Primary and Acquired Resistance to Immune Checkpoint Inhibitors in Metastatic Melanoma Tuba N

Published OnlineFirst November 10, 2017; DOI: 10.1158/1078-0432.CCR-17-2267

Review Clinical Cancer Research Primary and Acquired Resistance to Immune Checkpoint Inhibitors in Metastatic Melanoma Tuba N. Gide1,2, James S. Wilmott1,2, Richard A. Scolyer1,2,3, and Georgina V. Long1,2,4,5

Abstract

Immune checkpoint inhibitors have revolutionized the treat- involves various components of the cancer immune cycle, ment of patients with advanced-stage metastatic melanoma, as and interactions between multiple signaling molecules and well as patients with many other solid cancers, yielding long- pathways. Due to this complexity, current knowledge on resis- lasting responses and improved survival. However, a subset of tance mechanisms is still incomplete. Overcoming therapy patientswhoinitiallyrespondtoimmunotherapy,laterrelapse resistance requires a thorough understanding of the mechan- and develop therapy resistance (termed "acquired resistance"), isms underlying immune evasion by tumors. In this review, we whereas others do not respond at all (termed "primary resis- explore the mechanisms of primary and acquired resistance to tance"). Primary and acquired resistance are key clinical barriers immunotherapy in melanoma and detail potential therapeutic to further improving outcomes of patients with metastatic strategies to prevent and overcome them. Clin Cancer Res; 24(6); melanoma, and the known mechanisms underlying each 1260–70. 2017 AACR.

Introduction Drugs targeting the programmed cell death receptor 1 (PD-1, PDCD1) showed a further increase in response rates, PFS (2), and Immune checkpoint inhibitors have revolutionized the treat- OS (14–16) compared with anti–CTLA-4 blockade. PD-1 is also ment of advanced melanoma (1–5) and have signiﬁcant clinical expressed on the surface of activated T cells and binds to the activity across an increasing range of many other solid malignan- programmed cell death ligand 1 (PD-L1, CD274) to negatively cies, including non–small cell lung cancer (6, 7), renal cell regulate T-cell activation and differentiation. PD-L1 is constitu- carcinoma (8), head and neck cancer (9), Merkel cell carcinoma tively expressed by T cells, macrophages, and dendritic cells (DC), (10), and bladder cancer (11, 12). Understanding the biology as well as by some tumor cells including melanoma (17). Follow- behind response and resistance to immune checkpoint blockade up data from phase I clinical trials of the fully human IgG4 is critical to further improving outcomes of patients with meta- monoclonal antibody, nivolumab, showed a median OS of static melanoma. 17.3 months, with a 5 year OS rate of 34% (18). In a phase III The ﬁrst immune checkpoint to be clinically targeted, the study of nivolumab versus dacarbazine in patients with BRAF cytotoxic T-lymphocyte antigen 4 (CTLA-4), is expressed on the wild-type metastatic melanoma, the median OS was not reached surface of activated T cells and binds to its ligands, B7-1 and B7-2, for nivolumab at the most recent analysis, versus 11.2 months for on antigen-presenting cells (APC), resulting in the transmission of dacarbazine [hazard ratio (HR), 0.43, P < 0.001], and the 1- and inhibitory signals to T cells. In patients with metastatic melano- 2-year OS rates were 73% and 58%, respectively, for nivolumab ma, phase III clinical trials of ipilimumab, a fully human IgG1 (1, 14). Pembrolizumab, a humanized IgG4 monoclonal anti- monoclonal antibody inhibiting CTLA-4, demonstrated a signif- body against PD-1, also showed 1-, 2-, and 3-year OS rates icant improvement in progression-free survival (PFS) and overall of 67%, 50%, and 40%, respectively, in a phase I trial of survival (OS) when compared with a gp100 vaccine (13) or ipilimumab-treated and ipilimumab-na€ve patients with dacarbazine chemotherapy (4). advanced melanoma (3). Furthermore, in a phase III trial of pembrolizumab versus ipilimumab, the 2-year OS rates were 55% versus 43%, respectively (5, 15). More recently, combined anti–CTLA-4 and anti–PD-1 immu- 1Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. notherapies have shown improved response rates and clinical 2Sydney Medical School, The University of Sydney, Sydney, NSW, Australia. outcomes in comparison to ipilimumab monotherapy (the study 3 4 Royal Prince Alfred Hospital, Sydney, NSW, Australia. Royal North Shore was not powered to compare the two nivolumab treating arms: 5 Hospital, Sydney, NSW, Australia. Mater Hospital, North Sydney, NSW, nivolumab plus ipilimumab and nivolumab alone). A phase III Australia. study showed an increase in the median PFS of patients treated Note: Supplementary data for this article are available at Clinical Cancer with nivolumab and ipilimumab (11.5 months; HR, 0.42, P < Research Online (http://clincancerres.aacrjournals.org/). 0.001) and nivolumab alone (6.9 months; HR, 0.57, P < 0.001) Corresponding Author: Georgina V. Long, Melanoma Institute Australia, The compared with ipilimumab alone (2.9 months; ref. 2). At a Poche Centre, 40 Rocklands Road, North Sydney, NSW, 2060, Australia. Phone: minimum follow-up of 28 months, the median OS had not been 612-9911-7336; Fax: 612-9954-9290; E-mail: [email protected] reached in the combination or nivolumab-alone groups and doi: 10.1158/1078-0432.CCR-17-2267 was 20 months for ipilimumab alone [HR: combination vs. 2017 American Association for Cancer Research. ipilimumab, 0.55 (P < 0.0001); nivolumab vs. ipilimumab,

