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Translational Cancer Mechanisms and Therapy

Targeting Cancer Cells and TumorMicroenvironment in Preclinical and ClinicalModels of Hodgkin Lymphoma Using the DualPI3Kd/g Inhibitor RP6530Silvia L. Locatelli1, Giuseppa Careddu1, Simone Serio1, Francesca M. Consonni2,Akihiro Maeda2, Srikant Viswanadha3, Swaroop Vakkalanka4, Luca Castagna1,Armando Santoro1,5, Paola Allavena2,5, Antonio Sica2,6, and Carmelo Carlo-Stella1,5

Abstract

Purpose: Tumor-associated macrophages (TAMs) and thehyperactivation of the PI3K/AKT pathway are involved inthe pathogenesis of Hodgkin lymphoma and affect diseaseoutcome. Because the d and g isoforms of PI3K are over-expressed in Hodgkin/Reed–Sternberg (HRS) cells andthe tumor microenvironment (TME), we propose thatthe PI3Kd/g inhibitor RP6530 might affect both HRS cellsand TME, ultimately leading to an enhanced antitumorresponse.

Experimental Design: Hodgkin lymphoma cell lines(L-540, KM-H2, and L-428) and primary humanmacrophageswere used to investigate the activity of RP6530 in vitro and invivo in Hodgkin lymphoma cell line xenografts.

Results: In vitro, RP6530 besides killing and inhibitingthe proliferation of Hodgkin lymphoma cells, downregu-lated lactic acid metabolism, switching the activation ofmacrophages from an immunosuppressive M2-like pheno-type to a more inflammatory M1-like state. By RNA

sequencing, we define tumor glycolysis as a specificPI3Kd/g-dependent pathway implicated in the metabolicreprogramming of cancer cells. We identify the metabolicregulator pyruvate kinase M2 as the main mediator oftumor-induced immunosuppressive phenotype of macro-phages. Furthermore, we show in human tumor xenograftsthat RP6530 repolarizes TAMs into proinflammatorymacrophages and inhibits tumor vasculature, leading totumor regression. Interestingly, patients with Hodgkinlymphoma experiencing objective responses (completeresponse and partial response) in a phase I trial usingRP6530 showed a significant inhibition of circulating mye-loid-derived suppressor cells and an average mean reduc-tion in serum thymus and activation-regulated chemokinelevels of 40% (range, 4%–76%).

Conclusions: Our results support PI3Kd/g inhibition asa novel therapeutic strategy that targets both malignant cellsand the TME to treat patients with Hodgkin lymphoma.

IntroductionPrimary refractory and early-relapsed patients with Hodgkin

lymphoma experience poor responses to salvage chemotherapyand dismal long-term disease control (1–3). Despite the variety ofnovel therapeutic options, they represent an unmet medical needurgently requiring novel therapeutic agents to overcome thechemo-refractory phenotype (4–8).

The PI3K/Akt pathway is implicated in the pathogenesis ofHodgkin lymphoma (9). The d isoform of PI3K is highlyexpressed in tissues of hematopoietic origin and is involvedin the activation, proliferation, survival, homing, and retentionof B-cells in lymphoid tissues (10). Idelalisib is the first PI3Kdinhibitor to be approved for follicular lymphoma and chroniclymphocytic leukemia (11, 12), and we previously reportedthat PI3Kd isoform inhibition results in direct Hodgkin lym-phoma cell killing (13). The PI3Kg isoform, although highlyexpressed in leukocytes, may play a more crucial role in theimmune system than that in oncogenesis (10). To date, mucheffort has been devoted to PI3Kg as a target in inflammatorydiseases driven by leukocytes (14). Inflammation driven bytumor-associated macrophages (TAMs) is now considered ahallmark of cancer, contributing to both cancer cell expansionand angiogenesis (15). Recent data in solid tumors show thatselectively targeting the g isoform of PI3K in TAMs modulatesthe immunosuppressive tumor microenvironment (TME),resulting in tumor regression (16–18).

TAMs have been implicated in the pathogenesis of Hodgkinlymphoma and have been suggested to negatively impactclinical outcome (19–23). Both the d and g isoforms of PI3Kare overexpressed in HRS cells and cell of the microenviron-ment, respectively, thereby representing attractive therapeutic

1Department of Oncology and Hematology, Humanitas Cancer Center, Rozzano,Milan, Italy. 2Department of Inflammation and Immunology, Humanitas Clinicaland Research Center, Rozzano, Milan, Italy. 3Incozen Therapeutics, Hyderabad,India. 4Rhizen Pharmaceuticals S A, La Chaux-de-Fonds, Switzerland. 5Depart-ment of Biomedical Sciences, Humanitas University, Rozzano, Milan, Italy.6Department of Pharmaceutical Sciences, Universit�a del Piemonte Orientale"Amedeo Avogadro", Novara, Italy.

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

Corresponding Authors: Carmelo Carlo-Stella, Humanitas Cancer Center andHumanitas University, Via Manzoni, 56, Rozzano, Milan 20089, Italy. Phone: 3902 8224 4577; Fax: 39 02 8224 4590; E-mail: [email protected];and Silvia Laura Locatelli, [email protected]

doi: 10.1158/1078-0432.CCR-18-1133

�2018 American Association for Cancer Research.

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targets (24). Here, we describe the effects of RP6530, a novelPI3Kd/g inhibitor currently in phase I to II clinical trials inEurope and the United States. RP6530 exhibits high antipro-liferative and cytotoxic activity in Hodgkin lymphoma cell linesin vitro and potent efficacy in vivo in preclinical xenograft mousemodels. We show that RP6530 downregulates lactic acidmetabolism in Hodgkin lymphoma cells, reducing M2-likepolarization of macrophages. Furthermore, we demonstratethat this effect is mediated by the metabolic regulator pyruvatekinase M2 (PKM2). RP6530 reshapes the TME, inhibits angio-genesis, and switches TAM activation from an immunosup-pressive M2-like phenotype to a more inflammatory M1-likestate. We propose that dual pharmacologic targeting of the dand g PI3K isoforms affects both malignant tumor cells and theTME, ultimately leading to an enhanced antitumor response.The ability of RP6530 to affect both Hodgkin lymphoma tumorcells and the Hodgkin lymphoma TME indicates that a novelunique therapeutic opportunity may be achievable for treat-ment of patients with Hodgkin lymphoma.

Materials and MethodsReagents

Rhizen Pharmaceuticals, SA (La Chaux-de-Fonds) kindlyprovided the PI3Kd/g inhibitor RP6530. RP6530 had highpotency against PI3Kd (IC50 ¼ 24.5 nmol/L) and g (IC50 ¼33.2 nmol/L) enzymes with selectivity over a (>300-fold) and b(>120-fold) isoforms with specificity being similar in isoform-specific cell-based assays as well (25). For in vitro experiments,RP6530 was reconstituted in 100% DMSO and further dilutedin RPMI1640 to final concentrations of 0.05% and 0.1%DMSO(v/v). For in vivo experiments, RP6530 was dissolved in 0.5%methylcellulose (pH 2.2).

