Primary and Acquired Resistance to Immune Checkpoint … · Review Primary and Acquired Resistance...

Review

Primary and Acquired Resistance to ImmuneCheckpoint Inhibitors in Metastatic MelanomaTuba N. Gide1,2, James S.Wilmott1,2, Richard A. Scolyer1,2,3, andGeorgina V. Long1,2,4,5

Abstract

Immune checkpoint inhibitors have revolutionized the treat-ment of patients with advanced-stage metastatic melanoma, aswell as patients with many other solid cancers, yielding long-lasting responses and improved survival. However, a subset ofpatients who initially respond to immunotherapy, later relapseand develop therapy resistance (termed "acquired resistance"),whereas others do not respond at all (termed "primary resis-tance"). Primary and acquired resistance are key clinical barriersto further improving outcomes of patients with metastaticmelanoma, and the known mechanisms underlying each

involves various components of the cancer immune cycle,and interactions between multiple signaling molecules andpathways. Due to this complexity, current knowledge on resis-tance mechanisms is still incomplete. Overcoming therapyresistance requires a thorough understanding of the mechan-isms underlying immune evasion by tumors. In this review, weexplore the mechanisms of primary and acquired resistance toimmunotherapy in melanoma and detail potential therapeuticstrategies to prevent and overcome them. Clin Cancer Res; 24(6);1260–70. �2017 AACR.

IntroductionImmune checkpoint inhibitors have revolutionized the treat-

ment of advanced melanoma (1–5) and have significant clinicalactivity across an increasing range of many other solid malignan-cies, including non–small cell lung cancer (6, 7), renal cellcarcinoma (8), head and neck cancer (9), Merkel cell carcinoma(10), and bladder cancer (11, 12). Understanding the biologybehind response and resistance to immune checkpoint blockadeis critical to further improving outcomes of patients with meta-static melanoma.

The first immune checkpoint to be clinically targeted, thecytotoxic T-lymphocyte antigen 4 (CTLA-4), is expressed on thesurface of activated T cells and binds to its ligands, B7-1 and B7-2,on antigen-presenting cells (APC), resulting in the transmission ofinhibitory signals to T cells. In patients with metastatic melano-ma, phase III clinical trials of ipilimumab, a fully human IgG1monoclonal antibody inhibiting CTLA-4, demonstrated a signif-icant improvement in progression-free survival (PFS) and overallsurvival (OS) when compared with a gp100 vaccine (13) ordacarbazine chemotherapy (4).

Drugs targeting the programmed cell death receptor 1 (PD-1,PDCD1) showed a further increase in response rates, PFS (2), andOS (14–16) compared with anti–CTLA-4 blockade. PD-1 is alsoexpressed on the surface of activated T cells and binds to theprogrammed cell death ligand 1 (PD-L1, CD274) to negativelyregulate T-cell activation and differentiation. PD-L1 is constitu-tively expressed by T cells, macrophages, and dendritic cells (DC),as well as by some tumor cells includingmelanoma (17). Follow-up data from phase I clinical trials of the fully human IgG4monoclonal antibody, nivolumab, showed a median OS of17.3 months, with a 5 year OS rate of 34% (18). In a phase IIIstudy of nivolumab versus dacarbazine in patients with BRAFwild-type metastatic melanoma, the median OS was not reachedfor nivolumab at themost recent analysis, versus 11.2months fordacarbazine [hazard ratio (HR), 0.43, P < 0.001], and the 1- and2-year OS rates were 73% and 58%, respectively, for nivolumab(1, 14). Pembrolizumab, a humanized IgG4 monoclonal anti-body against PD-1, also showed 1-, 2-, and 3-year OS ratesof 67%, 50%, and 40%, respectively, in a phase I trial ofipilimumab-treated and ipilimumab-na€�ve patients withadvanced melanoma (3). Furthermore, in a phase III trial ofpembrolizumab versus ipilimumab, the 2-year OS rates were55% versus 43%, respectively (5, 15).

More recently, combined anti–CTLA-4 and anti–PD-1 immu-notherapies have shown improved response rates and clinicaloutcomes in comparison to ipilimumabmonotherapy (the studywas not powered to compare the two nivolumab treating arms:nivolumab plus ipilimumab and nivolumab alone). A phase IIIstudy showed an increase in the median PFS of patients treatedwith nivolumab and ipilimumab (11.5 months; HR, 0.42, P <0.001) and nivolumab alone (6.9 months; HR, 0.57, P < 0.001)compared with ipilimumab alone (2.9 months; ref. 2). At aminimum follow-up of 28 months, the median OS had not beenreached in the combination or nivolumab-alone groups andwas 20 months for ipilimumab alone [HR: combination vs.ipilimumab, 0.55 (P < 0.0001); nivolumab vs. ipilimumab,

1Melanoma Institute Australia, TheUniversity of Sydney, Sydney, NSW,Australia.2Sydney Medical School, The University of Sydney, Sydney, NSW, Australia.3Royal Prince Alfred Hospital, Sydney, NSW, Australia. 4Royal North ShoreHospital, Sydney, NSW, Australia. 5Mater Hospital, North Sydney, NSW,Australia.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Georgina V. Long, Melanoma Institute Australia, ThePoche Centre, 40 Rocklands Road, North Sydney, NSW, 2060, Australia. Phone:612-9911-7336; Fax: 612-9954-9290; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-2267

�2017 American Association for Cancer Research.