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Primary resistance Primary resistance • Poor immunogenicity • Downregulation of • Impaired DC chemokines maturation • Upregulation of Figure 1. Acquired resistance Antigen T-cell endothelin B receptor The cancer immune cycle. The • Loss of B2M presentation trafficking • Overexpression of VEGF induction of an effective antitumor and T-cell and tumor immune response requires activation infiltration successful (i) antigen presentation Therapeutic strategies Therapeutic strategies and T-cell activation, (ii) T-cell • Radiotherapy • Oncolytic viruses trafficking and tumor infiltration, • Oncolytic viruses • HDAC inhibitors and (iii) T-cell killing activity within • CTLA-4 inhibitors the tumor microenvironment. • HDAC inhibitors T-cell killing activity Various immune escape within the tumor mechanisms present at each of microenvironment these stages can result in primary or acquired resistance to immunotherapy. Potential therapeutic strategies can be used at each stage to overcome Primary resistance immunotherapy resistance. • Upregulation of PD-L1 A2AR, A2A receptor; B2M, • Induction of IDO beta-2-microglobulin; HDAC, • Upregulation of Tregs histone deacetylase; JAK1/JAK2, • Upregulation of Therapeutic strategies janus kinases 1 and 2; IDO, CD73/adenosine • PD-1/PD-L1 inhibitors indoleamine 2,3-dioxygenase; • Expression of IPRES • IDO inhibitors – IPRES, innate anti PD-1 resistance • Loss-of-function mutations • LAG-3 inhibitors signature; LAG-3, lymphocyte • TIM-3 inhibitors activation gene 3; TIM-3, T-cell Acquired resistance • CD73/A2AR inhibitors immunoglobulin and mucin domain • Mutations in JAK1/JAK2 3; Tregs, regulatory T cells; VEGF, • Upregulation of PD-L1 vascular endothelial growth factor. • Upregulation of immune checkpoint markers

0.63 (P < 0.0001); ref. 16]. The two-year OS rates were 64%, 59%, tumors evade the immune system, and strategies to overcome or and 45% in the combination, nivolumab, and ipilimumab prevent resistance in the future. groups, respectively (16). The results of these clinical trials highlight the significant impact immunotherapies have had on the clinical management Primary Resistance of patients with advanced-stage metastatic melanoma. However, Primary resistance to immune checkpoint blockade occurs although approximately 35% to 60% of patients have a RECIST in approximately 40% to 65% of patients with melanoma treated response (10%–12% a complete response) to anti–PD-1-based with anti–PD-1 based therapy (Fig. 2), depending on whether immunotherapy (2, 14, 15), 40% to 65% have shown minimal or anti–PD-1 is given upfront or after progression on other therapies no RECIST response at the outset, and 43% of responders develop (2, 14, 15), and >70% of those treated with anti–CTLA-4 therapy acquired resistance by 3 years (3). The underlying mechanisms (4, 13). This key unsolved clinical problem occurs when there is driving these variations in response are not yet well understood. failure to induce an effective antitumor immune response at any For an immunotherapy to elicit an efficient antitumor immune of the three stages of the cancer immune cycle (Fig. 1). To date, the response, the cancer immune cycle must be initiated and the clinicopathologic factors that have been associated with primary subsequent steps successfully completed. This involves efficient resistance are elevated levels of baseline serum LDH (23), (i) antigen presentation and T-cell activation, (ii) T-cell trafficking increased baseline tumor burden (24), lack of PD-L1 expression and tumor infiltration, and (iii) T-cell killing activity within the in baseline melanoma tissue samples (Fig. 3; ref. 25), lack of T-cell tumor microenvironment (Fig. 1). Studies examining possible infiltration (Fig. 3; ref. 21), the absence of PD-1 T cells and PD-L1 predictive biomarkers of response to immunotherapy have macrophages in melanoma biopsies taken early during treatment reported a higher density of preexisting cytotoxic T lymphocytes (22), insufficient neoantigens and low mutational burden (26), in tumor biopsies of patients who displayed a greater response to the presence of an innate anti–PD-1 resistance signature (IPRES) anti–PD-1/PD-L1 immunotherapy (19–21), and more signifi- transcriptional signature (27), or absence of an interferon signa- þ cantly, an increased influx of T cells and PD-L1 macrophages ture (28). It is currently unknown whether these measures are early during treatment (22). In this review, we discuss the different surrogates for resistance or have a direct mechanistic role in forms of immunotherapy resistance, the mechanisms by which preventing response. Here, we discuss the immune escape