Cell death and cell proliferation assayHodgkin lymphoma cell lines (4� 105mL�1) were cultured in

the absence or presence of RP6530 (ranging between 1.25 and10 mmol/L) for 24, 48, and 72 hours. Dead and proliferating cellswere detected by annexin-V/propidium iodide double staining(Immunostep) and WST assay (BioVision), respectively, accord-ing to the manufacturer's instructions. See SupplementaryInformation for further information.

Cell-cycle analysisHodgkin lymphoma cell lines were cultured in the absence or

presence of RP6530 (5 or 10 mmol/L) for 48 hours, fixed in 70%ethanol, and then stained with 2.5 mg/mL propidium iodide (PI)(Calbiochem). Cell-cycle status was measured using a FACSCa-libur flow cytometry system (BD Biosciences) and analyzed usingFlowJo software (Treestar).

RNA preparation and sequencingpolyA-RNA-seq was performed on Hodgkin lymphoma cell

lines (L-540 and KM-H2) after RP6530 100 nmol/L for 6 hoursand after RP6530 10 mmol/L for 6 and 24 hours. Two biologicalreplicates were profiled for each experimental condition. RNAwas purified using an RNeasy Mini Kit (Qiagen) and treatedwith DNase according to the manufacturer's protocol. TotalRNA quality was evaluated using an Agilent Bioanalyzer and anRNA Nano Kit. Only samples with an RIN score �8 wereprocessed further. RNA-seq libraries were sequenced using aTruSeq stranded mRNA Kit (Illumina) according to the man-ufacturer's instructions. See Supplementary Information forfurther information.

RNA-Seq analysisRNA-Seq samples were demultiplexed, and FASTQ files were

created from BCL files using bcl2fastq (Illumina). Quality controland assessmentwere performed using FastQCv0.11 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). STAR v2 wasused to align each sample's single-end reads to the GencodeHuman reference genome (build GRCh38). The raw read countswere normalized with TMM using edgeR package in R/Biocon-ductor. Differentially expressed transcripts were calculated inRPKM using edgeR. Significant genes were selected based on anFDR �0.1 and logCPM �0.5. Hierarchical clustering of the log2fold change (log2FC) between the vehicle and RP6530-treatedsamples at 6 and 24 hours was performed with cluster 3.0 usingthe complete linkage method and visualized using Java TreeView(NCBI Gene Express Omnibus website, GSE105439).

Pathway analysisPathway analysis was performed using QIAGEN's commercial

Ingenuity Pathway Analysis (IPA, www.qiagen.com/ingenuity)software. GSEA was used in preranked mode. The rank metricwas calculated as the sign of log2FCs multiplied by the inverseof adjusted P values. The gene sets used for this analysiswere the C2:CP:KEGG and C2:CP:PID Curated Molecular Signa-tures Database (MSigDB) gene sets. Enrichr was used to findsignificantly enriched (adjusted P value �0.1) KEGG and PIDterms for the gene set continuously modulated during the timecourse. PPI networks for the select MSigDB gene set pathways(KEGG_GLYCOLYSIS_GLUCONEOGENESIS and PID_HIF1_-TFPATHWAY) were constructed using the STRING database(http://www.string-db.org/). See Supplementary Information forfurther information.

Human macrophage differentiation and cultureHuman leukocytes from apheresis blood products were

obtained from the Pavia Blood Bank. Cells were diluted in PBSand centrifuged over Histopaque 1077 to purify mononuclearcells. An EasySep Human Monocyte Enrichment Kit (StemcellTechnologies) was used to isolate monocytes from peripheralblood mononuclear cells by negative selection. Purified

Translational Relevance

In preclinical and clinical models of Hodgkin lymphoma,the PI3Kd/g inhibitor RP6530 exerts a direct cytotoxic effect onHodgkin lymphoma cells and reprogrammes the tumor-asso-ciated macrophages (TAMs) from a protumor M2 phenotypeto an antitumor M1 phenotype, thus reshaping the interplaybetween cancer cells and their tumor microenvironment(TME). As a consequence of TME reprogramming induced bythe PI3Kd/g inhibition, Hodgkin lymphoma tumor cells andtumor vasculature are effectively reduced. Our data establishthe first evidence of the translational potential of PI3Kd/ginhibition in suppressingmalignant cell growth and reshapingthe microenvironment of Hodgkin lymphoma, suggestingthat a novel unique therapeutic opportunity may be achiev-able for treatment of patients with Hodgkin lymphoma.

PI3Kd/g Inhibition Targets HL TME and Cancer Cells

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http://www.bioinformatics.babraham.ac.uk/projects/fastqc

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monocytes were cultured in RPMI supplemented with 10% FBSand 50 ng/mL human mCSF (PeproTech). Nonadherent cellswere removed after 2 hours by washing, and adherent cells werecultured for 6 days to fully differentiate macrophages.

Macrophage polarization assayPeripheral blood-derived macrophages were polarized

toward an M1 phenotype with the addition of IFNg (20 ng/mL;Peprotech) and LPS (100 ng/mL; Alexis) or toward an M2phenotype with the addition of IL4 (20 ng/mL; Peprotech) for24 hours. Polarized macrophages were incubated with RP6530(10 mmol/L) for 24 hours, and mRNA expression and flowcytofluorimetric analyses were then performed. RNA was har-vested from the cells (Qiagen RNeasy), and SYBR green-basedqPCR was performed using primers for human CXCL9,IL12p40, CXCL11, CXCL10, CD80, CCL17, CCL22, IL10,CD301, and CD163. mRNA levels were normalized to actinexpression (DCt ¼ Ct

gene of interest � CtActin) and reported as

relative mRNA expression (DDCt ¼ 2�(DCtsample�DCtcontrol)).Primary antibodies to cell surface markers directed againstCD14 (M5E2) and CD80 (L307) were from BD Pharmingen,and primary antibodies to cell surface markers directed againstCD40 (HB14), CD209 (9E9A8), and CD301 (H037G3) werefrom eBioscience.

siRNA-mediated gene silencingHodgkin lymphoma cell lines (3� 105) were transfected using

Lipofectamine RNAiMAX transfection reagent (Thermo FisherScientific) with 1 nmol PKM2 siRNA (#285 and #286) or SilencerSelectNegativeControl siRNA (#AM4621 and#AM4611; ThermoFisher Scientific) for 48 hours according to the manufacturer'sinstructions.

