macrophage syk pi3kg inhibits antitumor …...macrophage syk–pi3kg inhibits antitumor immunity:...

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Macrophage Syk–PI3Kg Inhibits Antitumor Immunity:SRX3207, a Novel Dual Syk–PI3K Inhibitory ChemotypeRelieves Tumor Immunosuppression A C

Shweta Joshi1, Kevin X. Liu1, Muamera Zulcic1, Alok R. Singh1, Dylan Skola2, Christopher K. Glass2,P. Dominick Sanders3, Andrew B. Sharabi3, Timothy V. Pham1,4, Pablo Tamayo4, Daniel Shiang1,Huy Q. Dinh5, Catherine C. Hedrick5, Guillermo A. Morales6, Joseph R. Garlich6, and Donald L. Durden1,6

ABSTRACT◥

Macrophages (M�) play a critical role in tumor growth, immu-nosuppression, and inhibition of adaptive immune responses incancer. Hence, targeting signaling pathways in M�s that promotetumor immunosuppression will provide therapeutic benefit. PI3Kghas been recently established by our group and others as a novelimmuno-oncology target. Herein, we report that an M� Syk–PI3Kaxis drives polarization of immunosuppressive M�s that establishan immunosuppressive tumor microenvironment in in vivo syn-geneic tumor models. Genetic or pharmacologic inhibition of Sykand/or PI3Kg inM�s promotes a proinflammatoryM� phenotype,restores CD8þ T-cell activity, destabilizes HIF under hypoxia, and

stimulates an antitumor immune response. Assay for transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses onthe bone marrow–derived macrophages (BMDM) show that inhi-bition of Syk kinase promotes activation and binding of NF-kBmotif in SykMC-KO BMDMs, thus stimulating immunostimulatorytranscriptional programming in M�s to suppress tumor growth.Finally, we have developed in silico the “first-in-class” dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Sykand PI3K in one small molecule. This chemotype demonstratesefficacy in multiple tumor models and represents a novel combi-natorial approach to activate antitumor immunity.

IntroductionMacrophages (M�) play a broad role in host defense but can also

serve as major drivers of tumor growth, metastasis, and immunosup-pression, observed in the tumor microenvironment (TME; refs. 1, 2).In response to various environmental signals produced by tumor andstromal cells, proinflammatory M�s shift to immunosuppressivephenotype that block antitumor immunity (3). Hence, targeting themolecular pathways/signaling nodes in the M�s that regulate transi-tion of protumorigenic M�s into anti-tumorigenic M�s will activateimmune response in cancer. Although recent studies shed some lighton the molecular entities involved in the activation of protumorigenicM�s (4–6), the identification of potential druggable targets and smallmolecules to controlM� immunosuppression of antitumor immunityare still needed.

Syk kinase is a well-established cytoplasmic protein tyrosine kinaseimplicated in inflammation and hematopoietic cell responses, includ-

ing integrin and immunoreceptor tyrosine activation motif (ITAM)signaling (7–9). As a modulator of tumorigenesis, the role of Syk ishighly controversial, it acts as tumorpromoter in some cancers (10, 11),and in others it is tumor suppressor (12, 13). Syk kinase has beenextensively studied in adaptive immune responses, but its role in M�-mediated innate immune responses remains unclear (14). Previousstudies by our group revealed Syk kinase as a novel component ofa4b1integrin–Rac2 signaling axis that encodes myeloid and endothelialRac2 specificity in vivo (8, 15). Within the TME, Syk kinase functionsupstream of Rac2 GTPase and PI3K to modulate integrin (avb3/avb5&a4b1)-mediated migration andmetastasis in vivo (16) and thesereports suggest a role for Syk kinase in regulatingM� polarization andimmunosuppression.

Another target for negative regulation of antitumor immunityrecently discovered and reported by our group and other labora-tories is the PI3K signaling pathway, in particular p110g inM�s (6, 17). Our laboratory has reported that p110g in M�spromotes expression of protumorigenic M�s in TME and our panPI3K/BRD4 inhibitor SF1126 or SF2523, blocked tumor growth andM�-mediated immunosuppression in tumors (17, 18). Further-more, Syk kinase is required for activation of PI3K in M�s and Bcells (19, 20). These reports lead us to propose a hypothesis, thattargeting two crucial signaling entities that promote M�-mediatedimmunosuppression viz. Syk kinase and PI3K will activate theantitumor immune response. With this aim, using computationalchemistry methods, our laboratory has developed a novel chemo-type, SRX3207 that inhibits both PI3K and Syk, with a singlemolecule for maximal activation of adaptive immune responses.