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Figure 2. Primary resistance in metastatic melanoma. CT scans of a 55-year-old patient with metastatic melanoma treated with combined anti–CTLA-4 and anti–PD-1 immunotherapy at baseline (A) and after 12 weeks of therapy (B), demonstrating widespread metastatic disease, including signiﬁcant liver metastases, who did not respond, indicating primary resistance to therapy. The patient did not respond to subsequent chemotherapy and died 156 days after commencing combined immunotherapy.

mechanisms that can occur at each stage of the cancer immune outcome of patients with metastatic melanoma (26, 35). Circu- þ þ cycle (Fig. 1), thereby promoting both the growth and metastasis lating CD8 PD-1 lymphocytes in peripheral blood of patients of tumors and resistance to immune checkpoint inhibitor with melanoma can target patient-specific neoantigens, and therapies. neoantigen-specific T cells can in turn recognize autologous tumors (36). The immunogenicity of neoantigens can be pre- Antigen presentation and T-cell activation dicted by combining exome sequencing and mass spectrometry Upon encountering and engulfing foreign antigens, such as that data, thereby facilitating the identification of antigens that can be of cancer cells, DCs migrate from the tumor to regional lymph used to generate active T-cell responses (37). nodes where they present the antigens on major histocompati- Analysis of The Cancer Genome Atlas (TCGA) data from þ bility complex (MHC) class I molecules to CD8 T cells, resulting melanoma cases revealed that cutaneous melanoma displays a in activation of the latter. Barriers at this stage of the cancer high mutational burden and UV signature (38). In addition to immune cycle prevent optimal T-cell priming and activation, neoantigen recognition, a high mutational load was also found to hence resulting in evasion of the immune system by the tumor correlate with clinical benefit to immune checkpoint blockade (Table 1). (26). Similarly, a positive correlation was observed between a þ higher mutational load and increased CD8 T-cell infiltration Poor immunogenicity. Some tumors lack sufficient antigen pre- (39). Furthermore, an increased mutational burden is associated sentation by the immune system (29, 30) or do not present with elevated PD-L1 expression in advanced melanoma (40). In a antigens that can be recognized as foreign (31, 32). The process pooled analysis of 832 patients with melanoma, an increased PFS of distinguishing tumor cells from normal cells depends on T-cell was observed in PD-L1–positive patients treated with nivolumab recognition of tumor-specific or tumor-associated antigens (TAA; and ipilimumab combined immunotherapy, as well as in patients ref. 33). Tumor immune evasion by TAA-negative cells was treated with nivolumab alone, compared with PD-L1–negative reported in patients with melanoma who relapsed after respond- patients (2, 41). Similarly, PD-L1–positive patients treated with ing to peptide vaccinations (34). Recognition of tumor neoanti- pembrolizumab had increased PFS, OS, and overall response rate gens by T cells has been associated with increased and durable (ORR), highlighting PD-L1 as a potential biomarker of response response to immunotherapies and increased tumor regression, (42). As PD-L1 positivity is associated with improved response indicating a significant role for neoantigens in improving the in patients with melanoma, a lack of PD-L1 correlates with

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Anti–PD-1 responder Anti–PD-1 nonresponder AB

Figure 3.

Variable expression of immune Intratumor markers in patients with metastatic melanoma treated with anti–PD-1 immunotherapy. Multiplex immunoﬂuorescent images illustrating baseline PD-L1 expression and tumor-inﬁltrating lymphocytes (TIL) in a 59-year-old patient who responded to anti–PD-1 monotherapy (A and C), and lack thereof in a 55-year-old patient who did not respond to immunotherapy (B and D). Peritumor

primary resistance (Fig. 3). Nevertheless, some patients with Impaired DC maturation. In order to efficiently activate T cells, DCs PD-L1–positive tumors do not respond to PD-1 blockade, and must undergo a process called maturation, where they increase conversely, some patients with PD-L1–negative tumors respond. their capacity to stimulate T cells by displaying increased expres- For these reasons, intratumoral PD-L1 expression is a suboptimal sion of various costimulatory molecules required for T-cell acti- predictive biomarker (2, 41). Together, the aforementioned data vation, such as MHC class I/II, CD80, CD86, and CD40 (43). The indicate that PD-L1 expression is a possible surrogate for lack density of DCs strongly correlates with activated T cells in mela- of immunogenicity, as well as other failures further down the noma (44). The function of DCs can be impaired via multiple immune cycle. pathways. Interleukin (IL) 37b, a protein with a critical role in the inhibition of the innate immune response, suppresses DC maturation and function by decreasing CD80 and CD86 expression via Table 1. Mechanisms of resistance to immune checkpoint blockade the ERK/S6K/NF-kB signaling pathways (45). Furthermore, DC Mechanisms of resistance Contributing factors References maturation and tumor infiltration increased significantly in mel- fi Insuf cient antigen presentation Low mutational burden (26) anoma following the inhibition of signal transducer and activator and recognition Lack of neoantigen (26, 35) oftranscription3(STAT3),a transcriptionfactorthatisrequired for recognition Loss of B2M (99, 100) tumor growth and metastasis (46). STAT3 is also involved in the Loss of MHC class I (99) cross-talk between melanoma cells and immune cells, resulting in Insufficient T-cell activation Lack of mature DCs (44, 45) the induction of other immunosuppressive factors such as the STAT3 expression (46, 49) vascular endothelial growth factor (VEGF), IL10, regulatory T cells (Treg), and transforming growth factor b (TGF-b), all of which Absence of T cells from tumor Lack of chemokines (50, 51, 54) – microenvironment VEGF overexpression (63, 64) have inhibitory effects on DC maturation (47 49).