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0.63 (P < 0.0001); ref. 16]. The two-year OS rates were 64%, 59%,and 45% in the combination, nivolumab, and ipilimumabgroups, respectively (16).

The results of these clinical trials highlight the significantimpact immunotherapies have had on the clinical managementof patients with advanced-stage metastatic melanoma. However,although approximately 35% to 60% of patients have a RECISTresponse (10%–12% a complete response) to anti–PD-1-basedimmunotherapy (2, 14, 15), 40% to 65%have shownminimal orno RECIST response at the outset, and 43%of responders developacquired resistance by 3 years (3). The underlying mechanismsdriving these variations in response are not yet well understood.For an immunotherapy to elicit an efficient antitumor immuneresponse, the cancer immune cycle must be initiated and thesubsequent steps successfully completed. This involves efficient(i) antigen presentation and T-cell activation, (ii) T-cell traffickingand tumor infiltration, and (iii) T-cell killing activity within thetumor microenvironment (Fig. 1). Studies examining possiblepredictive biomarkers of response to immunotherapy havereported a higher density of preexisting cytotoxic T lymphocytesin tumor biopsies of patients who displayed a greater response toanti–PD-1/PD-L1 immunotherapy (19–21), and more signifi-cantly, an increased influx of T cells and PD-L1þ macrophagesearly during treatment (22). In this review,wediscuss the differentforms of immunotherapy resistance, the mechanisms by which

tumors evade the immune system, and strategies to overcome orprevent resistance in the future.

Primary ResistancePrimary resistance to immune checkpoint blockade occurs

in approximately 40% to 65% of patients withmelanoma treatedwith anti–PD-1 based therapy (Fig. 2), depending on whetheranti–PD-1 is given upfront or after progression on other therapies(2, 14, 15), and >70% of those treated with anti–CTLA-4 therapy(4, 13). This key unsolved clinical problem occurs when there isfailure to induce an effective antitumor immune response at anyof the three stages of the cancer immune cycle (Fig. 1). To date, theclinicopathologic factors that have been associated with primaryresistance are elevated levels of baseline serum LDH (23),increased baseline tumor burden (24), lack of PD-L1 expressionin baselinemelanoma tissue samples (Fig. 3; ref. 25), lack of T-cellinfiltration (Fig. 3; ref. 21), the absence of PD-1 T cells and PD-L1macrophages in melanoma biopsies taken early during treatment(22), insufficient neoantigens and low mutational burden (26),the presence of an innate anti–PD-1 resistance signature (IPRES)transcriptional signature (27), or absence of an interferon signa-ture (28). It is currently unknown whether these measures aresurrogates for resistance or have a direct mechanistic role inpreventing response. Here, we discuss the immune escape

Primary resistance• Poor immunogenicity• Impaired DC maturation

Acquired resistance• Loss of B2M

Primary resistance• Upregulation of PD-L1• Induction of IDO• Upregulation of Tregs• Upregulation of CD73/adenosine• Expression of IPRES• Loss-of-function mutations

Acquired resistance• Mutations in JAK1/JAK2• Upregulation of PD-L1• Upregulation of immune checkpoint markers

Primary resistance• Downregulation of chemokines• Upregulation of endothelin B receptor• Overexpression of VEGF

Antigenpresentation

and T-cellactivation

T-cell killing activitywithin the tumor

microenvironment

T-celltraffickingand tumorinfiltration

Therapeutic strategies• Radiotherapy• Oncolytic viruses• CTLA-4 inhibitors• HDAC inhibitors

Therapeutic strategies• Oncolytic viruses• HDAC inhibitors

Therapeutic strategies• PD-1/PD-L1 inhibitors• IDO inhibitors• LAG-3 inhibitors• TIM-3 inhibitors• CD73/A2AR inhibitors

© 2017 American Association for Cancer Research

Figure 1.

The cancer immune cycle. Theinduction of an effective antitumorimmune response requiressuccessful (i) antigen presentationand T-cell activation, (ii) T-celltrafficking and tumor infiltration,and (iii) T-cell killing activity withinthe tumor microenvironment.Various immune escapemechanisms present at each ofthese stages can result in primaryor acquired resistance toimmunotherapy. Potentialtherapeutic strategies can beused at each stage to overcomeimmunotherapy resistance.A2AR, A2A receptor; B2M,beta-2-microglobulin; HDAC,histone deacetylase; JAK1/JAK2,janus kinases 1 and 2; IDO,indoleamine 2,3-dioxygenase;IPRES, innate anti–PD-1 resistancesignature; LAG-3, lymphocyteactivation gene 3; TIM-3, T-cellimmunoglobulin andmucin domain3; Tregs, regulatory T cells; VEGF,vascular endothelial growth factor.