In this article, using a genetic approach and pharmacologic block-ade, we provide evidence that Syk kinase plays a crucial role in thecontrol of M�-mediated immunosuppression and the inhibition ofantitumor immunity. Moreover, our novel dual inhibitory chemotype,SRX3207 blocks both PI3K and Syk in M�s thereby activating innateand adaptive antitumor immunity in vivo.

1UCSD Department of Pediatrics, University of California, San Diego, San Diego,California. 2UCSD School of Medicine, University of California, San Diego, SanDiego, California. 3MooresCancerCenter, Department ofRadiationMedicine andApplied Sciences, University of California, San Diego, San Diego, California.4Office of Cancer Genomics, University of California, San Diego, San Diego,California. 5La Jolla Institute of Allergy and Immunology, La Jolla, California.6SignalRx Pharmaceuticals, Omaha, Nebraska.

S. Joshi and D.L. Durden contributed equally to this article.

Corresponding Authors: Shweta Joshi, Moores Cancer Center, Universityof California San Diego, 3855 Health Sciences Drive, San Diego, CA 92093.Phone: 858-822-7580; E-mail: [email protected]; and Donald L. Durden,SignalRx Pharmaceuticals, 8330 Loveland Drive, Omaha, NE 68124.E-mail: [email protected]

Mol Cancer Ther 2020;19:755–64

doi: 10.1158/1535-7163.MCT-19-0947

�2020 American Association for Cancer Research.

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Materials and MethodsMice, murine M�s, and hypoxia experiments

All procedures involving animals were approved by the UCSDAnimal Care Committee, which serves to ensure that all federalguidelines concerning animal experimentation are met. Floxed Sykmice and lysozyme M (LysM) Cre recombinase transgenic micewere purchased from The Jackson Laboratory. Integrin a4Y991Amice and normal littermates in C57BL/6J genetic background havebeen described before (16, 21). BMDMs were isolated as describedpreviously (16). For hypoxia experiments, M�s were placed in amodulator incubator chamber (Billups-Rothenberg) as describedbefore (16).

In vivo tumor experimentsLewis lung carcinoma (LLC), B16 melanoma, and CT26 cells

were obtained from the ATCC and were cultured in DMEM orRPMI media containing 10% FBS. B16-OVA cells were obtainedfrom Dr. A. Sharabi (University of California, San Diego, CA). Allcell lines were tested for Mycoplasma and mouse pathogens andchecked for authenticity against the International Cell Line Authen-tication Committee (ICLAC; http://iclac.org/databases/cross-con

taminations/) list. LLC or B16 or B16-OVA or CT26 (1 � 105)cells were injected subcutaneously into syngeneic mice and weretreated with 40 mg/kg R788 administered orally or 10 mg/kg IPI549or SRX3207 orally, starting from day 10 when tumors reached 100mm3 until tumors were harvested on day 21. In another experiment,B16-OVA cells were injected in C57BL/6 WT mice and whentumors reached 100 mm3, mice were treated with 200 mg anti-PDL1 antibodies either alone or in combination with 40 mg/kgR788 as described in Supplementary Methods. CD8 depletion andM� depletion experiments were performed as described earlier (18)and in Supplementary Methods.

Isolation of single cells from tumors, flow cytometry, and masscytometry

Tumors were isolated, minced, and then enzymatically dissociatedin collagenase digestion cocktail at 37�C for 30–45 minutes and cellswere prepared for magnetic bead purification of CD11b, or CD90.2cells or for flow cytometry as reported before (16, 18). For masscytometry, cells were subsequently stained with metal-labeled anti-bodies cocktail and run on mass cytometer (CyTOF, Fluidigm) asdescribed in Supplementary Methods.

Figure 1.