Upregulation of immunosuppressive PD-L1 (69, 70) T-cell trafficking and tumor infiltration markers IDO (80, 81) Tregs (82–85) Tumors can use a number of immune evasive mechanisms to prevent T-cell trafficking and infiltration into tumors. Assuming Decreased sensitivity to IFN- Mutations in the (93, 101) g that the T cells were successfully activated in the previous steps, signaling JAK/STAT pathway disruption during this stage is a likely cause for lack of response to Immune checkpoint markers TIM-3 (105) immunotherapy. LAG-3 (106) Abbreviations: B2M, beta-2-microglobulin; IDO, indoleamine 2,3-dioxygenase; Downregulation of chemokines required for T-cell recruitment. The IFN-g, interferon gamma; STAT3, signal transducer and activator of transcription 3; TIM-3, T-cell immunoglobulin and mucin domain 3; Tregs, regulatory T cells; differential expression of chemokine receptors is required for VEGF, vascular endothelial growth factor. effective T-cell homing and recruitment in cancer. In particular,

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CXCR3 has been identified as an important chemokine receptor an absence of intratumoral TILs and a shorter OS time in patients critical for T-cell infiltration. In mouse melanoma models, CXCR3 with ovarian carcinoma (66). Inhibition of VEGF resulted in an was highly expressed on a number of T-cell subsets, and trans- increase in T-cell infiltration into B16 melanoma tumors via the fection with its ligand, CXCL9, resulted in a significant increase in upregulation of CXCL10 and CXCL11 chemokines (67). Addi- þ þ both CD4 and CD8 T-cell infiltration (50). Similarly, human tionally, VEGF-A, along with prostaglandin E2 (PGE2) and IL10, þ þ melanoma samples with high CD8 T-cell expression were asso- upregulates the Fas ligand, resulting in CD8 T-cell death, and þ ciated with increased levels of CXCL9 and CXCL10 (51). Inter- subsequent inhibition of VEGF and PGE2 increased CD8 T-cell feron gamma (IFN-g, IFNG) has previously been shown to medi- infiltration (68). Corresponding with these data, in melanoma ate trafficking of Tregs, T helper cells, and cytotoxic T cells (52, 53). tumor biopsies, increased levels of VEGFA were observed in STAT3 inhibits CXCL10 production by tumor-associated myeloid nonresponders to anti–CTLA-4 and anti–PD-1 immunotherapies cells and T-cell recruitment into tumors by downregulating IFN-g in comparison to responders (19). þ production by CD8 T cells (54). Conversely, Stat3 ablation þ increases CXCR3 expression on CD8 T cells, allowing T-cell T-cell killing activity within the tumor microenvironment tumor infiltration (54). Primary resistance to immunotherapy also occurs when T cells Epigenetic alterations including DNA methylation and histone become successfully activated and infiltrate the tumor; however, modifications have also been identified as important mechanisms their function is hindered by the presence of immunosuppressive of chemokine repression and tumor progression. Epigenetic molecules within the tumor microenvironment (31). modifications are heritable modifications to DNA that result in changes to the gene expression profiles of tumor cells, thereby Upregulation of PD-L1. Primary resistance can be driven by the allowing them to evade the immune system (55). Epigenetic constitutive expression of PD-L1 through oncogenic signaling silencing resulted in the suppression of CXCL9 and CXCL10 in (69, 70). The increased expression of PD-L1 by cells in the tumor ovarian cancer, and treatment with epigenetic modulators microenvironment results in decreased function of cytotoxic T increased chemokine expression and T-cell infiltration into cells and apoptosis, hence providing an immune escape mecha- tumors (56). Increased expression of chemokines, T-cell recruit- nism for tumor cells. ment, and tumor regression was also observed in lung cancer cell Several studies have revealed a correlation between loss of the lines and mice treated with the histone deacetylase (HDAC) phosphatase and tensin homolog (PTEN) in cancer and the inhibitor romidepsin (57). upregulation of PD-L1, implicating PD-L1 in tumor immune evasion. PTEN is a tumor suppressor that negatively regulates the Upregulation of the endothelin B receptor. T-cell trafficking through P13K/AKT pathway. This pathway is responsible for the regula- the tumor and lymph nodes is controlled by a number of tion of cellular processes such as proliferation and survival. The endothelial signals, regulating T-cell homing, adhesion, and loss of PTEN and activation of the P13K/AKT pathway in human migration (58). The interaction between endothelin 1 (ET-1, glioma cell lines resulted in an increase in posttranscriptional EDN1) and the endothelin A receptor (ETAR, EDNRA) promotes CD274 expression (71). PD-L1 expression was also upregulated in tumorigenesis through various pathways including cell prolifer- lung squamous cell carcinoma following the simultaneous deple- ation, invasion, angiogenesis, bone remodeling, and inhibition of tion of Pten and Lkb1 [also known as Stk11 (serine–threonine apoptosis (59). The endothelin B receptor (ETBR, EDNRB) coun- kinase 11); ref. 72]. In melanoma, loss of PTEN led to a decrease in terregulates ET-1/ETAR activity via the