Cancer Cell

Previews S., Maclean, N., et al. (2015). Cancer Cell 27, this issue, 864–876. Corydon, T.J., Bross, P., Holst, H.U., Neve, S., Kristiansen, K., Gregersen, N., and Bolund, L. (1998). Biochem. J. 331, 309–316. Estey, E., Levine, R.L., and Lo¨wenberg, B. (2015). Blood 125, 2461–2466.

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Excluding T Cells: Is b-Catenin the Full Story? Siwen Hu-Lieskovan,1,4 Blanca Homet Moreno,1 and Antoni Ribas1,2,3,4,* 1Division

of Hematology-Oncology, Department of Medicine of Surgery 3Department of Medical and Molecular Pharmacology University of California Los Angeles (UCLA), Los Angeles, CA 90095, USA 4Jonsson Comprehensive Cancer Center (JCCC) at UCLA, Los Angeles, CA 90095, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2015.05.014 2Department

Spranger and colleagues reported recently in Nature an inverse relationship between melanoma intrinsic b-catenin signaling and intratumoral T cell infiltration, providing an explanation for potential mechanisms of T cell exclusion. Further insights are needed into the mechanisms leading to a lack of T cell infiltration of cancers and primary immune resistance. After decades of efforts, the war on cancer has reached an exciting new turning point with the promise of long-term remissions in patients with different cancer histologies treated with immune checkpoint inhibitors. It reflects a fundamental change by fully utilizing the power of the human immune system to control and eradicate cancer. Sustained responses have been reported beyond the traditionally ‘‘immunotherapy sensitive’’ tumor types such as melanoma or renal cell carcinoma, evidenced by the US Food and Drug Administration approval of the anti-PD-1 antibody nivolumab to treat the squamous subtype of non-small cell lung cancer as a second-line therapy. Full pipelines of immune modulatory agents are in different stages of clinical development for a wide variety of tumor types. It is still unclear, however, why only a percentage of patients within the same tumor type respond to the therapy and why certain tumors do not respond at all. Studying the immune resistance mechanisms in these patients and tumor types would pave the way to the rational design of combination strategies to improve efficacy.

The Wnt/b-catenin pathway is crucial in embryonic development and adult tissue homeostasis, including hematopoiesis, cell migration, and wound repair (Kahn, 2014). Aberrant regulation of this pathway has been linked to cancer development, more aggressive behavior, and worse prognosis in several types of cancers. Mutations of b-catenin have been described in melanoma cell lines (Rubinfeld et al., 1997), and active WNT/b-catenin signaling was reported in one third of melanoma tumors. However, its significance is a matter of debate, because melanomas with a b-catenin signature have been associated with lower proliferative index (Chien et al., 2009), more favorable prognosis, and less aggressive disease (Bachmann et al., 2005), adding to the complexity of this topic. In a recent publication in Nature, Spranger et al., (2015) demonstrated that tumor-intrinsic b-catenin signaling might result in the lack of T cell infiltration in melanoma models and in patient-derived biopsies. As a first step, the authors analyzed microarray gene expression data from 266 human cuta-

neous melanoma samples acquired from the public domain and categorized these samples into ‘‘T cell inflamed’’ and ‘‘nonT cell-inflamed’’ subtypes based on relevant gene expression. Pathway analysis suggested the association of active b-catenin signaling with the non-T cell-inflamed cohort, which was a novel observation. The authors then used previously established genetically engineered mouse models driven by conditional BRAFV600E activation and PTEN deletion (BRAFV600E/ PTEN / ), compared with an additional stabilized b-catenin (BRAFV600E/PTEN / / Bcat-STA) melanoma mouse model (Damsky et al., 2011). Melanomas in these mice can be induced by local application of tamoxifen, and the BRAFV600E/ PTEN / /Bcat-STA phenotype was previously reported to be more aggressive with high metastatic potential (Damsky et al., 2011). Melanomas arising from mice with active b-catenin were associated with almost complete absence of T cells, and the few remaining intratumoral T cells showed predominantly a naive phenotype with low PD-1, PD-L1, and LAG3 expression. When a neo-antigen (SIY) was

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Cancer Cell

Previews genetically engineered into the melanomas of these mice, adoptively transferred T cells with SIY T cell receptor (TCR) accumulated in the BRAFV600E/ tumors, but not in the PTEN / BRAFV600E/PTEN / /Bcat-STA tumors, indicating defective homing of target-specific T cells in the tumors. The authors concluded from this data that intrinsic tumor b-catenin signaling prevents early T cell priming, although alternative mechanisms could be provided. They then analyzed the Batf3-lineage dendritic cells (DC) in the tumors, given its crucial role in presenting tumor antigens to CD8+ T cells. Indeed, CD8a+ (skin-derived) and CD103+ (lymph node-derived) DCs were almost absent, with reduced IFN-b cytokine expression in tumors from the model, BRAFV600E/PTEN / /Bcat-STA but not in its counterpart without b-catenin stabilization. Interestingly, BRAFV600E/ PTEN / /Batf3 / bone marrow chimeras manifested similarly decreased T cell infiltration, and intratumoral injection of activated DCs by poly I:C could restore T cell infiltration in BRAFV600E/PTEN / / Bcat-STA tumors. Further gene expression profiling from tumors of these two genotypes revealed five chemokines differentially expressed, with four of these showing lower levels in the BRAFV600E/PTEN / /Bcat-STA tumors (CCL3, CXCL1, CXCL2, and CCL4) associated with a lack of expression of the corresponding chemokine receptor CCR5 by DCs isolated from these tumors. CCR5 was linked to migratory capacity of CD8a+ DCs. In vitro migration assays showed DC migration in response to recombinant murine CCL4 and the supernatant of cell lines derived from the BRAFV600E/PTEN / , but no DC migration was observed with the BRAFV600E/ PTEN / /Bcat-STA tumors. Further CHIP assay revealed that the mechanism by which b-catenin signaling prevents CCL4 expression might be through binding of ATF3 (downstream of b-catenin) to the CCL4 promoter region in BRAFV600E/ PTEN / /Bcat-STA cells. Knock-down of b-catenin or ATF3 restored CCL4 production in these cells. This observation was also confirmed in human melanoma