Resistance to Immunotherapy in Melanoma

www.aacrjournals.org Clin Cancer Res; 24(6) March 15, 2018 1261




mechanisms that can occur at each stage of the cancer immunecycle (Fig. 1), thereby promoting both the growth and metastasisof tumors and resistance to immune checkpoint inhibitortherapies.

Antigen presentation and T-cell activationUpon encountering and engulfing foreign antigens, such as that

of cancer cells, DCs migrate from the tumor to regional lymphnodes where they present the antigens on major histocompati-bility complex (MHC) class I molecules to CD8þ T cells, resultingin activation of the latter. Barriers at this stage of the cancerimmune cycle prevent optimal T-cell priming and activation,hence resulting in evasion of the immune system by the tumor(Table 1).

Poor immunogenicity. Some tumors lack sufficient antigen pre-sentation by the immune system (29, 30) or do not presentantigens that can be recognized as foreign (31, 32). The processof distinguishing tumor cells from normal cells depends on T-cellrecognition of tumor-specific or tumor-associated antigens (TAA;ref. 33). Tumor immune evasion by TAA-negative cells wasreported in patients with melanoma who relapsed after respond-ing to peptide vaccinations (34). Recognition of tumor neoanti-gens by T cells has been associated with increased and durableresponse to immunotherapies and increased tumor regression,indicating a significant role for neoantigens in improving the

outcome of patients with metastatic melanoma (26, 35). Circu-lating CD8þPD-1þ lymphocytes in peripheral blood of patientswith melanoma can target patient-specific neoantigens, andneoantigen-specific T cells can in turn recognize autologoustumors (36). The immunogenicity of neoantigens can be pre-dicted by combining exome sequencing and mass spectrometrydata, thereby facilitating the identification of antigens that can beused to generate active T-cell responses (37).

Analysis of The Cancer Genome Atlas (TCGA) data frommelanoma cases revealed that cutaneous melanoma displays ahigh mutational burden and UV signature (38). In addition toneoantigen recognition, a highmutational loadwas also found tocorrelate with clinical benefit to immune checkpoint blockade(26). Similarly, a positive correlation was observed between ahigher mutational load and increased CD8þ T-cell infiltration(39). Furthermore, an increased mutational burden is associatedwith elevated PD-L1 expression in advancedmelanoma (40). In apooled analysis of 832 patients withmelanoma, an increased PFSwas observed in PD-L1–positive patients treated with nivolumaband ipilimumab combined immunotherapy, aswell as in patientstreated with nivolumab alone, compared with PD-L1–negativepatients (2, 41). Similarly, PD-L1–positive patients treated withpembrolizumab had increased PFS, OS, and overall response rate(ORR), highlighting PD-L1 as a potential biomarker of response(42). As PD-L1 positivity is associated with improved responsein patients with melanoma, a lack of PD-L1 correlates with

A B


Figure 2.

Primary resistance in metastaticmelanoma. CT scans of a 55-year-oldpatient with metastatic melanomatreated with combined anti–CTLA-4and anti–PD-1 immunotherapy atbaseline (A) and after 12 weeks oftherapy (B), demonstratingwidespread metastatic disease,including significant livermetastases, who did not respond,indicating primary resistance totherapy. The patient did not respondto subsequent chemotherapy anddied 156 days after commencingcombined immunotherapy.

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primary resistance (Fig. 3). Nevertheless, some patients withPD-L1–positive tumors do not respond to PD-1 blockade, andconversely, some patients with PD-L1–negative tumors respond.For these reasons, intratumoral PD-L1 expression is a suboptimalpredictive biomarker (2, 41). Together, the aforementioned dataindicate that PD-L1 expression is a possible surrogate for lackof immunogenicity, as well as other failures further down theimmune cycle.

ImpairedDCmaturation. Inorder to efficiently activateT cells,DCsmust undergo a process called maturation, where they increasetheir capacity to stimulate T cells by displaying increased expres-sion of various costimulatory molecules required for T-cell acti-vation, such as MHC class I/II, CD80, CD86, and CD40 (43). Thedensity of DCs strongly correlates with activated T cells in mela-noma (44). The function of DCs can be impaired via multiplepathways. Interleukin (IL) 37b, a protein with a critical role in theinhibition of the innate immune response, suppresses DC matu-ration and function by decreasing CD80 and CD86 expression viathe ERK/S6K/NF-kB signaling pathways (45). Furthermore, DCmaturation and tumor infiltration increased significantly in mel-anoma following the inhibition of signal transducer and activatorof transcription3(STAT3),a transcription factor that is required fortumor growth and metastasis (46). STAT3 is also involved in thecross-talk betweenmelanoma cells and immune cells, resulting inthe induction of other immunosuppressive factors such as thevascular endothelial growth factor (VEGF), IL10, regulatory T cells(Treg), and transforming growth factor b (TGF-b), all of whichhave inhibitory effects on DC maturation (47–49).

T-cell trafficking and tumor infiltrationTumors can use a number of immune evasive mechanisms to

prevent T-cell trafficking and infiltration into tumors. Assumingthat the T cells were successfully activated in the previous steps,disruption during this stage is a likely cause for lack of response toimmunotherapy.