M� Syk and PI3Kg are required for tumor growth and metastasis. A, Tumor volume of LLC tumors implanted inWT and p110g �/�mice (n¼ 7), P < 0.001 comparedwithWT tumors, t test. B,mRNA analysis of immunostimulatory or immunosuppressive genes in TAMs sorted from tumors fromA. (n¼ 3), �, P < 0.05; ��, P < 0.001,t test.C,Western blot analysis showing deletion of Syk kinase in SykMC-KO BMDMs (n¼ 2).D,Western blot of Syk and b-actin in cell lines, CD19þB cells, CD90.2 T cellsand CD11bþF4/80þ TAMs. E, Tumor volume of LLC, or B16F10 cells implanted in SykMC-WT and SykMC-KO mice (n ¼ 8–10). ��, P < 0.001 compared with WT tumors,t test one-way ANOVA using Tukey multiple comparison tests. F, Top shows images of metastasized lungs of SykMC-WT and SykMC-KO mice (n ¼ 3) injectedintravenously with B16 melanoma and bottom shows quantitative analysis of the data (bottom). G,Mass cytometry analysis of CD45þ leukocytes isolated from LLCtumors inoculated in SykMC-WT and SykMC-KO. Figure represents uniform manifold approximation and projection (UMAP; ref. 43) plot of CD45þ leukocytes overlaidwith color coded clusters. H, Frequency of clusters of different immune cell infiltrates. Data are representative of mean � SEM (n ¼ 2 mice per group).

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http://iclac.org/databases/cross-contaminations/




Quantification of gene expression and RNA sequencingTotal RNA was isolated from BMDMs and TAMs using the Qiagen

RNAeasy kit (Qiagen) and cDNA was amplified by RT-PCR asdescribed before (16). For RNA sequencing, RNA libraries wereprepared and sequenced on Illumina HiSeq2000 using standardIllumina protocols described in Supplementary Methods.

ATAC-seqTo profile open chromatin, ATAC-seq was performed on LPS

and IL4-stimulated BMDMs. The cells were submitted to UCSDCenter for Epigenomics. The details are available in SupplementaryMethods.

Molecular modeling and in silico design, optimization, synthesisand PK/PD of SRX3188 and SRX3207 chemotypes

X-ray structures of human Syk, ZAP70 and PI3K/p110a andPI3K/p110g (PDB codes: 4XG9, 1U59, 4JPS and 4XZ4, respectively)were obtained from the Protein Data Bank. Detailed description ofin silico design of SRX3188 and SRX3207 is provided in Supple-mentary Methods. Detailed description of synthesis of SRX3188 andSRX3207 has been described before, where compound 1 and 3 refersto SRX3188 and SRX3207, respectively (22). PK/PD and ADMEproperties of the compounds were studied in collaboration withQuintara Discovery.

ResultsMacrophage Syk and PI3Kg drive tumor growth and metastasis

M�s play an important role in promoting tumor growth and inestablishing an immunosuppressive microenvironment that dam-pens effective T-cell responses in tumors (23). Recently, our lab-oratory and others reported that PI3Kg is one of the targets that is amajor driver of tumor growth and immunosuppression (Fig. 1Aand B; refs. 6, 17, 24). Herein, we identify that Syk in M�s promotestumor immunosuppression. Given our previous reports that M�-specific Syk kinase functions upstream of Rac2 and downstream ofa4b1 integrin receptor (16), we sought to evaluate the role ofmyeloid Syk in regulating tumor growth. We generated conditionalSyk knockout mice (SykMC-KO) in which Syk expression wascontrolled by LysM cre recombinase, a myeloid cell-specific pro-moter. Conditional deletion of Syk as confirmed by immunoblottingof BMDMs clearly demonstrate Syk expression only in wild-type(SykMC-WT) M�s and not in SykMC-KO M�s (Fig. 1C). Further-more, our results in Fig. 1C and D, show that Syk kinase isexpressed only in BMDMs, CD11bþ F4/80þ tumor-associated M�s(TAM), CD19þ B cells and minimally in CD90.2þ T cells, with noexpression of Syk in the murine syngeneic tumor cells used in theseexperiments. To investigate the functional role of M� Syk kinase intumor growth, we used 2 different syngeneic tumors that included:LLC and B16 melanoma. Tumor growth was reproducibly signif-icantly reduced in SykMC-KO animals compared with SykMC-WT

animals (Fig. 1E). Most notably, intravenous injection of B16 tumorcells resulted in significantly reduced lung tumor metastasis inSykMC-KO (Fig. 1F) and p110g�/� animals reported earlier by ourlaboratory (17).