Transwell coculture assayHodgkin lymphoma cells (3 � 105 cells) were plated in the

lower compartment of 6.5-mm polycarbonate Transwell inserts(pore size 0.4 mm; Corning) and exposed to RP6530 (10 mmol/L)or siRNA transfection as described in the previous section. Thenext day, 3 � 105 M2-polarized macrophages were placed in theupper compartment of the Transwell inserts and cocultured withthe Hodgkin lymphoma cells. After 24 to 48 hours of coculture,RNAwas extracted from themacrophages in the upper inserts, andHodgkin lymphoma cells were washed and then used for subse-quent experiments. Control wells contained either Hodgkin lym-phoma cells only in the lower compartment with RPMI in theupper compartment or RPMI in the lower compartment with onlyM2-polarized macrophages or M0 macrophages in the uppercompartment.

ImmunoblottingHodgkin lymphoma cell lines or polarized M1 and M2macro-

phages were treated with RP6530 as described in the figurelegends and proteins were detected with the indicated antibodies.See Supplementary Information for further information.

Biochemical assaysThe lactate concentration in Hodgkin lymphoma cell and

polarized macrophage lysates was measured after RP6530(10 mmol/L) treatment with an L-Lactate Assay Kit (Abcam)according to the manufacturer's instructions.

ELISASerum samples were centrifuged at 4,000 rpm for 10 minutes

and then kept at �80�C until ELISA assessment. Serum levels ofthymus and activation-regulated chemokine (TARC/CCL17)weredetermined according to the manufacturer's instructions (R&DSystems). The TARC/CCL17 level in patient sera samples wasdetermined by correlating each value duplicate with a standardcurve based on a 2-fold serial dilution of recombinant TARC/CCL17 with known concentration.

Flow cytometry staining and analysisSingle-cell suspensions (106 cells in 100 mL total volume) were

incubated with FcR-blocking reagent (BD Biosciences) at 4�C for30minutes. Staining of cell surfacemarkers was performed at 4�Cfor 20minuteswith the following primary antibodies: anti-mouseF4/80 (BM8), anti-mouse CD45 (30-F11), anti-mouse/humanCD11b (M1/70), anti-mouse CD86 (GL-1), anti-mouse CD301(LOM-14), anti-mouse MHC-II (M5/114.15.2), anti-mouseCD206 (C068C2), anti-mouse Ki-67 (16A8), anti-human HLA-DR (L243), anti-human CD14 (M5E2), anti-human CD33(WM53; Biolegend); and anti-mouse Ly6G (1A8) and anti-mouseLy6C (HK1.4; eBioscence). Unconjugated rabbit anti-mouseNOS2 (Abcam) was also used followed by incubation withsecondary goat anti-rabbit Alexa Fluor 647-conjugated antibody(Invitrogen,Molecular Probes). For the gating of the viable cells, aLIVE/DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher)was used. For intracellular staining, a Cytofix/Cytoperm andPermwash Staining Kit (BD Pharmigen) was used according tothe manufacturer's instructions. Multicolor FACS analysis wasperformed on aBDFACSCanto IIflow cytometer. All data analysiswas performed using the flow cytometry analysis program FlowJo(Treestar).

Tumor challenge and treatment experimentsSix- to 8-week-old NOD/SCID mice (20–25 g) were pur-

chased from Charles River Labs and xenografted with L-540 (25� 106 cells) and KM-H2 (20 � 106 cells) cells by subcutaneousinoculation into the right flank. The treatments started whenthe tumors were palpable (approximately 200 mm3). RP6530was administered by oral gavage twice per day at 100 and 150mg/kg for 3 weeks or at 150 mg/kg for 5 days. The controlgroups received a vehicle (0.5% methylcellulose, pH 2.2)without the active product. When appropriate, in vivo biotiny-lation of tumor vasculature (26) was performed 5 days afterRP6530 treatment and 3 hours after the last drug administra-tion. The animal experiments were performed according to EU86/109 Directive (D.L. 116/92 and following additions) andwere approved by the institutional Ethical Committeefor Animal Experimentation of the Humanitas Clinical andResearch Center. See Supplementary Information for furtherinformation.

Tumor-infiltrating myeloid cell analysisSix- to 8-week-old NOD/SCID mice were injected subcutane-

ouslywith L-540 (25�106 cells) andKM-H2 (20�106 cells) cellsin 400 mL of PBS in the right flank. On days 19 (for L-540) or 25(for KM-H2) after tumor injection, the tumor-bearing mice weregrouped and treated with RP6530 (150 mg/kg, twice per day,orally) or vehicle (0.5%methylcellulose, pH2.2) for 5 days. Threehours after the final treatment, the mice were euthanized, and thetumors were snap frozen or digested in a mixture of 0.5 mg/mL

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collagenase IV and 150 U/mL DNase I in RPMI1640 for 30minutes at 37�C. Tumor-infiltrating myeloid cells were analyzedby IHC and flow cytometry. Tumor macrophage enrichment wasperformed by plating cells in FBS-free RPMI containing 1%penicillin/streptomycin for 1 hour at 37 �C and 5% CO2. After1 hour, nonadherent cells were removed with 3 PBS washes, andRNA was harvested from tumor macrophages (Qiagen RNeasy).SYBR green-based qPCR was performed using primers for murineIL1b, CXCL10, iNOS, TNFa, CD80, CXCL11, IL6, Arg1, CCL22,CCL2, IL10,CD163,CD206, TGFb, PGF, IGF1, EGF, FGF2,VEGFA,HIF-1a, and PD-L1 (Sigma-Aldrich). mRNA levels were normal-ized to actin levels (DCt¼Ctgene of interest�CtActin) and reported asa fold change in the RP6530-treated mice over the vehicle-treatedcontrols.

ImmunofluorescenceThe in vivo biotinylated tumor nodules were incubated with

incubated with Alexa Fluor 568-streptavidin (Invitrogen),pAKT (Ser473; 736E11), pERK1/2 (197G2), and caspase-3(E-8) antibodies (Santa Cruz), PKM2 (D78A4) from CellSignaling Technology, CD163 (EPR19518) from Novus Biolo-gicals, MHC-II (OX-6) from Abcam and F4/80 (CI:A3-1)from Bio-Rad. See Supplementary Information for furtherinformation.

ImmunohistochemistrySections from FFPE lymph node biopsies obtained from

consenting patients at the time of diagnostic workup or Hodg-kin lymphoma cell line cytospins were stained with the fol-lowing antibodies: p110d (EPR986) from Novus Biologicals;p110g (#PA5-28070) from Thermo Fisher Scientific; and pAkt(S473) (736E11), pAkt (T308) (L32A4), pS6 (#2211), andpERK1/2 (20G11) from Cell Signaling Technology. Cryostatsections of in vivo biotinylated tumor nodules were stained withF4/80 (CI:A3-1) from Bio-Rad, HRP-conjugated streptavidin,Ki-67 (MIB-1) from Dako, and VEGF (ab46154) from Abcam.Tumor apoptosis and necrosis were detected via TUNEL stain-ing (Roche). See Supplementary Information for furtherinformation.