Downregulation of chemokines required for T-cell recruitment. Thedifferential expression of chemokine receptors is required foreffective T-cell homing and recruitment in cancer. In particular,

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

Perit

umor

Intr

atum

or

A B

C D


Figure 3.

Variable expression of immunemarkers in patients with metastaticmelanoma treated with anti–PD-1immunotherapy. Multipleximmunofluorescent imagesillustrating baseline PD-L1expression and tumor-infiltratinglymphocytes (TIL) in a 59-year-oldpatient who responded to anti–PD-1monotherapy (A and C), and lackthereof in a 55-year-old patientwho did not respond toimmunotherapy (B and D).

Table 1. Mechanisms of resistance to immune checkpoint blockade

Mechanisms of resistance Contributing factors References

Insufficient antigen presentationand recognition

Low mutational burden (26)Lack of neoantigenrecognition

(26, 35)

Loss of B2M (99, 100)Loss of MHC class I (99)

Insufficient T-cell activation Lack of mature DCs (44, 45)STAT3 expression (46, 49)

Absence of T cells from tumormicroenvironment

Lack of chemokines (50, 51, 54)VEGF overexpression (63, 64)

Upregulation of immunosuppressivemarkers

PD-L1 (69, 70)IDO (80, 81)Tregs (82–85)

Decreased sensitivity to IFN-gsignaling

Mutations in theJAK/STAT pathway

(93, 101)

Immune checkpoint markers TIM-3 (105)LAG-3 (106)

Abbreviations: B2M, beta-2-microglobulin; IDO, indoleamine 2,3-dioxygenase;IFN-g , interferon gamma; STAT3, signal transducer and activator of transcription3; TIM-3, T-cell immunoglobulin and mucin domain 3; Tregs, regulatory T cells;VEGF, vascular endothelial growth factor.






CXCR3 has been identified as an important chemokine receptorcritical for T-cell infiltration. Inmousemelanomamodels, CXCR3was highly expressed on a number of T-cell subsets, and trans-fection with its ligand, CXCL9, resulted in a significant increase inboth CD4þ and CD8þ T-cell infiltration (50). Similarly, humanmelanoma samples with high CD8þ T-cell expression were asso-ciated with increased levels of CXCL9 and CXCL10 (51). Inter-feron gamma (IFN-g , IFNG) has previously been shown to medi-ate trafficking of Tregs, T helper cells, and cytotoxic T cells (52, 53).STAT3 inhibits CXCL10 production by tumor-associatedmyeloidcells and T-cell recruitment into tumors by downregulating IFN-gproduction by CD8þ T cells (54). Conversely, Stat3 ablationincreases CXCR3 expression on CD8þ T cells, allowing T-celltumor infiltration (54).

Epigenetic alterations including DNAmethylation and histonemodifications have alsobeen identified as importantmechanismsof chemokine repression and tumor progression. Epigeneticmodifications are heritable modifications to DNA that result inchanges to the gene expression profiles of tumor cells, therebyallowing them to evade the immune system (55). Epigeneticsilencing resulted in the suppression of CXCL9 and CXCL10 inovarian cancer, and treatment with epigenetic modulatorsincreased chemokine expression and T-cell infiltration intotumors (56). Increased expression of chemokines, T-cell recruit-ment, and tumor regression was also observed in lung cancer celllines and mice treated with the histone deacetylase (HDAC)inhibitor romidepsin (57).

Upregulation of the endothelin B receptor. T-cell trafficking throughthe tumor and lymph nodes is controlled by a number ofendothelial signals, regulating T-cell homing, adhesion, andmigration (58). The interaction between endothelin 1 (ET-1,EDN1) and the endothelin A receptor (ETAR, EDNRA) promotestumorigenesis through various pathways including cell prolifer-ation, invasion, angiogenesis, bone remodeling, and inhibition ofapoptosis (59). The endothelin B receptor (ETBR, EDNRB) coun-terregulates ET-1/ETAR activity via the increased production ofnitric oxide, activation of apoptotic pathways, and clearanceof ET-1 (60). The endothelin system has been implicated in thepathogenesis of a number of cancers, including ovarian cancer,prostate cancer, and colon cancer. Interestingly, ETBR is upregu-lated in melanoma and has also been proposed as a marker ofmelanoma progression, suggesting a role for ETBR in melanomatumorigenesis (61). ETBR inhibition in 10 humanmelanoma celllines using the ETBR antagonist BQ788 resulted in an increase inapoptosis and cell death, as well as an increase in angiogenesis inthe tumors (62). In human ovarian cancers, ETBR was found tocorrelate with an absence of tumor-infiltrating lymphocytes (TIL)as well as decreased patient survival time. Administration ofBQ788 increased T-cell homing to tumors and improved theefficacy of immunotherapy (58), highlighting ETBR as a potentialimmune escape mechanism and future target in patients who failto respond to immunotherapy.