To evaluate the immunologic changes in the LLC tumors, CD45þ

hematopoietic cells were profiled from SykMC-WT and SykMC-KO miceusing mass cytometry (cytometry by time of flight, CyTOF). Analysisof the total CD45þ leukocytes using Phenograph clustering (25)revealed 18 different clusters (Fig. 1G and H; SupplementaryFig. S1). Among those clusters, we found slight expansion of CD4þ

(cluster C18), CD8þ (cluster C13) T cells in SykMC-KO tumors with nochange in B-cell population (cluster C17). Among myeloid cell pop-ulation, we observed no changes in the CD11bþF4/80þ M�s (clusterC2), or CD11bþF4/80þCD80þCD86þCX3CR1þCD64þ tissue resi-dent M�s (Cluster C16) or CD11bþ CD86þCD11cþMHCIIþ orCD11cþMHCIIþ dendritic cells (cluster C9, C15; Fig. 1H). Interest-ingly, we found slight expansion in CD11bþLy6Cþ classical mono-cytes (cluster C6), CD11bþF4/80þCD80þCD86þMHCIIþ immunos-timulatory M� population (cluster C5) in SykMC-KO tumors bycomparison with decrease in immunosuppressive M�s, CD11bþF4/80hiCD206þTGFbþCD64þ (cluster C11) and myeloid-derived sup-pressor cells or neutrophils, CD11bþLY6CþLy6Gþ (Cluster C8 andC14). Although, the changes in different myeloid populations inSykMC-WT and SykMC-KO tumors did not reach to any statisticalsignificance but it provides the evidence that deletion of myeloid Sykshowed shifting of immunosuppressive M� polarization towardimmunostimulatory M� population in SykMC-KO tumors that mightlead to inhibition of tumor growth and metastasis observed in theseKO mice.

Syk kinase promotes M� polarization and immunosuppressionWe next investigated whether Syk deletion in M�s alters expres-

sion of genes associated with immunosuppression and tumorprogression. The expression of genes that mediates immunosup-pression in TME (6, 18) were significantly higher in TAMs isolatedfrom LLC tumors grown in SykMC-WT animals compared with thatof SykMC-KO animals (Fig. 2A). In contrast, expression of immu-nostimulatory genes (6, 18) was significantly enhanced in the TAMsfrom SykMC-KO animals compared with that of SykMC-WT tumors(Fig. 2A). Moreover, increased arginase activity (P < 0.01) anddecreased nitrite production (NOS; P < 0.001) was observed inTAMs isolated from SykMC-WT LLC tumors compared with thoseisolated from SykMC-KO LLC tumors (Fig. 2B). RNA-seq data onLLC TAMs suggest that the expression of genes that mediatesimmunosuppression in TME, for example, Vegf, Tgfb, Ido2, Myc,Ccl8, Ccl28 are expressed high in SykMC-WT and on the contrary,genes involved in antigen presentation and innate immunity areexpressed high in SykMC-KO (Fig. 2C). Interestingly, deletion of Sykdid not affect the infiltration of TAMs in the LLC tumors (ClusterC2, Fig. 1H), but it increased the expression of major histocom-patibility complex (MHC) class II (Fig. 2D) and immunostimula-tory cytokines and decreased the expression of immunosuppressivegenes in TAMs (Fig. 2A and C), suggesting that Syk regulatesimmunosuppression.

To determine whether Syk kinase controls transcriptional changesin M�s, we tested the effect of Syk deletion on lipopolysaccharide(LPS) polarized and IL4 polarized bonemarrow–derivedmacrophages(BMDM). It is well documented that LPS induce M� expression ofTH1 cytokines, whereas IL4 signaling stimulates TH2 response (26).Genes associatedwith immune stimulationwere upregulated in LPS orIL4 stimulated SykMC-KO M�s, whereas genes associated with immu-nosuppression were downregulated in these M�s (SupplementaryFig. S2A–S2D). Taken together, these results confirm that Syk playsa major role in promoting the immunosuppressive M� epigenetic/transcriptional program.