Statistical analysisAnalysis for significance was performed using 1-way ANOVA

with a Tukey's post hoc test for multiple pairwise testing of morethan 2 groups andby parametric Student t test whenonly 2 groupswere compared. Two-way ANOVA with Tukey's post hoc test andDunnett post hoc test was performedwhen comparingmore than 2groups and 2 variables.

ResultsRP6530 inhibits the PI3K/AKT and ERK signaling pathways inHodgkin lymphoma cells

The PI3K/Akt pathway is consistently activated in Hodgkinlymphoma (24). The d and g isoforms of PI3K are highlyexpressed both in primary HRS cells and microenvironmentalcells (Supplementary Fig. S1A). The multiple roles of PI3Kd andPI3Kg in HRS cells and reactive cells of the microenvironmentsupport the hypothesis that blocking d and g isoforms mayprovide a therapeutic benefit. To investigate these hypothesis,Hodgkin lymphoma cell lines (L-540, KM-H2, and L-428) dis-playing an expression pattern of PI3K d and g isoforms similar to

that of primary HRS cells were used to assess the effects of thePI3Kd/g dual inhibitor RP6530 (Supplementary Fig. S1B). Basedon pharmacokinetic studies performed in patients enrolled inphase I trial (clinicaltrials.gov identifier NCT02017613) usingRP6530, drug concentrations up to 10 mmol/L are pharmacolog-ically achievable (C. Carlo-Stella and colleagues, manuscript inpreparation). Exposure of Hodgkin lymphoma cell lines toRP6530 (1.25–10 mmol/L) led to a dose-dependent inhibitionof phosphorylation of ERK1/2, Akt and its downstream targetproteins (Fig. 1A–C), indicating that targeting PI3Kd/g affectsboth the Akt and MAPK pathways.

RP6530 inhibits cell proliferation through induction of G0–G1

arrest and apoptosisIncubating L-540, KM-H2, and L-428 cell lines for up to 72

hours with RP6530 (1.25–10 mmol/L) resulted in a significantdose- and time-dependent decrease in cell proliferation down to30% (Fig. 1D). The cell-cycle data showed that treatment withRP6530 induced 2-fold accumulation of cells in the G0–G1 phasewith an accompanying 4-fold decrease in cells in the S phasecompared with vehicle controls (Fig. 1E). In addition, caspase-dependent cell death was increased (up to 50%) after RP6530treatment (Fig. 1F–H). These results showed that RP6530 inhibitscell proliferation through induction of G0–G1 arrest and apopto-sis in Hodgkin lymphoma cell lines.

Functional characterization of differentially expressed genes inRP6530-treated Hodgkin lymphoma cell lines

Next, we evaluated by RNA-Seq the anti-lymphoma functionsand the underlying genes affected by dual PI3Kd/g inhibition.Concordantly downregulated genes in L-540 and KM-H2 cells at6- or 24-hour time points were involved in cell proliferation,MAPK, JAK/STAT, IL2, IL4/STAT5, glycolysis, HIF1a, and MYCsignaling, whereas concordantly upregulated genes were involvedin cell death, apoptosis, and cell-cycle deregulation (Supplemen-tary Figs. S2A–S2D, S3, and S4).We confirmed these findingswitha much lower concentration of RP6530 (100 nmol/L), whichreduced the extent of off-target effects (Supplementary Fig. S5A). Acommon gene expression signature was created consisting ofgenes consistently up- and downregulated across all time pointsin both cell lines (Supplementary Fig. S2B and Fig. 2A). Althoughno significantly enriched pathways were assessed in the 39 upre-gulated genes, the 111 genes downregulated following RP6530treatment were highly enriched in tumor glycolysis and HIF1asignaling (Fig. 2A), a phenomenon already detectable at nmol/Lconcentration of RP6530 (Supplementary Fig. S5B and S5C),supporting the strong relevance of those 2 pathways in themechanism of action of RP6530. Among these genes, we identi-fied 28 highly connected hub genes likely to be key drivers in bothtumor glycolysis and HIF1a signaling (Fig. 2B). PKM2was select-ed as the most important hub gene (Fig. 2C; Supplementary Fig.S6A and S6B). PKM2 catalyzes the final and rate-limiting reactionof the aerobic glycolysis, thus regulating lactic acid production(27) and theWarburg effect in cancer cells (28). Because lactic acidsecreted by tumor cells also functions as a critical signaling factorfor M2-like polarization of macrophages (28), we hypothesizedthat by downregulating PKM2, RP6530might reduceM2markersexpression in M2 polarized macrophages (Fig. 3A). According tothe decreased expression of PKM2 after RP6530 and PKM2 siRNAtreatment (Supplementary Fig. S6C),wedetected a50%reductionin lactate levels in Hodgkin lymphoma cell lines (Fig. 3B) and






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significant downregulation of the expression of the M2 markersCCL17 and CCL22 in the M2-like macrophage population (Fig.3C), supporting an attenuation of the M2 phenotype. PKM2therefore appears to be a critical determinant of the RP6530-mediated crosstalk between Hodgkin lymphoma tumor cells andmacrophages (Fig. 3D).

RP6530 repolarizes macrophages to an M1-like phenotypeMany studies have implicated the PI3K/Akt pathway in mac-

rophage activation (29, 30). RP6530 reduced Akt phosphoryla-tion (Fig. 3E) in primary human M1 or M2 stimulated macro-phages (Supplementary Fig. S7A). Differential expression ofPI3Kd and PI3Kg isoforms was observed in M1 or M2 macro-phages; with abundant p110g in both M1 and M2 macrophages,whereas low levels of p110d in M1 macrophages compared withM2 macrophages (Fig. 3E). Indeed, M2 macrophages were moresensitive than M1 macrophages to RP6530-induced cell death(Supplementary Fig. S7B) after 48 hours, suggesting that theexpression of the d isoform of PI3K is a prerequisite for thecytotoxic activity of RP6530.

IRF/STAT signaling is crucial inmodulating macrophage polar-ization. The activation of IRF/STAT pathway by IFNs induces anM1-like phenotype (via STAT1), whereas IL4-induced IRF/STATpathway activation generates an M2-like phenotype (via STAT6;ref. 31). RP6530 sustained and activated STAT1 phosphorylationinM1 andM2macrophages, respectively, while inhibiting STAT6phosphorylation in M2 macrophages (Fig. 3F), suggesting thatRP6530 directly regulates macrophage polarization. Consistentwith these findings, RP6530 markedly inhibited PKM2 mRNAand protein expression (Supplementary Fig. S8A and S8B),leading to the inhibition of lactic acid production in bothM1 and M2-like macrophages (Supplementary Fig. S8C). Wefurther tested RNA and protein expression of M1 and M2markers in M1 or M2 macrophages after RP6530 treatment.The expression of representative M2 markers (CCL17, CCL22,CD163) was reduced, whereas M1 markers (CXCL9, CXCL10,CXCL11) were higher in RP6530-treated M2 macrophages (Fig.3G and Supplementary Fig. S8D). Taken together, these find-ings demonstrated that RP6530 switches the activation ofmacrophages from an immunosuppressive M2-like phenotypeto an inflammatory M1-like state.

