Nature 553, 446–454. Kargl, J., Busch, S.E., Yang, G.H., Kim, K.H., Hanke, M.L., Metz, H.E., Hubbard, J.J., Lee, S.M., Madtes, D.K., McIntosh, M.W., and Houghton, A.M. (2017). Neutrophils dominate the immune cell composition in non-small cell lung cancer. Nat. Commun. 8, 14381. Kiriu, T., Yamamoto, M., Nagano, T., Hazama, D., Sekiya, R., Katsurada, M., Tamura, D., Tachihara, M., Kobayashi, K., and Nishimura, Y. (2018). The time-series behavior of neutrophilto-lymphocyte ratio is useful as a predictive marker in non-small cell lung cancer. PLoS ONE 13, e0193018. €gelMollaoglu, G., Guthrie, M.R., Bo¨hm, S., Bra mann, J., Can, I., Ballieu, P.M., Marx, A., George, J., Heinen, C., Chalishazar, M.D., et al. (2017). MYC drives progression of small cell lung cancer to a variant neuroendocrine subtype with vulnerability to aurora kinase inhibition. Cancer Cell 31, 270–285. Mollaoglu, G., Jones, A., Wait, S.J., Mukhopadhyay, A., Jeong, S., Arya, R., Camolotto, S.A., Mosbruger, T.L., Stubben, C.J., Conley, C.J., et al. (2018). The lineage-defining transcription factors SOX2 and NKX2-1 determine lung-cancer cell fate and shape the tumor immune microenvironment. Immunity 49, this issue, 764–779. Mukhopadhyay, A., Berrett, K.C., Kc, U., Clair, P.M., Pop, S.M., Carr, S.R., Witt, B.L., and Oliver, T.G. (2014). Sox2 cooperates with Lkb1 loss in a mouse model of squamous cell lung cancer. Cell Rep. 8, 40–49. Singhal, S., Bhojnagarwala, P.S., O’Brien, S., Moon, E.K., Garfall, A.L., Rao, A.S., Quatromoni, J.G., Stephen, T.L., Litzky, L., Deshpande, C., et al. (2016). Origin and role of a subset of tumorassociated neutrophils with antigen-presenting cell features in early-stage human lung cancer. Cancer Cell 30, 120–135. Tanaka, H., Yanagisawa, K., Shinjo, K., Taguchi, A., Maeno, K., Tomida, S., Shimada, Y., Osada, H., Kosaka, T., Matsubara, H., et al. (2007). Lineage-specific dependency of lung adenocarcinomas on the lung development regulator TTF-1. Cancer Res. 67, 6007–6011.
Immunity 49, October 16, 2018 589
Previews underscore the importance of elucidating the detailed mechanism by which 20 30 cGAMP mounts the antitumor immune response. The data from Marcus et al. (2018) suggest that targeting cGAMP to effector populations that facilitate a robust type I IFN response capable of activating NK cells would be most effective in priming tumor clearance. As opposed to direct tumor injection, it is possible that more sophisticated approaches to retaining cGAMP in the tumor microenvironment and targeting it to certain cell populations will be more effective. In addition, this work also suggests that therapeutic strategies that naturally engage cGAMP-STING signaling, such as treatment with certain DNA damaging agents, are likely to prime innate immune responses, which could be tailored in combination therapies to maximize NK cell activation. Regardless, these findings provide a rationale to further our understanding of the mechanisms of 20 30 -cGAMP transport and modulation of cGAS activity in order to maximally augment tumor immunogenicity and broaden the therapeutic utility of cancer immunotherapy.