To investigate the mechanism by which Syk regulates M� immuneresponses, ATAC seq was performed. In the IL4 exposed group, mostsignificant immune-related motifs (AP-1, AR, C/EBPB, EGR1, EGR2,HIF2A) were found to be highly enriched in SykMC-WTM�s (Fig. 2E).Conversely in the LPS group, the significantly enriched motifs (AP-1,and NF-kB) were mostly found in the SykMC-KO M�s. Existing

Macrophage Syk–PI3Ky Axis in Tumor Immunity

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literature suggests that NF-kB promotes expression of proinflamma-tory cytokines whereas HIF1a andHIF2a are the transcription factorsinvolved in immunosuppressive M� differentiation and suppressionof T-cell function (27–30). Hence, we determined whether geneticdeletion of Syk affects the phosphorylation of p65 RelA or stability ofhypoxic HIF1a or HIF2a. In consistent with ATAC-seq results, wefound that deletion of Syk kinase stimulated and sustained p65 RelAphosphorylation, and destabilized hypoxic HIF1a andHIF2a (Fig. 2Fand G). Together, these results suggest that in addition to PI3Kg (6),Syk is another molecular switch that controls immunosuppression orimmune activation.

Syk kinase inhibits CD8þ T-cell recruitment and activationTo investigate whether the deletion of M� Syk promotes the

adaptive immune response, we evaluated the recruitment andactivation of CD8þ T cells that play an important role in antitumorimmune responses. Flow cytometric analysis of LLC tumorsdemonstrate that recruitment of total CD8þ T cells increased inSykMC-KO tumors (Fig. 3A and B), without significantly alteringT-cell recruitment in the spleen (Supplementary Fig. S3A and S3B).

To validate that M� Syk inhibits adaptive immune responses,TAMs isolated from SykMC-WT and SykMC-KO were mixed in 1:1ratio with LLC tumor cells and were adoptively transferred intodifferent SykMC-WT and SykMC-KO mice (Supplementary Fig. S3C).WT M�s adoptively transferred into SykMC-WT and SykMC-KO miceshowed increase in tumor growth, whereas adoptive transfer of KOM�s suppressed tumor growth in SykMC-WT and SykMC-KO mice(Fig. 3C). In addition, Syk inhibition did not reduce tumor growthin CD8-depleted mice, suggesting that Syk inhibition reduces tumorgrowth by recruiting and activating CD8þ T cells (Fig. 3D). More-over, we found that CD90þ T cells isolated from SykMC-KO LLCtumors expressed significantly more Ifng and Gzmb as comparedwith that fromWT tumors (Fig. 3E), suggesting that deletion of Sykkinase activates T-cell–mediated antitumor immune responsein vivo. RNA-seq analysis performed on LLC tumors implantedin SykMC-KO tumors showed an increase in Prf, Gzm, Klre1 andseveral other genes that are involved in activation of cytotoxicT cells (Fig. 3F). We did not observe any difference in theproliferative capacity of T cells between SykMC-WT and SykMC-KO

animals (Supplementary Fig. S4D). Taken together, our results

Figure 2.

Syk kinase promotes M� polarization and immune suppression. A, RTPCR analysis of cDNAs reflecting TAMs isolated from LLC tumors grown in SykMC-WT andSykMC-KO animals (n ¼ 3), Student t test. B, Arginase (left) and nitrite production (right) was assayed in the TAMs isolated from the tumors (n ¼ 3). C, Heatmap ofimmune relatedmRNAexpression in SykMC-WT versus SykMC-KO TAMs isolated fromLLC tumors implanted in thesemice (n¼ 3).D,MHC II expression on SykMC-WT andSykMC-KO TAMs gated on CD11bþF4/80þ population (n¼ 4). E,Differential enrichment heatmap of Log2 fold change for immune-related transcription factor–bindingmotifs in open chromatin segments, found via ATAC-seq, of cre and flox M�s exposed to IL4 and LPS. Blue-colored cells represent higher enrichment in flox(SykMC-WT)mice and red-colored cells represent higher enrichment in cre (SykMC-KO)mice. F, Immunoblotting of p-p65 (pRelA), p-65 (RelA), in LPS or IL4 stimulatedBMDMs fromSykMC-WT versus SykMC-KOmice.G,Western blot analysis of nuclear extracts for HIF1a or HIF2a from SykMC-WT versus SykMC-KOBMDMs incubated underhypoxic conditions (1% O2). All experiments were performed 2 to 3 times. Graphs inA, B andD represent mean� SEM, where � , P < 0.05; ��, P < 0.01; ��, P < 0.0001,analyzed by t test.

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demonstrate that Syk kinase inhibition in M�s indirectly promotescytotoxic-adaptive immune responses in the tumor.