RP6530 induces functional reprogramming of TAMs anddecreases MDSCs in preclinical models and clinical samplesfrom a phase I study

Based on previously published data showing that PI3Kgis predominantly expressed in the myeloid cell compartment

(32), we reasoned that RP6530, as a dual PI3Kd/g inhibitor,might affect the accumulation of TAMs in vivo in Hodgkinlymphoma tumors. Indeed, we showed a significant reductionof F4/80þ TAMs in L-540 or KM-H2 tumors after RP6530treatment (P < 0.0001; Fig. 4A and B). In addition, RP6530skewed the macrophage phenotype toward classically activatedmacrophages (M1) in vivo. In RP6530-treated Hodgkin lym-phoma xenograft, a shift within the macrophage populationtowards fewer CD206þ and CD301þ macrophages (M2)and more CD86þ and MHC-IIþ macrophages (M1) wasdetected (Fig. 4C). We further investigated whether RP6530could influence the function of TAMs by directly modulatingtheir activity, and indeed we observed a significant downregu-lation of common M2-related genes such as arginase-1 (Arg1),IL10, and CCL2, and a concomitant upregulation of M1-relatedgenes such as inducible nitric oxide synthase (iNOS), CD80and CXCL11 (Fig. 4D; Supplementary Fig. S9), resulting in asignificantly increased M1:M2 ratio and in a less immunosup-pressive TME. To strengthen this observation, we examinedwhether RP6530 affected the myeloid-derived suppressor cell(MDSC) compartment in vivo. Besides reducing the percentageof tumor-infiltrating and splenic MDSCs (Fig. 4E), RP6530was found to downregulate the expression of iNOS byM-MDSCs, thereby inhibiting their suppressive function inHodgkin lymphoma tumors (Fig. 4F). Furthermore, inhibitionof circulating M-MDSCs was correlated with the clinical out-comes of patients with Hodgkin lymphoma treated withRP6530 (Fig. 4G).

Our current findings demonstrate a PI3Kd/g-dependent inhi-bition of several macrophage-attracting chemokines, such ascolony-stimulating factor-1 (CSF-1), CC chemokine ligand5 (CCL5), and thymus and activation-regulated chemokine(TARC/CCL17; Supplementary Fig. S3). TARC is highlyexpressed by HRS cells (33), suggesting its role as a biomarkerfor response evaluation (34). We therefore investigated wheth-er serum TARC levels were correlated with the clinical outcomesof patients with Hodgkin lymphoma enrolled in a phase I trial(clinicaltrials.gov identifier NCT02017613) using RP6530(35). Upon RP6530 treatment, serum TARC levels were eval-uated in 14 patients with Hodgkin lymphoma. Patients achiev-ing complete or partial remission (n ¼ 4) experienced anaverage mean reduction in serum TARC levels of 40% (range,4–76%) after 1 month of therapy, whereas the levels wereunchanged in patients experiencing SD (n ¼ 7) or PD (n ¼3), suggesting that the ability of RP6530 to reduce TARC islikely a result of HL tumor cell death and TAMs repolarizationto an M1-like phenotype (Fig. 4H and I).

Figure 1.RP6530 reduces Hodgkin lymphoma cell proliferation and increases cell death.A andB, Immunoblots of L-540, KM-H2, and L-428 cells treatedwith increasing dosesof RP6530 (1.25–10 mmol/L) for 2 hours. All experiments were performed 2 or more times. C, Cytospin preparation showing pAkt (S473) and pERK1/2 expressionin L-540, KM-H2, and L-428 cells treated with RP6530 (10 mmol/L) for 30 minutes. Scale bar, 20 mm. D, Cell proliferation inhibition by RP6530 (1.25–10 mmol/L)for 48 and72 hours inHodgkin lymphoma cells. �� ,P<0.0001; �� ,P<0.001; �� ,P<0.01; 2-sidedANOVAwithDunnettpost hoc test.E,Cell-cycle analysis of RP6530(5–10 mmol/L)-treated L-540, KM-H2, and L-428 cells or vehicle-treated controls after 48 hours. �� , P < 0.0001; �� , P < 0.001; �� , P < 0.01; � , P < 0.05; 2-sidedANOVA with Dunnett post hoc test, compared with vehicle-treated cells. All data are shown as mean � SEM and all experiments were performed 3 times. F,Cell death induction by RP6530 (1.25–10 mmol/L) for 24, 48, and 72 hours in Hodgkin lymphoma cells. Data are shown asmean� SEM. �� , P <0.0001; �� , P <0.001;�� , P < 0.01; � , P < 0.05; 2-sided ANOVA with Dunnett post hoc test. G, Immunoblots of Hodgkin lymphoma cells treated with RP6530 or vehicle-treated controlsfor 48 hours. H, Hodgkin lymphoma cells were pretreated with 50 mmol/L Z-VADfmk for 1 hour and then treated with 10 mmol/L RP6530 for 48 hours. Celldeathwas assayedby flow cytometry usingAnnexin V/PI double staining. SU-DHL-8 cell line exposed to sTRAIL 10 ng/mLwas used as positive control (Ctrlþ) for celldeath inhibition after Z-VADfmk treatment. �� , P < 0.0001; 2-sided ANOVA with Tukey post hoc test, in comparison to RP6530 alone. All data are shown asmean � SEM and all experiments were performed 2 or more times.






Figure 2.

RP6530 inhibits tumor glycolysis and HIF1a pathways.A, Heatmap of the unsupervised hierarchical clustering of 1,303 genes that underwent a change in expression(log2FC) inHodgkin lymphoma cell lines (left). Pathway analysis for KEGGandPIDof 111 continuously downregulated genes in the time course (right). Non-significantoutcome was obtained in pathway analysis of the 39 continuously upregulated genes at both 6 and 24 hours. B, The PPI network was generated using thestringApp Cytoscape plug-in. We show only gene sets involved in the HIF1a TF network and glycolysis/gluconeogenesis, which are labeled with the appropriatepathway term. The center of each circle corresponds to the 6-hour time point, and the border corresponds to the 24-hour time point. The intensity of the nodecolor is proportional to the log2FC. Yellow indicates upregulation in RP6530-treated samples, and blue indicates downregulation in RP6530-treated samples.Gray nodes are unmodulated or not significantly modulated (adjusted P value > 0.1) genes. The node size is proportional to the score of betweennesscentrality. C, Heatmap of the 4 centrality measures of DEGs sorted by the combination score reflecting their important role in the network.