Overexpression of VEGF. Increased levels of the proangiogenicfactor VEGF in plasma and tissue samples have also been asso-ciated with the growth and progression of melanoma (63, 64).VEGF-Adownregulates T-cell adhesion to the endotheliumvia thesuppression of intercellular adhesion molecule 1 (ICAM-1) andvascular cell adhesion molecule 1 (VCAM-1) on endothelial cells(65). Increased expression of VEGF in tumor was associated with

an absence of intratumoral TILs and a shorter OS time in patientswith ovarian carcinoma (66). Inhibition of VEGF resulted in anincrease in T-cell infiltration into B16 melanoma tumors via theupregulation of CXCL10 and CXCL11 chemokines (67). Addi-tionally, VEGF-A, along with prostaglandin E2 (PGE2) and IL10,upregulates the Fas ligand, resulting in CD8þ T-cell death, andsubsequent inhibition of VEGF and PGE2 increased CD8þ T-cellinfiltration (68). Corresponding with these data, in melanomatumor biopsies, increased levels of VEGFA were observed innonresponders to anti–CTLA-4 and anti–PD-1 immunotherapiesin comparison to responders (19).

T-cell killing activity within the tumor microenvironmentPrimary resistance to immunotherapy also occurs when T cells

become successfully activated and infiltrate the tumor; however,their function is hindered by the presence of immunosuppressivemolecules within the tumor microenvironment (31).

Upregulation of PD-L1. Primary resistance can be driven by theconstitutive expression of PD-L1 through oncogenic signaling(69, 70). The increased expression of PD-L1 by cells in the tumormicroenvironment results in decreased function of cytotoxic Tcells and apoptosis, hence providing an immune escape mecha-nism for tumor cells.

Several studies have revealed a correlation between loss of thephosphatase and tensin homolog (PTEN) in cancer and theupregulation of PD-L1, implicating PD-L1 in tumor immuneevasion. PTEN is a tumor suppressor that negatively regulates theP13K/AKT pathway. This pathway is responsible for the regula-tion of cellular processes such as proliferation and survival. Theloss of PTEN and activation of the P13K/AKT pathway in humanglioma cell lines resulted in an increase in posttranscriptionalCD274 expression (71). PD-L1 expressionwas also upregulated inlung squamous cell carcinoma following the simultaneous deple-tion of Pten and Lkb1 [also known as Stk11 (serine–threoninekinase 11); ref. 72]. Inmelanoma, loss of PTEN led to a decrease inT-cell trafficking, infiltration, and T-cell activity (73). However,silencing PTEN did not significantly alter the expression of PD-L1in melanoma cell lines in vitro or in xenograft models in vivo,indicating that PD-L1 regulation may not be the principal mech-anism of immune resistance resulting from a loss of PTEN (73).

Other mechanisms that have been shown to have a role in theconstitutive upregulation of PD-L1 include the transcriptionfactor interferon regulatory factor 1 (IRF-1) and mutations in theepidermal growth factor receptor (EGFR). IRF-1 is responsible forthe regulation of cell proliferation, apoptosis, and immunity (74).The knockdown of IRF-1 using siRNA resulted in the decrease intranscription and translation of PD-L1 in a lung carcinoma cellline (75). Similarly, activationof the EGFRpathway resulted in theincreased expression of PD-L1 in lung cancer cell lines (76, 77)and tissue (76). Increased expression of markers of T-cell exhaus-tion, such as PD-1 and FOXP3, was also observed in the tumormicroenvironment (76). PD-1 blockade increased cytotoxic T-cellnumbers as well as effector T-cell function (76), highlighting therole of the PD-1/PD-L1 axis in immune evasion and its mani-pulation as a therapeutic strategy.

Induction of IDO. Another molecule proposed to play a criticalrole in the negative regulation of T-cell function is indoleamine2,3-dioxygenase (IDO, IDO1). IDO is expressed in awide range ofhuman cancers and is the rate-limiting enzyme responsible for the

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degradation of tryptophan into kynurenine (78, 79). T lympho-cytes undergo arrest in response to this tryptophan depletion,resulting in the suppression of T-cell proliferation and activity(80). To understand the mechanism of immunosuppressioninduced by IDO, Holmgaard and colleagues (81) developed aB16 melanoma tumor model overexpressing IDO and revealed acorrelation between IDO expression and increased tumor-infil-trating myeloid-derived suppressor cells (MDSC), as well asCD4þFOXP3þ Tregs. This association demonstrated that IDOsuppresses T-cell activity through the recruitment and activationof MDSCs in a Treg-dependent fashion (81). Systemic inhibitionof IDO in mice using a tryptophan analogue, 1-methyl-L-trypto-phan (1MT), reduced tumor progression in a T-cell–dependentmanner (79). Similarly, administration of 1MT in combinationwith anti–CTLA-4 immunotherapy in B16F10 mouse melanomamodels resulted in a significant delay in tumor growth and anincrease in OS (78). These findings provide a strong rationale fortargeting IDO to improve the efficacy of immunotherapies inpatients with melanoma.