increased production of T-cell trafficking, infiltration, and T-cell activity (73). However, nitric oxide, activation of apoptotic pathways, and clearance silencing PTEN did not significantly alter the expression of PD-L1 of ET-1 (60). The endothelin system has been implicated in the in melanoma cell lines in vitro or in xenograft models in vivo, pathogenesis of a number of cancers, including ovarian cancer, indicating that PD-L1 regulation may not be the principal mech- prostate cancer, and colon cancer. Interestingly, ETBR is upregu- anism of immune resistance resulting from a loss of PTEN (73). lated in melanoma and has also been proposed as a marker of Other mechanisms that have been shown to have a role in the melanoma progression, suggesting a role for ETBR in melanoma constitutive upregulation of PD-L1 include the transcription tumorigenesis (61). ETBR inhibition in 10 human melanoma cell factor interferon regulatory factor 1 (IRF-1) and mutations in the lines using the ETBR antagonist BQ788 resulted in an increase in epidermal growth factor receptor (EGFR). IRF-1 is responsible for apoptosis and cell death, as well as an increase in angiogenesis in the regulation of cell proliferation, apoptosis, and immunity (74). the tumors (62). In human ovarian cancers, ETBR was found to The knockdown of IRF-1 using siRNA resulted in the decrease in correlate with an absence of tumor-infiltrating lymphocytes (TIL) transcription and translation of PD-L1 in a lung carcinoma cell as well as decreased patient survival time. Administration of line (75). Similarly, activation of the EGFR pathway resulted in the BQ788 increased T-cell homing to tumors and improved the increased expression of PD-L1 in lung cancer cell lines (76, 77) efficacy of immunotherapy (58), highlighting ETBR as a potential and tissue (76). Increased expression of markers of T-cell exhaus- immune escape mechanism and future target in patients who fail tion, such as PD-1 and FOXP3, was also observed in the tumor to respond to immunotherapy. microenvironment (76). PD-1 blockade increased cytotoxic T-cell numbers as well as effector T-cell function (76), highlighting the Overexpression of VEGF. Increased levels of the proangiogenic role of the PD-1/PD-L1 axis in immune evasion and its mani- factor VEGF in plasma and tissue samples have also been asso- pulation as a therapeutic strategy. ciated with the growth and progression of melanoma (63, 64). VEGF-A downregulates T-cell adhesion to the endothelium via the Induction of IDO. Another molecule proposed to play a critical suppression of intercellular adhesion molecule 1 (ICAM-1) and role in the negative regulation of T-cell function is indoleamine vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells 2,3-dioxygenase (IDO, IDO1). IDO is expressed in a wide range of (65). Increased expression of VEGF in tumor was associated with human cancers and is the rate-limiting enzyme responsible for the

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degradation of tryptophan into kynurenine (78, 79). T lympho- Loss-of-function mutations. Mutations in the janus kinases 1 and 2 cytes undergo arrest in response to this tryptophan depletion, (JAK1/2) have also been shown to be involved in primary resis- resulting in the suppression of T-cell proliferation and activity tance to anti–PD-1 immunotherapy. JAK1/2 loss-of-function (80). To understand the mechanism of immunosuppression mutations identified in one of 23 melanoma tumor biopsies, induced by IDO, Holmgaard and colleagues (81) developed a and two of 48 human melanoma cell lines via whole-exome B16 melanoma tumor model overexpressing IDO and revealed a sequencing, resulted in a lack of PD-L1 expression due to an correlation between IDO expression and increased tumor-infil- inability to respond to IFN-g signaling (93). Furthermore, the trating myeloid-derived suppressor cells (MDSC), as well as recent development of a two cell type-CRISPR (2CT-CRISPR) þ þ CD4 FOXP3 Tregs. This association demonstrated that IDO screening assay revealed an important role for apelin receptor suppresses T-cell activity through the recruitment and activation (APLNR) loss-of-function mutations in disturbing effector T-cell of MDSCs in a Treg-dependent fashion (81). Systemic inhibition function (94). Retroviral overexpression of APLNR correlated of IDO in mice using a tryptophan analogue, 1-methyl-L-trypto- with an increase in JAK1 as well as tumor sensitivity to effector phan (1MT), reduced tumor progression in a T-cell–dependent T-cell function (94). Conversely, APLNR-knockout cells demon- manner (79). Similarly, administration of 1MT in combination strated decreased activation of the JAK/STAT pathway following with anti–CTLA-4 immunotherapy in B16F10 mouse melanoma IFN-g treatment, and Aplnr knockout in mouse melanoma in vivo models resulted in a significant delay in tumor growth and an resulted in a decrease in the efficacy of anti–CTLA-4 immuno- increase in OS (78). These findings provide a strong rationale for therapy (94). These findings provide a strong rationale for further targeting IDO to improve the efficacy of immunotherapies in investigating APLNR as a potential target to prevent immune patients with melanoma. evasion by tumors.