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Figure 3.

RP6530-mediated regulation ofmacrophage polarization.A,Descriptionof coculture scheme. Coculture betweenM2-polarized macrophages (topchamber) and RP6530- or PKM2 siRNA-treated HL cell lines (bottom chamber)was performed for 24 and 48 hours.B, L-Lactate production in HL cell lines(bottom chamber) was then measured.C, mRNA expression of selected M2markers in M2-polarized macrophages(top chamber) was determined byRT-PCR after 48 hours. The data werenormalized to b-actin expression andare expressed relative to the mean ofvehicle-treated M2-polarizedmacrophage cell populations.�� , P < 0.0001; �� , P < 0.001;�� , P < 0.01, and � , P < 0.05 according to2-sided ANOVA with Tukey post hoctest. The data are shown as the mean �SEM, and the experiments wereperformed 3 times. D, Schematicshowing the mode of action of RP6530,which inhibitsmetabolic reprogrammingin HL cells, thereby leading to reductionof the M2-like phenotype inmacrophages. E–F, Immunoblotting ofM1 and M2 macrophages treated withvehicle or RP6530 (10 mmol/L) for theindicated time points. The relative levelsof phospho/total Akt, phospho/totalSTAT1 and phospho/total STAT6 in M1and M2 macrophages are expressed asthe percentage of variation incomparison with vehicle-treatedmacrophages. All the experimentswere performed 2 or more times.G, mRNA expression of selected M1 andM2 markers in RP6530-treated(10 mmol/L for 24 hours) M1- andM2-polarized macrophages asdetermined by RT-PCR. The data werenormalized tob-actin expression and areexpressed relative to the mean of thevehicle-treated M1- or M2-polarizedmacrophage cell populations.�� , P < 0.0001; �� , P < 0.001, and�� , P < 0.01 according to t test. The dataare shown as the mean � SEM,and the experiments were performed3 times.






Figure 4.

RP6530 repolarizes TAMs and reduces MDSCs in Hodgkin lymphoma xenografts. A, Representative images of F4/80 expression in Hodgkin lymphoma tumors toidentify TAMs. Scale bar, 20 mm. n ¼ 3 biological replicates; 1-sided ANOVA with Tukey post hoc test. All the data are shown as the mean � SEM, andall the experiments were performed 2 times. B, Histogram bars show the percentage change in each cell population (CD45þ and TAM) in the RP6530-treatedgroup compared with those in the vehicle-treated controls. C, Flow cytometric analysis and quantification of CD11bþF4/80þ (TAM) cell populations invehicle- and RP6530-treated L-540 and KM-H2 tumors and expression of CD86 andMHC-II (M1) aswell as CD206 and CD301 (M2) in CD11bþF4/80þ cell populations.n ¼ 3 biological replicates; �� , P < 0.001; �� , P < 0.01, and � , P < 0.05 according to t test. All the data are shown as the mean � SEM, and all the experimentswere performed 2 times. (Continued on the following page.)

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RP6530 reduces tumor angiogenesis and TAM expression ofproangiogenic factors

In addition to being immunosuppressive, pro-tumor TAMscontribute to abnormal tumor vasculature (15, 36). Re-polariza-tionof the TAMsphenotype towardM1byPI3Kd/g inhibitionwasassociated with a marked reduction of pro-angiogenic factors(EGF, VEGFA, HIF1a; Fig. 5A). Given the notion that VEGF is aprimary activating factor of angiogenesis and a macrophagechemotactic protein (37, 38), we showed that RP6530 treatmentalmost completely decreased the expression of VEGF in L-540and KM-H2 tumors (Fig. 5B). In addition, microvessel density(average 80% inhibition of endothelial areas, P<0.0001; Fig. 5C),and endothelial and tumor Akt and ERK1/2 phosphorylation(Fig. 5D) were reduced in RP6530-treated mice. In line with thestrong inhibition of ERK1/2 and/or Akt phosphorylation onvascular cells, we detected a severe increase in tumor endothelialcell apoptosis manifested by increased expression of caspase-3(Supplementary Fig. S10).

RP6530 suppresses tumor growth in an Hodgkin lymphomaxenograft model and exerts in vivo antiproliferative, apoptotic,and necrotic effects

The effect of RP6530 on Hodgkin lymphoma tumor growthwas determined. RP6530 significantly (P < 0.0001) reducedthe in vivo growth of L-540 and KM-H2 xenografts [tumorgrowth inhibition (TGI) ¼ 52% and 46% at 150 mg/kg,respectively; Fig. 6A and B]. These findings were associated witha strong decrease in Ki-67 expression in tumor cells (Fig. 6C),suggesting that RP6530 inhibits tumor cell proliferation. In addi-tion, the antiproliferative effect was associated with 12-foldincrease in tumor cell apoptosis in the KM-H2nodules, comparedwith that in the vehicle-treated controls (Fig. 6D), suggesting thatthe effect of RP6530 on tumor cell growth is both cytostatic andcytotoxic. Because apoptosis was a prominent feature of tumor orendothelial cells in RP6530-treated mice, tumors from theseanimals even showed large areas of nonhemorrhagic tumornecrosis by hematoxylin/eosin and TUNEL staining (Fig. 6E),suggesting that hypoxic conditions after tumor vessel inhibitionmight have triggered tumor destruction. RP6530 significantlyincreased necrotic areas in mice bearing L-540 (3% vs. 20%,P < 0.01) and KM-H2 (4% vs. 26%, P < 0.0001) xenograftscompared with those in the vehicle-treated controls (Fig. 6E).

DiscussionIn this study, we demonstrate that the dual PI3Kd/g inhibitor

RP6530 directly targets Hodgkin lymphoma tumor cells and actsas a critical regulator of signals involved in communication

between tumor cells andmacrophages. Our data show that PI3Kdand PI3Kg are expressed in Hodgkin lymphoma tumor andmicroenvironmental cells and that inhibition of these isoformsnot only suppresses tumor growth and tumor vasculature but alsorepolarizes tumor-promoting M2-like TAMs toward tumor-sup-pressive M1-like TAMs by downregulating the limiting glycolyticenzyme PKM2 (39).