Upregulation of Tregs. The upregulation of FOXP3-expressingTregs has been observed in a number of melanoma studies,revealing a possible role for Tregs in melanoma tumorigenesis(82–84). Tregs promote tumor growth by inhibiting the activityof T-cell subsets, either through direct cell-to-cell contact orindirectly through the secretion of anti-inflammatory cyto-kines, such as IL10 and TGF-b (85, 86). The presence ofCD4þCD25þ FOXP3 Tregs was observed amongst TILs inmetastatic melanoma (87) and the transfer of CD25 (IL2RA)-depleted splenic T cells into B16 mouse melanoma modelsresulted in the suppression of tumor growth in vivo (82). Popula-tions of tumor-infiltrating Tregs also significantly correlated withincreased tumor growth in B16BL6 mice (88). Furthermore, adecrease in FOXP3þ Tregs significantly correlated with increasedtumor control and survival in patients with melanoma treatedwith ipilimumab (89), highlighting the immunosuppressivefunction of Tregs in melanoma.

Upregulation of the CD73/adenosine pathway. Elevated levels ofextracellular adenosine and CD73 (NT5E) have also been impli-cated in immune suppression and tumor progression. Adenosineis produced via the conversion of extracellular ATP by the ectoen-zymes CD39 (ENTPD1) and CD73 and binds to the adenosineA2A receptor (A2AR,ADORA2A) to inhibit effector T-cell function(90). Increased CD73 expression correlated with advanced-stagedisease in melanoma (91), and an upregulation in CD73 wasobserved in patients who progressed following treatment withanti–PD-1 immunotherapy (92). A2AR inhibition increasedCD8þ T-cell infiltration and significantly reduced tumor growthinmousemelanomamodels (91), suggesting a role for the CD73/adenosine axis in promoting immune escape.

Expression of IPRES signature. The expression of IPRES has recent-ly been identified as a mechanism of primary resistance toimmunotherapy. Transcriptomal analyses of responding andnonresponding pretreatment melanoma biopsies from patientstreated with anti–PD-1 immunotherapy revealed the coenrich-ment of genes associated with mesenchymal transition, woundhealing, and angiogenesis in nonresponding patient samples(27). This was observed not only in metastatic melanoma butalso in other major cancer types such as pancreatic cancer (27).

Loss-of-function mutations.Mutations in the janus kinases 1 and 2(JAK1/2) have also been shown to be involved in primary resis-tance to anti–PD-1 immunotherapy. JAK1/2 loss-of-functionmutations identified in one of 23 melanoma tumor biopsies,and two of 48 human melanoma cell lines via whole-exomesequencing, resulted in a lack of PD-L1 expression due to aninability to respond to IFN-g signaling (93). Furthermore, therecent development of a two cell type-CRISPR (2CT-CRISPR)screening assay revealed an important role for apelin receptor(APLNR) loss-of-function mutations in disturbing effector T-cellfunction (94). Retroviral overexpression of APLNR correlatedwith an increase in JAK1 as well as tumor sensitivity to effectorT-cell function (94). Conversely, APLNR-knockout cells demon-strated decreased activation of the JAK/STAT pathway followingIFN-g treatment, and Aplnr knockout in mouse melanoma in vivoresulted in a decrease in the efficacy of anti–CTLA-4 immuno-therapy (94). These findings provide a strong rationale for furtherinvestigating APLNR as a potential target to prevent immuneevasion by tumors.

Mechanisms under investigationThe composition of the gut microbiome. Recent studies havehighlighted a possible role for the gut microbiome in patientresponse to immunotherapy. Dysbiosis (an imbalance of themicrobiota) involves decreases in the diversity and stability ofthe microbiome, thereby promoting tumorigenesis (95).Sequencing of the oral and gut microbiome of patients withmetastatic melanoma revealed a correlation between higher gutmicrobiome diversity and response to anti–PD-1 monotherapy(96). Responders also had a significantly different gut micro-biome composition in comparison with nonresponders, and thiscorrelated with differences in PFS (96). The increased abundanceof specific bacteria in the gut microbiome also correlated with ahigher CD8þ T-cell density in responders to anti–PD-1 immuno-therapy (96). Similarly, the composition of the baseline gutmicrobiome inpatientswithmetastaticmelanomawas associatedwith response to ipilimumab, and improved PFS and OS wereassociatedwith specific groups of bacteria such as Faecalibacteriumand other Firmicutes (97). Additionally, a significant decrease inTILs and lack of response to CTLA-4 blockade was observed intumors of mice housed in germ-free conditions (98). The anti-cancer therapeutic effects of the anti–CTLA-4 antibody wererestored upon oral feeding of the germ-free mice with Bacteroidesfragilis (B. fragilis), aBacteroides isolate, aswell aswith the adoptivetransfer of B. fragilis–specific memory T cells (98). The mechan-isms through which the gut microbiome influences the immuneresponse are currently being investigated.

Acquired ResistanceAcquired resistance occurs in patients who relapse after exhibit-

ing an initial response to immunotherapy (Fig. 4). Currently, littleis understood about the mechanisms that give rise to acquiredresistance, and many are likely to be similar to those underlyingprimary resistance (Table 1).