Pharmacologic inhibition of Syk kinase blocks tumor growth,immunosuppression and increases CD8þ T-cell activation

Given that the conditional deletion of Syk kinase inhibits tumorgrowth and intratumoral immunosuppression, we speculated thatpharmacologic inhibitors of Syk could similarly inhibit tumor growthand induce an antitumor response. To test this, we used commerciallyavailable specific Syk kinase inhibitor, Fostamatinib (R788; ref. 31).Tumor growth and metastasis were significantly suppressed in themice treated with Syk inhibitor compared with vehicle-treated tumors(Fig. 4A). Importantly, R788 had no effect on viability of LLC cells(Supplementary Fig. S4). Pharmacologic blockade of Syk kinasesignificantly inhibited expression of immunosuppressive genes andincreased expression of immunostimulatory genes in TAMs purifiedfrom LLC tumors (Fig. 4B). Moreover, R788-treated LLC tumorsshowed increased infiltration of CD8þ T cells with no significantreduction in number of regulatory T cells (Fig. 4D). Interestingly, weobserved increased CD44hiCD62Llo effector T cells (gated on CD8þTcells) and cytotoxic T cells in R788-treated tumors (Fig. 4E and F), andthese findings are not due to differences in T-cell proliferation as weobserved no effect of R788 on T-cell proliferation ex vivo (Supple-mentary Fig. S5A).

To determine whether Syk suppresses antitumor adaptive immuneresponse in vivo, we injected B16-OVA cells in C57BL/6 mice andcharacterized OVA-specific CD8þ T cells, in R788-treated tumors.Using H-2Kb tetramers containing the OVA protein-derived peptideSIINFEKL, we found that R788 treatment significantly blocked tumorgrowth and increased OVA-specific CD8þ T cells in the tumordraining lymph nodes (Supplementary Fig. S5B and S5C). Theseresults provide evidence that Syk kinase suppress the antigen-specific adaptive immune response in the TME in vivo.

To explore the possibility that Syk inhibitors might synergize withT-cell check point inhibitors, we determined the effect of R788 incombination with anti-PDL1 in B16 melanoma cells. Both anti-PDL1and R788 substantially inhibited B16-OVA tumor growth. Althoughthe combination of R788 and anti-PDL1 showed no significantadditive effect on Syk inhibition (Supplementary Fig. S5B) in resistantB16 model, but CD44hi and CD62Llo effector T cells were significantlyincreased in the combination treated group compared with mono-therapy-treated groups (Supplementary Fig. S5C–S5E). Although, thecombination of R788 and anti-PDL1 did not work well in B16 model,further in vivo synergistic studies are ongoing in our laboratory toexplore the efficacy of this combination regimen in other cancermodels. Taken together, these results validate Syk as an immuno-oncology drug candidate with equivalent in vivo activity comparedanti-PDL1 blockade in this immune competent model.

Figure 3.

Syk kinase inhibits CD8þ T-cell recruitment and activation.A andB, Flow cytometric representation of percentage of CD4þ and CD8þ T cells (gated on CD3þ cells;A,left) and quantification of these cells (A, right) and CD3þ cells (B) in the LLC tumors implanted in SykMC-WT and SykMC-KO mice. (n¼ 3, � indicates P < 0.05, t test) C,Tumor volumes of LLC tumors implanted in SykMC-WT and SykMC-KO animals adoptively transferred with TAMs from SykMC-WT and SykMC-KO animals. Data wereanalyzed by one-way ANOVA using Tukey multiple comparisons, ��, P < 0.01 compared with SykMC-WT. D, LLC tumor volume from SykMC-WT and SykMC-KO micetreated with anti-CD8 or isotype control antibodies (n ¼ 5). E,mRNA expression of Ifng and Gzmb in LLC tumors implanted in SykMC-WT and SykMC-KO mice (n¼ 5,� indicates P < 0.05, t test). F, Heatmap of log2 fold differences from sample wise mean expression for selected T-cell activation genes that were differentiallyexpressed in LLC tumors from SykMC-WT versus SykMC-KO mice conditions (n ¼ 2). Graphs in A–E represent mean � SEM.






Syk and PI3Kg twomolecular targets in TAMs: In silico design ofSRX3207, a novel dual Syk/PI3K inhibitory chemotype

The concept of combinatorial therapeutics recently emergedas effective strategy to synergistically activate antitumor immuni-ty (18, 32–34). Our laboratory has developed a drug discoveryparadigm that uses computational methods to engineer a singlechemotype to inhibit two separate targets in one small moleculeand demonstrated proof of concept for greater antitumor efficacyin vivo (18, 34). Herein, we apply this technology to two immuno-oncology targets, PI3K (6) and Syk kinase (data presented in thisarticle).