Over the past decade, new biological insights have revealedthe key role of the TME in the pathogenesis of Hodgkinlymphoma (19). The cross-talk between HRS cells and the cellsof the Hodgkin lymphoma microenvironment sustains tumorgrowth and survival (40). TAMs and MDSCs release immune-suppressive factors that inhibit T-cell-mediated antitumorresponses (41), and therapeutic approaches that alter theHodgkin lymphoma microenvironment, changing it from pro-tective to cytotoxic, hold some promise as novel therapeuticsfor patients with Hodgkin lymphoma (42). In addition, geno-mic advances in Hodgkin lymphoma have provided insightsinto deregulation of key nodal signaling pathways, includingthe PI3K, NF-kB, and JAK/STAT pathways, which are amenableto small-molecule targeting (43). Among these pathways, thePI3K/Akt pathway and its downstream targets have emerged ascentral regulators of M2 phenotype activation in macrophages(29). In this context, we considered the high therapeutic poten-tial of targeting the PI3K/Akt pathway to kill Hodgkin lym-phoma tumor cells and circumvent the supportive Hodgkinlymphoma microenvironment.

Recent studies have revealed that PKM2-dependent lacticacid production by tumor cells has an important role in theWarburg effect (44). We found that RP6530 inhibits PKM2 inHodgkin lymphoma cells, preventing its function in regulatingthe M2-like macrophage polarization through lactate produc-tion. Indeed, RP6530 indirectly downregulated the expressionof M2 markers in macrophages via Hodgkin lymphoma cells,thus identifying the therapeutic value of reprogrammingmacrophages in Hodgkin lymphoma. In addition, using spe-cific PKM2 siRNAs, we showed that inhibition of PKM2 iscritical for lactate production in HL cells and for stabilizationof the M2 phenotype in macrophages; these findings agreewith previous studies demonstrating that the activation ofPKM2 attenuates the LPS-induced proinflammatory M1 mac-rophage phenotype while promoting traits typical of an M2macrophage (45). The effects on lactate production by tumorcells and on cytokines secretion by macrophages were earlyevents detected at 24 hours of coculture and were not influ-enced by RP6530-induced apoptosis of Hodgkin lymphomacell lines, an event appearing only after 48 hours of RP6530exposure (Fig. 1F; Supplementary Fig. S6B). In addition, RNA-

(Continued.)D,mRNAexpression of selectedM1 andM2markers in the vehicle- andRP6530-treated L-540- andKM-H2-derived TAMs as determinedby RT-PCR. n¼3 biological replicates. The data were normalized to b-actin expression and are expressed relative to the mean of vehicle-treated tumors. All the data are shownas the mean � SEM, and all the experiments were performed 2 times. E and F, Flow cytometric analysis and quantification of tumor and splenic M-MDSC cellpopulation in vehicle and RP6530-treated L-540 tumors; expression of CD11bþLy6Chigh (M-MDSC), CD11bþLy6Gþ (G-MDSC) in CD45þ cell population and iNOSin M-MDSC. Mean fluorescence intensity (MFI). n¼ 5 biological replicates; ��, P < 0.001; �� , P < 0.01; t test. All data are shown as mean� SEM. and all experimentswere performed 2 times. G, Fold change over baseline (BL) of the percentage of M-MDSC in PBLs after 1 month (C2D1), 2 months (C3D1), and 3 months (C4D1) ofRP6530 administration in patients with Hodgkin lymphoma. Human M-MDSCwere identified within the gate of HLA-DRlow-neg cells as CD33highCD14þ cells. Data arerepresented as box plots displaying the median, 25th and 75th percentiles as boxes and the 10th and 90th percentiles as whiskers. CR ¼ Complete Responseand PR ¼ Partial Response (n ¼ 4); SD, stable disease (n ¼ 7); PD, progressive disease (n ¼ 5). H, Percentage of variations over baseline of TARC levels(pg/mL) in Hodgkin lymphoma patient sera after 1 to 12 months (C2D1 to C14D1) of RP6530 administration. I, Fold change (FC) over baseline (BL) of TARC levels(pg/mL) in Hodgkin lymphoma patient sera after 1 month (C2D1) of RP6530 administration. CR, complete response and PR, partial response (n ¼ 4); SD,stable disease (n ¼ 7); PD, progressive disease (n ¼ 3).






Figure 5.

RP6530 inhibits tumor vasculature in Hodgkin lymphoma xenografts. A, mRNA expression of selected angiogenesis markers in the vehicle- and RP6530-treatedL-540-derived TAMs as determined by RT-PCR. n ¼ 3 biological replicates. The data were normalized to b-actin expression and are expressed relative to themeanof the vehicle-treated tumors. All thedata are shownas themean�SEM, and all the experimentswere performed2 times.B,Representative histological imagesof VEGFA staining of the vehicle- and RP6530-treated L-540 and KM-H2 tumors. Scale bar, 20 mm. C, Representative histologic images and quantification of tumorvasculature (streptavidin staining) in the vehicle- and RP6530-treated HL tumors. Scale bar, 20 mm. n ¼ 3 biological replicates; � , P < 0.001 accordingto t test. D, Immunofluorescence analysis of pAkt and pERK1/2 in the vehicle- and RP6530-treated L-540 and KM-H2 tumors. Arrows and arrowheads indicate pAktand pERK1/2 expression in endothelial cells and tumor cells, respectively. Scale bar, 50mm. E,Model depicting the effect of RP6530 onHL tumor cells and theHL TME.RP6530 converts TAMs into proinflammatory macrophages, leading to the inhibition of tumor vasculature and the suppression of tumor growth.

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Figure 6.

RP6530 reduces tumor growth and increases tumor necrosis.A, Therapy regimen (top; PO, per os;WK,week; BID, twice a day) and themean� SEM tumor volume ofsubcutaneous L-540andKM-H2 implants in themice treatedwith the vehicle orRP6530 (100and 150mg/kgadministered orally twice a day; bottom).n¼ 10mice pergroup. � , P < 0.0001 according to 2-sided ANOVA with Dunnett post hoc test. B, Mean weight (�SEM) values were assessed. C–E, L-540 and KM-H2tumors treatedwith RP6530 (150mg/kg twice a day for 5 days) or vehicle control.C,Ki-67 expression based on IHC of L-540 andKM-H2 tumor sections at day 5 aftertreatment with RP6530 or vehicle. Scale bar, 100 mm.D, Images (left) and quantification (right) of apoptotic tumor cells detected by TUNEL staining. Scale bar, 1 mm.n¼ 3mice per group. Each dot represents the value obtained from the analysis of a single tissue field, and the lines indicate themean� SEM. � , P < 0.0001 accordingto t test. E, Representative TUNEL and hematoxylin and eosin-stained sections (H&E) of L-540 and KM-H2 tumors at day 5 after treatment with RP6530or the vehicle (left). Quantification of tumor necrosis (right). n¼ 3mice per group. At least 3 sections from different animalswere analyzed per treatment group. Theboxes extend from the 25th to the 75th percentiles, the lines indicate the median values, and the whiskers indicate the range of the values. � , P < 0.001 and� , P < 0.0001 according to t test. All the experiments were performed 2 or more times.