Acquired resistance to immunotherapy can developwhen thereis Darwinian selection of subpopulations of tumor cells withgenetic and epigenetic traits that allow them to evade the immunesystem (32). An example of one such trait is beta-2-microglobulin(B2M), a component of MHC class I molecules that is necessaryfor their functional expression. The loss of B2M expression was






reported in melanoma cell lines from five patients who had beentreated with immunotherapy and cytokine–gene therapy (99).This resulted in a loss of MHC class I expression and, therefore, asubsequent decrease in recognition by CD8þ T cells. Archivaltissues taken prior to immunotherapy from three of these patientswere B2M positive, suggesting loss of B2M expression as a mech-anism of acquired resistance (99). Similarly, the loss of B2M hasbeen observed in sequential lesions obtained from a patient withmetastatic melanoma following immunotherapy treatment withDCs transfected with autologous tumor mRNA (100).

JAK1/2 mutations have also recently been identified as geneticmarkers of acquired resistance to immunotherapy in melanoma.These mutations in tumor cells lead to decreased sensitivity toIFN-g , ultimately preventing IFN-g–induced cell growth arrest(101). Upon exposure to IFN-g (produced by activated T cells),JAK1/2 become activated and subsequently phosphorylate atyrosine residue present on STATs (102). This JAK/STAT signalingpathway is responsible for cell proliferation, differentiation, cellmigration, and apoptosis (102). However, IFN-g also results inthe upregulation of PD-L1 on tumor cells, thus inactivatingantitumor T cells (70). Loss-of-function mutations in the genesencoding JAK1 or JAK2 were found in relapsed tumors in two offour patients following whole-exome sequencing of baseline andprogression biopsies; all patients had an objective response totreatment with pembrolizumab and then progressed aftera median of 1.8 years (101). The anti–PD-1-resistant cells har-boring JAK mutations were derived from cells clonally selectedfrom the baseline tumor. These findings demonstrate the roleof the JAK/STAT pathway in promoting acquired resistanceto immunotherapy.

In addition, acquired resistance can also occur on the level ofthe individual cells, whereby tumor cells alter their gene expres-sion in response to immune molecules within the tumor micro-environment (32). For example, PD-L1 can be upregulated bytumor cells in response to immune cytokines, such as IFN-greleased by T cells, hence limiting T-cell function (70), and canoccur in both primary and acquired resistance (32, 103).

Other immune checkpoint markers such as lymphocyte acti-vation gene 3 (LAG-3) and T-cell immunoglobulin and mucindomain 3 (TIM-3, HAVCR2) have also been revealed to interferewith the activity of T cells (70, 104), resulting in acquiredresistance to immunotherapy. In a recent study, TIM-3 upregula-tion was observed in patients who developed adaptive immuneresistance to anti–PD-1 immunotherapy (105). Furthermore,

TIM-3 blockade in mice resulted in a significant increase insurvival time, as well as increased production of IFN-g andproliferation of CD8þ T cells (105). LAG-3 is also overexpressedinPD-L1–positivemelanoma, suggesting LAG-3upregulation as apotential immune evasion mechanism (106).

OvercomingMechanismsofTumor ImmuneEvasion

From the above, it is clear that there exist multiple immuneevasive mechanisms that can be utilized by tumors at each of thedifferent stages of the cancer immune cycle thatmay induce eitherprimary or acquired resistance to immunotherapy. Determiningthe specificmechanisms underlying resistance to immunotherapyin these patients is a crucial step toward effective treatment andultimately producing durable responses for them.

Combinatorial therapiesThe immune escapemechanisms discussed above do not act in

isolation. Together, the overlap between various signaling path-ways and the interactions between several of the immunosup-pressive molecules leads to resistance. It is likely that combina-tions of therapy will be more effective than single-agent therapiesfor a given patient. However, the challenge remains to determinewhich of these combinations are most effective and in whichpatient given interpatient heterogeneity. Currently, multiple clin-ical trials are underway examining the activity and toxicity ofcombined immunotherapies, particularly using an anti–PD-1drug in combination with an agent targeting a complimentarypart of the immune system (Supplementary Table S1).

Dual blockade of PD-1 and LAG-3 in mouse cancer modelsresulted in the regression of tumors in most mice, as well as anincreased survival rate (107). In a recent study, all mice that weretreated with a triple combination of LAG-3 blockade, PD-1blockade, and poxvirus-based immunotherapy demonstratedcomplete tumor regression (108). Phase I clinical trials involvingLAG-3 blockade with and without PD-1 blockade in solid tumorsare currently underway, and in patients who had progressed onanti–PD-1 monotherapy, a response rate of 16% (any tumorreduction in 45%) was observed in those patients whose tumorsexpressed LAG-3 (109). Similarly, in mouse models, combinedTIM-3 and PD-L1 blockade significantly reduced tumor growth incomparison with single-agent immunotherapy (110). Further-more, dual blockade of TIM-3 and PD-L1 increased the ability of

A C DB


Figure 4.