Upregulation of Tregs. The upregulation of FOXP3-expressing Mechanisms under investigation Tregs has been observed in a number of melanoma studies, The composition of the gut microbiome. Recent studies have revealing a possible role for Tregs in melanoma tumorigenesis highlighted a possible role for the gut microbiome in patient (82–84). Tregs promote tumor growth by inhibiting the activity response to immunotherapy. Dysbiosis (an imbalance of the of T-cell subsets, either through direct cell-to-cell contact or microbiota) involves decreases in the diversity and stability of indirectly through the secretion of anti-inflammatory cyto- the microbiome, thereby promoting tumorigenesis (95). kines, such as IL10 and TGF-b (85, 86). The presence of þ þ Sequencing of the oral and gut microbiome of patients with CD4 CD25 FOXP3 Tregs was observed amongst TILs in metastatic melanoma revealed a correlation between higher gut metastatic melanoma (87) and the transfer of CD25 (IL2RA)- microbiome diversity and response to anti–PD-1 monotherapy depleted splenic T cells into B16 mouse melanoma models (96). Responders also had a significantly different gut micro- resulted in the suppression of tumor growth in vivo (82). Popula- biome composition in comparison with nonresponders, and this tions of tumor-infiltrating Tregs also significantly correlated with correlated with differences in PFS (96). The increased abundance increased tumor growth in B16BL6 mice (88). Furthermore, a þ of specific bacteria in the gut microbiome also correlated with a decrease in FOXP3 Tregs significantly correlated with increased þ higher CD8 T-cell density in responders to anti–PD-1 immuno- tumor control and survival in patients with melanoma treated therapy (96). Similarly, the composition of the baseline gut with ipilimumab (89), highlighting the immunosuppressive microbiome in patients with metastatic melanoma was associated function of Tregs in melanoma. with response to ipilimumab, and improved PFS and OS were associated with specific groups of bacteria such as Faecalibacterium Upregulation of the CD73/adenosine pathway. Elevated levels of and other Firmicutes (97). Additionally, a significant decrease in extracellular adenosine and CD73 (NT5E) have also been impli- TILs and lack of response to CTLA-4 blockade was observed in cated in immune suppression and tumor progression. Adenosine tumors of mice housed in germ-free conditions (98). The anti- is produced via the conversion of extracellular ATP by the ectoen- cancer therapeutic effects of the anti–CTLA-4 antibody were zymes CD39 (ENTPD1) and CD73 and binds to the adenosine restored upon oral feeding of the germ-free mice with Bacteroides A2A receptor (A2AR, ADORA2A) to inhibit effector T-cell function fragilis (B. fragilis), a Bacteroides isolate, as well as with the adoptive (90). Increased CD73 expression correlated with advanced-stage transfer of B. fragilis–specific memory T cells (98). The mechan- disease in melanoma (91), and an upregulation in CD73 was isms through which the gut microbiome influences the immune observed in patients who progressed following treatment with response are currently being investigated. anti–PD-1 immunotherapy (92). A2AR inhibition increased þ CD8 T-cell infiltration and significantly reduced tumor growth in mouse melanoma models (91), suggesting a role for the CD73/ Acquired Resistance adenosine axis in promoting immune escape. Acquired resistance occurs in patients who relapse after exhibit- ing an initial response to immunotherapy (Fig. 4). Currently, little Expression of IPRES signature. The expression of IPRES has recent- is understood about the mechanisms that give rise to acquired ly been identified as a mechanism of primary resistance to resistance, and many are likely to be similar to those underlying immunotherapy. Transcriptomal analyses of responding and primary resistance (Table 1). nonresponding pretreatment melanoma biopsies from patients Acquired resistance to immunotherapy can develop when there treated with anti–PD-1 immunotherapy revealed the coenrich- is Darwinian selection of subpopulations of tumor cells with ment of genes associated with mesenchymal transition, wound genetic and epigenetic traits that allow them to evade the immune healing, and angiogenesis in nonresponding patient samples system (32). An example of one such trait is beta-2-microglobulin (27). This was observed not only in metastatic melanoma but (B2M), a component of MHC class I molecules that is necessary also in other major cancer types such as pancreatic cancer (27). for their functional expression. The loss of B2M expression was