Seq identified downregulation of PKM2 as a key player in themodulation of macrophages occurring at 6 hours ofincubation.

Clinical evidences have shown that an increased numberof M2-like TAMs is correlated with treatment failure andpoor prognosis in Hodgkin lymphoma (21, 22). Therefore,TAM-targeting immunotherapies represent a promising cancertherapeutic approach (41). In addition to preferentially induc-ing apoptosis in M2-like macrophages, RP6530 inhibits theexpression of several macrophage-attracting chemokines, suchas CSF-1, CCL5, and TARC/CCL17 in Hodgkin lymphoma celllines. These in vitro findings were further validated by in vivoexperiments in human tumor xenografts showing that RP6530not only exerted potent antitumor effects as shown by signif-icant TGI, but also reprogrammed TAMs to an M1-like pheno-type. RP6530 directly downregulated the immune-suppressivetranscriptional signature of tumor-derived macrophages, thussuppressing the expression of Arg1, TGFb, and IL10 and stim-ulating the expression of IL1b and CXCL11. Moreover, RP6530reduced the percentage of tumor-infiltrating and splenicMDSCs in Hodgkin lymphoma xenografts. Our system usingHodgkin lymphoma cell lines, which are by their very naturemicroenvironment-independent, represents clearly a limitationto investigate the interaction between the TME and tumor cells.However, the effects that we observed on macrophage repro-gramming in vitro and the reduction of MDSCs in mice byRP6530, have been validated in a phase I trial using RP6530. Inthis trial, responsive patients with Hodgkin lymphoma expe-rienced significant reduction of circulating TARC levels andinhibition of circulating MDSCs. TARC has been implicatedin the recruitment of Th2 lymphocytes (46), and in the sup-pression of classically activated M1 macrophages (47). Reduc-tions in TARC levels were observed in patients with Hodgkinlymphoma under standard chemotherapy (33), as well asin patients with Hodgkin lymphoma treated with Akt andmultikinase inhibitors, supporting its role as a biomarker forresponse evaluation (34).

Notably, recent data in solid tumors show that PI3Kg sig-naling regulates the switch between macrophage polarizationand that selectively targeting the g isoform of PI3K in TAMsinhibited their immunosuppressive phenotype resulting intumor regression (16, 17). Our previous finding on the anti-lymphoma efficacy of the PI3Kd inhibitor TGR-1202 alsohighlighted the potential of targeting the d isoform of PI3K inHodgkin lymphoma (13). Thus, our current work confirmedand expanded these observations by using a dual PI3Kd/ginhibitor. In addition, we demonstrated the effects of PI3Kd/ginhibition on tumor vasculature and identified TARC as apotential biomarker of response in patients with HL receivingPI3Kd/g inhibitor. These findings are particularly relevant to HLpatients as the survival, proliferation, and immune escape ofmalignant HRS cells are highly dependent on the interactionswith immune microenvironment.

Macrophage recruitment and reprogramming by tumor cellsare well known to produce several angiogenic factors thatmediate tumor angiogenesis (37). In addition to its criticalrole in tumor cell survival and cellular metabolism, the PI3Kpathway is also involved in angiogenesis (32). Thus, we spec-ulate that targeting PI3Kd/g as a common regulator of angio-genesis in macrophages and of tumor cell survival will likelyprovide a more effective strategy for HL treatment. RP6530

downregulated VEGF expression in tumor cells and TAMs,which led to tumor angiogenesis inhibition and tumor growthreduction. Thus, these findings demonstrated that RP6530 hasantiangiogenic effects in Hodgkin lymphoma.

In conclusion, our findings reveal the important signalingrole of PI3Kd/g in the induction of TAMs polarization and thesubsequent promotion of tumor growth in Hodgkin lympho-ma. We show that modulating the suppressive phenotypeof these cells towards a more inflammatory one can beachieved by targeting PI3Kd/g with RP6530. As a conse-quence, Hodgkin lymphoma tumor burden and tumor vas-culature were effectively reduced. Our data establish the firstevidence of the translational potential of PI3Kd/g inhib-ition in targeting malignant cells and reshaping the TME inHodgkin lymphoma.

Disclosure of Potential Conflicts of InterestA. Santoro is a consultant/advisory board member for Bristol-

Myers Squibb, Eisai, Gilead, Novartis, Pfizer, Roche, Sandoz, and Servier.C. Carlo-Stella reports receiving commercial research grants from RhizenPharmaceuticals. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: S.L. Locatelli, S. Viswanadha, A. Santoro, A. Sica,C. Carlo-StellaDevelopment of methodology: S.L. Locatelli, G. CaredduAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.L. Locatelli, G. Careddu, F.M. Consonni, A. Maeda,A. SicaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.L. Locatelli, S. Serio, A. Maeda, P. Allavena, A. Sica,C. Carlo-StellaWriting, review, and/or revision of the manuscript: S.L. Locatelli, S. Viswa-nadha, L. Castagna, A. Santoro, A. Sica, C. Carlo-StellaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.L. Locatelli, S. SerioStudy supervision: S.L. Locatelli, S. Vakkalanka, C. Carlo-StellaOther (I have performed ELISA assay for the samples from patients.):A. Maeda

AcknowledgmentsAuthors thank Alberto Mantovani (Humanitas University, Rozzano,

Milan, Italy), and Giorgio Inghirami (Department of Pathology andLaboratory Medicine, Weill Cornell Medical College, Cornell University,NJ) for review of the manuscript and discussion. This work was supportedin part by grants from the Italian Association for Cancer Research, Milan,Italy (AIRC, project No. 20575 to C. Carlo-Stella); Fondazione RegionaleRicerca Biomedica, Milan, Italy (FRRB project No. 2015-0042 to A. Sica);Worldwide Cancer Research, UK (grant #15-1346 to P. Allavena); Minis-tero dell'Istruzione dell'Universit�a e della Ricerca (MIUR), Milan, Italy(PRIN, project No. C52F16000940001 to A. Sica); Irish Research Council2017, Ireland (IRC, project No. 19885 to A. Sica). S.L. Locatelli issupported by Fondazione Regionale Ricerca Biomedica, Milan, Italy (FRRBproject No. 2015-0042). A. Maeda is recipient of a Marie Skøodowska-Curie Individual European Fellowship-H2020-MSCA-IF-2015-EF-ST (No.706557).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 16, 2018; revised August 31, 2018; acceptedOctober 19, 2018;published first October 23, 2018.

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2019;25:1098-1112. Published OnlineFirst October 23, 2018.Clin Cancer Res Silvia L. Locatelli, Giuseppa Careddu, Simone Serio, et al. Inhibitor RP6530

γ/δand Clinical Models of Hodgkin Lymphoma Using the Dual PI3KTargeting Cancer Cells and Tumor Microenvironment in Preclinical

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