DECLARATION OF INTERESTS D.A.B. is a consultant for N-of-One and has received honoraria from Loxo Oncology and Madalon Consulting and research grants from Bristol-Myers Squibb and Novartis. REFERENCES Bridgeman, A., Maelfait, J., Davenne, T., Partridge, T., Peng, Y., Mayer, A., Dong, T., Kaever, V., Borrow, P., and Rehwinkel, J. (2015). Viruses transfer the antiviral second messenger cGAMP between cells. Science 349, 1228–1232. Chen, Q., Boire, A., Jin, X., Valiente, M., Er, E.E., Lopez-Soto, A., Jacob, L., Patwa, R., Shah, H., Xu, K., et al. (2016). Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer. Nature 533, 493–498. €ck, S., Guey, B., Gulen, M.F., Wolter, K., Kang, Glu T.W., Schmacke, N.A., Bridgeman, A., Rehwinkel, J., Zender, L., and Ablasser, A. (2017). Innate immune sensing of cytosolic chromatin fragments through cGAS promotes senescence. Nat. Cell Biol. 19, 1061–1070. Lam, A.R., Bert, N.L., Ho, S.S., Shen, Y.J., Tang, L.F., Xiong, G.M., Croxford, J.L., Koo, C.X., Ishii, K.J., Akira, S., et al. (2014). RAE1 ligands for the NKG2D receptor are regulated by STING-dependent DNA sensor pathways in lymphoma. Cancer Res. 74, 2193–2203. Marcus, A., Mao, A.J., Lensink-Vasan, M., Wang, L., Vance, R.E., and Raulet, D.H. (2018). Tumor-
derived cGAMP triggers a STING-mediated interferon response in non-tumor cells to activate the NK cell response. Immunity 49, this issue, 754–763. Ng, K.W., Marshall, E.A., Bell, J.C., and Lam, W.L. (2018). cGAS-STING and cancer: Dichotomous roles in tumor immunity and development. Trends Immunol. 39, 44–54. Sade-Feldman, M., Jiao, Y.J., Chen, J.H., Rooney, M.S., Barzily-Rokni, M., Eliane, J.P., Bjorgaard, S.L., Hammond, M.R., Vitzthum, H., Blackmon, S.M., et al. (2017). Resistance to checkpoint blockade therapy through inactivation of antigen presentation. Nat. Commun. 8, 1136. Woo, S.R., Fuertes, M.B., Corrales, L., Spranger, S., Furdyna, M.J., Leung, M.Y., Duggan, R., Wang, Y., Barber, G.N., Fitzgerald, K.A., et al. (2014). STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity 41, 830–842. Xia, T., Konno, H., and Barber, G.N. (2016). Recurrent loss of STING signaling in melanoma correlates with susceptibility to viral oncolysis. Cancer Res. 76, 6747–6759. Zaretsky, J.M., Garcia-Diaz, A., Shin, D.S., EscuinOrdinas, H., Hugo, W., Hu-Lieskovan, S., Torrejon, D.Y., Abril-Rodriguez, G., Sandoval, S., Barthly, L., et al. (2016). Mutations associated with acquired resistance to PD-1 blockade in melanoma. N. Engl. J. Med. 375, 819–829.
Lineage Specifiers in Lung Cancer Are Ahead of Their TIME Kelli A. Connolly,1 Brittany Fitzgerald,1 and Nikhil S. Joshi1,* 1Yale University School of Medicine, Department of Immunobiology, New Haven, CT 06519, USA *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2018.10.004
The factors that shape the distinctive tumor-immune landscapes of various types and subtypes of cancer remain poorly understood. In this issue of Immunity, Mollaoglu et al. (2018) reveal a mechanistic link between the function of lineage specifiers SOX2 and NKX2-1 and the presence of neutrophils in the tumor-immune microenvironment of lung cancer. Non-small-cell lung cancer (NSCLC) is a complex disease that accounts for approximately 85% of lung cancer. NSCLC is composed of two primary subtypes, lung adenocarcinoma (LADC) and lung squamous-cell carcinoma (LSCC). NSCLC patient tumors contain a variety of driver mutations that contribute to the heterogeneity between subtypes.
LADC commonly contains alterations in KRAS and EGFR and in tumor suppressors TP53, LKB1, KEAP1, and NF1. In contrast, LSCC frequently harbors mutations in TP53 and CDKN2A and has some EGFR mutations that do not respond to small-molecule therapy (Herbst et al., 2018). The cell-lineagespecifying transcription factors NKX2-1
and SOX2 are often overexpressed in LADC (Tanaka et al., 2007) and LSCC (Bass et al., 2009), respectively. The growing understanding of the molecular heterogeneity of patient NSCLC over the past two decades has led to a shift from general chemotherapy treatments to first-line precision medicine, which ranges from administration of
Immunity 49, October 16, 2018 ª 2018 Elsevier Inc. 587
Immunity
Previews
Figure 1. The Mechanisms of Genetic Alterations in Cancer Could Inform More Personalized Therapy SOX2 recruits tumor-associated neutrophils via regulation of chemoattractants CXCL5 (in mice) and CXCL6 (in humans), and high TAN infiltration correlates with squamous subtype and poorer prognosis. NKX2-1 has an inverse effect and leads to lower TAN recruitment. The SOX2/NKX2-1 axis described by Mollaoglu et al. in this issue of Immunity reveals the therapeutic potential of using high SOX2 and low NKX2-1 gene expression as a marker for identifying patients who might benefit from combining checkpoint therapy.