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ACB DFigure 4. Acquired resistance in metastatic melanoma. Images of a 74-year-old patient with metastatic melanoma treated with anti–PD-1 monotherapy, showing complete resolution of melanoma in right-neck lymph nodes from baseline (A), week 4 (B), and week 12 (C). However, acquired resistance developed in the right preauricular region at 12 months after the commencement of anti– PD-1 monotherapy (D). The patient subsequently received radiotherapy © 2017 American Association for Cancer Research and ipilimumab in addition to continuing anti–PD-1, with an excellent ongoing response 12 months after acquired resistance.

reported in melanoma cell lines from five patients who had been TIM-3 blockade in mice resulted in a significant increase in treated with immunotherapy and cytokine–gene therapy (99). survival time, as well as increased production of IFN-g and þ This resulted in a loss of MHC class I expression and, therefore, a proliferation of CD8 T cells (105). LAG-3 is also overexpressed þ subsequent decrease in recognition by CD8 T cells. Archival in PD-L1–positive melanoma, suggesting LAG-3 upregulation as a tissues taken prior to immunotherapy from three of these patients potential immune evasion mechanism (106). were B2M positive, suggesting loss of B2M expression as a mechanism of acquired resistance (99). Similarly, the loss of B2M has Overcoming Mechanisms of Tumor Immune been observed in sequential lesions obtained from a patient with Evasion metastatic melanoma following immunotherapy treatment with DCs transfected with autologous tumor mRNA (100). From the above, it is clear that there exist multiple immune JAK1/2 mutations have also recently been identified as genetic evasive mechanisms that can be utilized by tumors at each of the markers of acquired resistance to immunotherapy in melanoma. different stages of the cancer immune cycle that may induce either These mutations in tumor cells lead to decreased sensitivity to primary or acquired resistance to immunotherapy. Determining fi IFN-g, ultimately preventing IFN-g–induced cell growth arrest the speci c mechanisms underlying resistance to immunotherapy (101). Upon exposure to IFN-g (produced by activated T cells), in these patients is a crucial step toward effective treatment and JAK1/2 become activated and subsequently phosphorylate a ultimately producing durable responses for them. tyrosine residue present on STATs (102). This JAK/STAT signaling pathway is responsible for cell proliferation, differentiation, cell Combinatorial therapies migration, and apoptosis (102). However, IFN-g also results in The immune escape mechanisms discussed above do not act in the upregulation of PD-L1 on tumor cells, thus inactivating isolation. Together, the overlap between various signaling path- antitumor T cells (70). Loss-of-function mutations in the genes ways and the interactions between several of the immunosup- encoding JAK1 or JAK2 were found in relapsed tumors in two of pressive molecules leads to resistance. It is likely that combina- four patients following whole-exome sequencing of baseline and tions of therapy will be more effective than single-agent therapies progression biopsies; all patients had an objective response to for a given patient. However, the challenge remains to determine treatment with pembrolizumab and then progressed after which of these combinations are most effective and in which a median of 1.8 years (101). The anti–PD-1-resistant cells har- patient given interpatient heterogeneity. Currently, multiple clin- boring JAK mutations were derived from cells clonally selected ical trials are underway examining the activity and toxicity of from the baseline tumor. These findings demonstrate the role combined immunotherapies, particularly using an anti–PD-1 of the JAK/STAT pathway in promoting acquired resistance drug in combination with an agent targeting a complimentary to immunotherapy. part of the immune system (Supplementary Table S1). In addition, acquired resistance can also occur on the level of Dual blockade of PD-1 and LAG-3 in mouse cancer models the individual cells, whereby tumor cells alter their gene expres- resulted in the regression of tumors in most mice, as well as an sion in response to immune molecules within the tumor micro- increased survival rate (107). In a recent study, all mice that were environment (32). For example, PD-L1 can be upregulated by treated with a triple combination of LAG-3 blockade, PD-1 tumor cells in response to immune cytokines, such as IFN-g blockade, and poxvirus-based immunotherapy demonstrated released by T cells, hence limiting T-cell function (70), and can complete tumor regression (108). Phase I clinical trials involving occur in both primary and acquired resistance (32, 103). LAG-3 blockade with and without PD-1 blockade in solid tumors Other immune checkpoint markers such as lymphocyte acti- are currently underway, and in patients who had progressed on vation gene 3 (LAG-3) and T-cell immunoglobulin and mucin anti–PD-1 monotherapy, a response rate of 16% (any tumor domain 3 (TIM-3, HAVCR2) have also been revealed to interfere reduction in 45%) was observed in those patients whose tumors with the activity of T cells (70, 104), resulting in acquired expressed LAG-3 (109). Similarly, in mouse models, combined resistance to immunotherapy. In a recent study, TIM-3 upregula- TIM-3 and PD-L1 blockade significantly reduced tumor growth in tion was observed in patients who developed adaptive immune comparison with single-agent immunotherapy (110). Further- resistance to anti–PD-1 immunotherapy (105). Furthermore, more, dual blockade of TIM-3 and PD-L1 increased the ability of