cell lines with low or high b-catenin expression, and gene expression analysis of human melanoma metastasis showed inverse correlation of Batf3-lineage DC marker expression and gain-of-function mutations in b-catenin. In the end of the study, the authors explored the therapeutic relevance by treating both mouse models with a combination of anti-CTLA-4 and anti-PD-L1 antibodies, which induced a significant delay in tumor growth in the BRAFV600E/ model, but not in the PTEN / model. BRAFV600E/PTEN / /Bcat-STA Intratumoral injection of activated DCs into the BRAFV600E/PTEN / /Bcat-STA tumors, on the other hand, could partially restore the therapeutic effect of antiCTLA-4 and anti-PD-L1 antibodies. It is unclear, however, why the authors did not include the therapeutic effects of single agent controls. Based on this work, the lack of efficacy may stem from the priming phase (which is the primary target of CTLA-4 blockade) or the effector phase of an anti-tumor T cell cytotoxic response (the primary target of PD-1 blockade). The inverse relationship of active b-catenin signaling and T cell infiltration in both human melanoma samples and transgenic melanoma mouse models by Spranger and colleagues provides a first insight into a potential new mechanism of immune resistance. Further evidence to support this notion, but not provided by this paper, would be a correlation of the b-catenin signaling with a differential responsiveness with immune modulatory therapies in humans. Although the therapeutic potential was demonstrated in a transgenic mouse model, similar observations need to be confirmed in animal models of different genetic background (such as BRAFWT/PTEN / /Bcat-STA, BRAFV600E/PTENWT/Bcat-STA, or others) before generalized application. The Wnt signaling pathway has been shown to be critical in lymphopoiesis and hematopoiesis (Staal et al., 2008), including T cell development and DC maturation. One potential concern of transgenic mouse models is if the tissue-specific promoter is leaky or if there was disrup-

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tion of the genome at the embryonic level when inserting the transgene, it might compromise the development of an intact immune system. Spranger and collaborators described a lack of CCR5 expression on DCs in BRAFV600E/ PTEN / /Bcat-STA tumors, but no data indicated whether DC isolated from the spleens of the same mice had the same defect. Using syngeneic mouse models with cell lines derived from these transgenic tumors would be helpful in this situation. Finally, given the previous observation that active WNT/b-catenin signaling was associated with a lower proliferative index and more favorable prognosis in human samples, but had the opposite effect in this mouse model, the exact role of this pathway plays in the tumor immune microenvironment remains to be fully determined. ACKNOWLEDGEMENTS S.H-L. is supported by the ASCO Young Investigator Award and a Tower Foundation Research Grant. A.R. is funded by NIH grants R01 CA199205, P01 CA168585, P01 CA132681, U54 CA119347, and R01 CA170689, the Dr. Robert Vigen Memorial Fund, and the Ressler Family Fund.

REFERENCES Bachmann, I.M., Straume, O., Puntervoll, H.E., Kalvenes, M.B., and Akslen, L.A. (2005). Clin Cancer Res 11, 8606–8614. Chien, A.J., Moore, E.C., Lonsdorf, A.S., Kulikauskas, R.M., Rothberg, B.G., Berger, A.J., Major, M.B., Hwang, S.T., Rimm, D.L., and Moon, R.T. (2009). Proc. Natl. Acad. Sci. USA 106, 1193– 1198. Damsky, W.E., Curley, D.P., Santhanakrishnan, M., Rosenbaum, L.E., Platt, J.T., Gould Rothberg, B.E., Taketo, M.M., Dankort, D., Rimm, D.L., McMahon, M., and Bosenberg, M. (2011). Cancer Cell 20, 741–754. Kahn, M. (2014). Nat. Rev. Drug Discov. 13, 513–532. Rubinfeld, B., Robbins, P., El-Gamil, M., Albert, I., Porfiri, E., and Polakis, P. (1997). Science 275, 1790–1792. Spranger, S., Bao, R., and Gajewski, T.F. (2015). Nature. Published online May 11, 2015. http://dx. doi.org/10.1038/nature14404. Staal, F.J., Luis, T.C., and Tiemessen, M.M. (2008). Nat. Rev. Immunol. 8, 581–593.

Excluding T Cells: Is β-Catenin the Full Story?

Spranger and colleagues reported recently in Nature an inverse relationship between melanoma intrinsic β-catenin signaling and intratumoral T cell inf...
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