small-molecule inhibitors in tyrosine-kinase-receptor-driven disease to the use of checkpoint-inhibitor immunotherapy in PDL1-expressing cancers. The lungcancer field has been a clinical leader for matching treatment to specific markers of disease. Accordingly, the search for molecular markers useful for therapeutic prediction remains fervent. In this issue of Immunity, Mollaoglu et al. (2018) provide insight into the molecular mechanisms driving LSCC and LADC and the corresponding impacts on the tumor immune microenvironment (TIME). The success of immunotherapies in producing long-term regression in cancer patients has made clear the immune system’s impact on cancer progression and treatment outcomes, and recent studies have shown effectiveness of checkpointblockade therapies in NSCLC patients. Nevertheless, efficacy of immunotherapies has been restricted to only a portion of NSCLC patients. Investigations into the mechanism(s) underlying responsiveness to immunotherapy have highlighted how much remains unknown about the TIME and its determinants. In NSCLC, these studies have led to the discovery that the immune landscapes of LSCC and LADC are distinct, suggesting a link between histologic subtypes and the quality and makeup of the TIME. Still, a direct relationship between molecular drivers 588 Immunity 49, October 16, 2018
of lung-cancer subtypes and the TIME has remained elusive. One reason for this is the level of difficulty required in experimentally uncoupling the functions of lineage-determining transcription factors from their associated histologic cancer subtypes. Oliver and colleagues overcome this challenge through the manipulation and development of various genetically engineered mouse models (GEMMs) that recapitulate the molecular characteristics of histologically distinct lung-cancer subtypes; such characteristics include the expression of lineage specifiers SOX2 and NKX2-1. Previous work by the Oliver group and others demonstrated that tumor-cell genetics can impact the differentiation of lung cancers into distinct histologic subtypes, for example by affecting Myc expression in a neuroendocrine-low subtype of small-cell lung cancer (Mollaoglu et al., 2017). More specifically, the combination of SOX2 overexpression and LKB1 deletion promotes LSCC histopathology (Mukhopadhyay et al., 2014). Following up on these findings, Mollaoglu et al. (2018) developed a novel GEMM of LSCC-like disease in which the overexpression of SOX2 and deletion of LKB1 were achieved by the expression of Cre recombinase in cells. LSCC-like disease from these Rosa26LSL-Sox2-IRES-GFP; Lkb1fl/fl (SL) mice share molecular drivers
with other previously established GEMMs of LSCC, such as Lkb1fl/fl;Ptenfl/fl (LP) and Sox2LSL/LSL;Ptenfl/fl;Cdkn2abfl/fl (Sox2PC) mice, but also with adenosquamous tumors from KrasLSL-G12D/+;Lkb1fl/fl (KL) mice. Tumors across all four of these GEMMs are abundant in neutrophils, suggesting a role for the shared molecular drivers in determining aspects of the TIME. Direct comparison of tumors from these models facilitated a deeper understanding of the molecular mechanism of LSCC tumorigenesis. Furthermore, the authors interrogated the precise functions of the lineage specifiers SOX2 and NKX2-1 in both tumor-cell fate and the overall TIME by temporally deleting NKX2-1 (highly associated with the adenocarcinoma subtype) and overexpressing SOX2 (highly associated with the squamous-cell subtype) in a mouse model of LADC. Although the inhibition of neutrophil infiltration in lung cancer has shown promise in preclinical studies, little is known of the mechanism(s) of recruitment or precise functions in various malignant settings. By combining their advanced genetic models with ChIP-seq and gene expression data, the authors outline a mechanism linking the expression of the transcription factors SOX2 and NKX2-1 in tumor cells and the chemotaxis of tumor-associated neutrophils (TAN). Using an unbiased comprehensive approach, Mollaoglu et al. (2018) uncovered direct transcriptional regulation of the neutrophil chemokine CXCL5, by both SOX2 and NKX2-1. They also found binding sites for both SOX2 and NKX2-1 in the promoter region and first exon of Cxcl5 and showed that CXCL5 expression is inversely regulated by these two lineage specifiers. Although specific genetic or pharmacologic targeting of CXCL5 is not explored in this report, these studies raise the possibility that neutrophil chemoattractants might mediate the histological determination of malignancies and therefore present an attractive therapeutic option for patients with LSCC. Furthermore, analysis of TCGA data of patients across all lungcancer subtypes revealed that CXCL6, the human ortholog of mouse CXCL5, correlated with squamous subtype and high SOX2 and low NKX2-1 expression in human NSCLC samples, highlighting the translational applicability of the findings. Thus, this elegant investigation takes