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þ CD8 T cells to produce IFN-g, thereby restoring their function point therapies is currently being tested in various human cancers (110). Consistent with this, anti–TIM-3 and anti–PD-1/anti– (Supplementary Table S1). CTLA-4 combined immunotherapy resulted in a significant These findings provide a strong rationale for the development decrease in the tumor sizes of multiple cancers (111). of combinations of different treatment strategies in clinical trials Phase I/II clinical trials testing the combination of an IDO as an effective means of melanoma treatment. inhibitor with PD-1/PD-L1 inhibitors in metastatic melanoma are also currently underway. These include the administration of Conclusions nivolumab and a PD-L1/IDO peptide vaccine (NCT03047928), Checkpoint inhibitor immunotherapy has revolutionized the and indoximod (IDO inhibitor) in combination with CTLA-4 or treatment of multiple cancer types. However, responses in PD-1 inhibitors (NCT02073123). Furthermore, the combination patients with metastatic melanoma are diverse, with many of epacadostat (an IDO1 inhibitor) plus pembrolizumab was patients displaying primary or acquired resistance. An important generally well tolerated and correlated with improved response in ongoing, major unmet clinical need remains to identify predictors various cancers (112–114), leading to the initiation of phase III and causes of this resistance and strategies to overcome them. A studies such as that in patients with treatment-na€ve advanced key element of effective immunotherapy is identifying the various melanoma (NCT02752074). mechanisms by which the tumors evade the immune system on Many studies have also demonstrated the increased efficacy of an individual basis. Improving our understanding of these combined radiotherapy and immune checkpoint blockade. mechanisms and determining which immune markers to target Radiotherapy activates the inflammatory pathways and induces in each patient will then allow for the administration of the most DNA damage, resulting in the release of antigens from irradiated appropriate form of therapy to achieve optimal response and cells and an increase in tumor sensitization to T-cell immune improve the overall outcomes of patients with metastatic responses (115). Radiotherapy in patients with advanced mela- melanoma. noma who had progressed following treatment with ipilimumab led to an abscopal effect in some patients, whereby irradiation of Disclosure of Potential Conflicts of Interest the primary tumor induced regression in other nonirradiated fi G.V. Long reports receiving speakers bureau honoraria from Bristol-Myers metastases, which correlated with longer OS (116). Signi cant Squibb, Incyte, Merck, Novartis, and Roche, and is a consultant/advisory board tumor regression was also observed in patients with metastatic member for Amgen, Array, Bristol-Myers Squibb, Incyte, Merck, Novartis, Pierre melanoma who received combined radiotherapy and anti–CTLA- Fabre, and Roche. No potential conflicts of interest were disclosed by the other 4 immunotherapy, and experiments in mice showed that a authors. combination of anti–CTLA-4 immunotherapy, anti–PD-L1 immunotherapy, and radiotherapy was required to achieve opti- Acknowledgments mum response (117). This work was supported by Melanoma Institute Australia, the New South Wales Ministry of Health, NSW Health Pathology, National Health and The combination of epigenetic modulators and immune check- Medical Research Council of Australia (NHMRC), and Cancer Institute NSW. point blockade has recently been shown to be more effective than J.S. Wilmott, R.A. Scolyer, and G.V. Long are supported by NHMRC Fellow- single-agent immunotherapy in patients with melanoma. The ships. J.S. Wilmott is also supported by a CINSW Fellowship. G.V. Long is inhibition of HDAC using LBH589 in combination with PD-1 also supported by the University of Sydney Medical Foundation. T.N. Gide is blockade in B16F10 mouse melanoma models resulted in delayed supported by The University of Sydney and Melanoma Institute Australia tumor growth and increased survival compared with the control Scholarships. group and PD-1 blockade alone (118). Similarly, a reduction in tumor size and Tregs was observed in B16F10 mouse melanoma The costs of publication of this article were defrayed in part by the payment – of page charges. This article must therefore be hereby marked advertisement in models treated with a combination of anti CTLA-4 immunother- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. apy and epigenetic modulation of trimethylation of lysine 27 on histone H3 (H3K27me3) compared with CTLA-4 blockade alone Received August 12, 2017; revised October 15, 2017; accepted November 6, (119). The efficacy of combined epigenetic and immune check- 2017; published OnlineFirst November 10, 2017.

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1270 Clin Cancer Res; 24(6) March 15, 2018 Clinical Cancer Research

Primary and Acquired Resistance to Immune Checkpoint Inhibitors in Metastatic Melanoma

Tuba N. Gide, James S. Wilmott, Richard A. Scolyer, et al.

Clin Cancer Res 2018;24:1260-1270. Published OnlineFirst November 10, 2017.

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