Acquired resistance in metastaticmelanoma. Images of a 74-year-oldpatient with metastatic melanomatreatedwith anti–PD-1monotherapy,showing complete resolution ofmelanoma in right-neck lymphnodes from baseline (A), week 4 (B),and week 12 (C). However, acquiredresistance developed in the rightpreauricular region at 12 monthsafter the commencement of anti–PD-1 monotherapy (D). The patientsubsequently received radiotherapyand ipilimumab in addition tocontinuing anti–PD-1, with anexcellent ongoing response 12months after acquired resistance.

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CD8þ T cells to produce IFN-g , thereby restoring their function(110). Consistent with this, anti–TIM-3 and anti–PD-1/anti–CTLA-4 combined immunotherapy resulted in a significantdecrease in the tumor sizes of multiple cancers (111).

Phase I/II clinical trials testing the combination of an IDOinhibitor with PD-1/PD-L1 inhibitors in metastatic melanomaare also currently underway. These include the administration ofnivolumab and a PD-L1/IDO peptide vaccine (NCT03047928),and indoximod (IDO inhibitor) in combination with CTLA-4 orPD-1 inhibitors (NCT02073123). Furthermore, the combinationof epacadostat (an IDO1 inhibitor) plus pembrolizumab wasgenerally well tolerated and correlated with improved response invarious cancers (112–114), leading to the initiation of phase IIIstudies such as that in patients with treatment-na€�ve advancedmelanoma (NCT02752074).

Many studies have also demonstrated the increased efficacy ofcombined radiotherapy and immune checkpoint blockade.Radiotherapy activates the inflammatory pathways and inducesDNA damage, resulting in the release of antigens from irradiatedcells and an increase in tumor sensitization to T-cell immuneresponses (115). Radiotherapy in patients with advanced mela-noma who had progressed following treatment with ipilimumabled to an abscopal effect in some patients, whereby irradiation ofthe primary tumor induced regression in other nonirradiatedmetastases, which correlated with longer OS (116). Significanttumor regression was also observed in patients with metastaticmelanomawho received combined radiotherapy and anti–CTLA-4 immunotherapy, and experiments in mice showed that acombination of anti–CTLA-4 immunotherapy, anti–PD-L1immunotherapy, and radiotherapy was required to achieve opti-mum response (117).

The combination of epigeneticmodulators and immune check-point blockade has recently been shown to bemore effective thansingle-agent immunotherapy in patients with melanoma. Theinhibition of HDAC using LBH589 in combination with PD-1blockade inB16F10mousemelanomamodels resulted indelayedtumor growth and increased survival compared with the controlgroup and PD-1 blockade alone (118). Similarly, a reduction intumor size and Tregs was observed in B16F10 mouse melanomamodels treated with a combination of anti–CTLA-4 immunother-apy and epigenetic modulation of trimethylation of lysine 27 onhistone H3 (H3K27me3) compared with CTLA-4 blockade alone(119). The efficacy of combined epigenetic and immune check-

point therapies is currently being tested in various human cancers(Supplementary Table S1).

These findings provide a strong rationale for the developmentof combinations of different treatment strategies in clinical trialsas an effective means of melanoma treatment.

ConclusionsCheckpoint inhibitor immunotherapy has revolutionized the

treatment of multiple cancer types. However, responses inpatients with metastatic melanoma are diverse, with manypatients displaying primary or acquired resistance. An importantongoing,major unmet clinical need remains to identify predictorsand causes of this resistance and strategies to overcome them. Akey element of effective immunotherapy is identifying the variousmechanisms by which the tumors evade the immune system onan individual basis. Improving our understanding of thesemechanisms and determining which immune markers to targetin each patient will then allow for the administration of the mostappropriate form of therapy to achieve optimal response andimprove the overall outcomes of patients with metastaticmelanoma.

Disclosure of Potential Conflicts of InterestG.V. Long reports receiving speakers bureau honoraria from Bristol-Myers

Squibb, Incyte, Merck, Novartis, and Roche, and is a consultant/advisory boardmember for Amgen, Array, Bristol-Myers Squibb, Incyte, Merck, Novartis, PierreFabre, and Roche. No potential conflicts of interest were disclosed by the otherauthors.

AcknowledgmentsThis work was supported by Melanoma Institute Australia, the New South

Wales Ministry of Health, NSW Health Pathology, National Health andMedical Research Council of Australia (NHMRC), and Cancer Institute NSW.J.S. Wilmott, R.A. Scolyer, and G.V. Long are supported by NHMRC Fellow-ships. J.S. Wilmott is also supported by a CINSW Fellowship. G.V. Long isalso supported by the University of Sydney Medical Foundation. T.N. Gide issupported by The University of Sydney and Melanoma Institute AustraliaScholarships.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 12, 2017; revised October 15, 2017; accepted November 6,2017; published OnlineFirst November 10, 2017.

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Gide et al.




2018;24:1260-1270. Published OnlineFirst November 10, 2017.Clin Cancer Res Tuba N. Gide, James S. Wilmott, Richard A. Scolyer, et al. in Metastatic MelanomaPrimary and Acquired Resistance to Immune Checkpoint Inhibitors

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