Immunity
Previews great strides toward answering the question of how changes in tumor-cell-intrinsic factors impact cancer histopathology and quality of the TIME. The role of neutrophils in cancer remains a contentious topic and, accordingly, the mechanisms of intratumoral neutrophil recruitment and function are active areas of research. Although neutrophils are often a prevalent immune cell type in tumors of NSCLC patients, they have been found in greater quantities in LSCC than in LADC patients (Kargl et al., 2017). In a mouse model of NSCLC, neutrophils contributed directly and indirectly to cancer progression by establishing immune exclusion and modifying blood vessels, resulting in increased hypoxia and stabilization of the transcription factor HIF1a (Faget et al., 2017). Additionally, data suggest that a rising neutrophil-tolymphocyte ratio in patients during nivolumab treatment is potentially predictive of a shorter time to treatment failure (Kiriu et al., 2018). Conversely, intratumoral neutrophils are often heterogeneous, and evidence for neutrophils with antitumor functions have been observed. For example, Singhal et. al. have revealed the existence of a sub-population of intratumoral neutrophils capable of presenting antigen and augmenting anti-tumor T cell responses (Singhal et al., 2016). In the present study, through a combination of TAN-depletion studies and single-cell RNAseq, Mollaoglu et al. (2018) demonstrated largely pro-tumor effects of TANs and cooperation with genetic alterations, which promoted the development of tumors of the LSCC histologic subtype. Notably, neutrophil depletion not only resulted in the development of fewer LSCC tumors but also seemed to promote a LADC phenotype. These data suggest that the TIME, and the prevalence of neutrophils in particular, could promote the growth of histologically distinct tumor cells. These findings have interesting clinical implications because transdifferentiation of LADC to LSCC has been
documented in patients with relapsing disease after therapy. Taken together, these findings open a number of questions regarding the potential impacts of current therapies on intratumoral neutrophils and whether combination with neutrophiltargeted therapies could inhibit LADC to LSCC transdifferentiation and improve responses of LADC patients to therapy. The mechanisms for how tumor genetics regulate the quality of the TIME are understudied and have implications for improving patient selection for more personalized therapies. For example, understanding how specific genetic alterations impact TAN infiltration could help identify cancer patients who would benefit from combining checkpoint-blockade therapy with CXCL6 inhibitors, as illustrated in Figure 1. The push in cancer genetics by way of large-consortium-style analysis of human patient samples opens important questions about what these data mean biologically for the treatment of particular patient subgroups. Efforts made by the International Cancer Genomic Consortium (https://icgc.org) and The Cancer Genome Atlas (TCGA; https://cancergenome.nih.gov/) have resulted in an abundance of genomic and transcriptomic data with potential relevance to precision cancer treatment. By revealing a mechanistic link between lineage specifiers and the shaping of the TIME, the findings by Oliver and colleagues highlight the importance of a deeper understanding of the relationship between tumor-cell-intrinsic factors and the determinants of the anti-tumor immune response. Complimentary studies that examine the immunological context associated with genetic alterations present in human cancers should ultimately improve our ability to design effective therapies for difficult-to-treat malignancies. REFERENCES Bass, A.J., Watanabe, H., Mermel, C.H., Yu, S., Perner, S., Verhaak, R.G., Kim, S.Y., Wardwell,
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