We used multiple X-ray crystallographic datasets in the form ofPDB files of Syk, ZAP70, PI3K/p110a and PI3K/p110g (PDB 4XG9,1U59, 4JPS and 4XZ4, respectively) to engineer a small-moleculechemotype that will contain two distinct “warheads” within thethienopyranone scaffold (35) to bind to the ATP binding pockets ofPI3K isoforms and Syk kinase. Fig. 5A shows the docking ofSRX3188 in Syk active site that showed that this inhibitor is engagedvia hydrogen-bond interactions with the amide and carbonylgroups of Ala451, the sulfur atom of methionine Met450, and the

hydroxyl group and Arg498, whereas the azetidine nitrogen ismaking a charge interaction with the carboxylate group of Asp512in the same way the co-crystallized inhibitor in 4XG9 is observed,confirming the expected binding mode and necessary hydrogen-bond interactions. When modeling SRX3188 in ZAP70 it wasobserved that, contrary to staurosporine which in the crystalstructure of ZAP70 is found to make 2 key hydrogen bond inter-actions with Glu415 and Ala417, SRX3188 does not manifest thisinteraction predicting no affinity toward ZAP70, a tyrosine kinasehomologous to Syk.

Modeling of SRX3188 in PI3K/p110a indicates that the TP coreof SRX3188 binds as expected establishing a hydrogen bond interac-tion between the morpholine group and Val815, the pyrazole ringforming a p-p T-shaped interaction with Trp780, and the azetidinenitrogen is making a charge interaction with the carboxylate groupof Glu798 (Fig. 5B). Modeling of SRX3188 in PI3K/p110g alsoshowed an expected hydrogen bond interaction between the morpho-line group and Val882, and the predicted p-p T-shaped interaction ofthe pyrazole ring with Trp812 also observed with PI3K/p110a(Fig. 5C). It was also predicted the hydroxyl group of the azetidine

Figure 4.

Pharmacological inhibition of Syk kinase inhibits tumor growth and promotes CD8þ T-cell recruitment and activation. A, Tumor volume of LLC inoculatedsubcutaneously inWTmice treatedwith 40mg/kgR788 (n¼ 6).B,RelativemRNAexpression of TAMs from LLC tumors grown inWT animals and treatedwith R788.�� , P <0.01 and � , P <0.05, t test C. FACS quantification of percentage of CD4þ and CD8þ T cells (gated on CD3þ T cells) in R788-treated LLC tumors (n¼ 3, P < 0.05,t test).D, Flow cytometric representation of CD4þFoxP3þ regulatory T cells in the LLC tumors treated with R788. E, FACS analysis of CD44þCD62L� effector T cells(gated on CD8þ T cells) in R788-treated tumors (n ¼ 3), �� , P < 0.05, t test. F, In vitro tumor cell cytotoxicity induced by T cells isolated from R788-treated tumors(n ¼ 3), �� , P < 0.001, two-way ANOVA using Bonferroni test.

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group occupies a region where no additional interactions are engagedwith this kinase.

Because the proximity of the hydroxyl group of the azetidine groupto the Syk's backbone and the nonfavorable electrostatic microenvi-ronment surrounding the hydroxyl group were a concern, the hydrox-yl group was removed leading to the design and synthesis of SRX3207.In silico docking studies of SRX3207 against Syk indicated this analogwould bind to Syk in the sameway as SRX3188 does. Similarly, dockingresults of SRX3207 against PI3K/p110a and PI3K/p110g indicatesimilar binding modes while exhibiting no affinity toward ZAP70.The final chemotype designed was synthesized and tested in cell freesystems for potency against Syk, ZAP70 and PI3K isoforms p110a,p110b, p110g and p110d (Fig. 5D). The first-generation chemotype,SRX3188 had excellent Syk inhibitory activity (IC50 of 20 nmol/L) butpoor PI3K inhibitory potency. Extensive modeling of the p110a andp110g crystal structures (PDB 4JPS and PDB 4XZ4, respectively)enabled a structure activity relation analysis (SAR) to develop thesecond-generation prototype inhibitor, SRX3207 with potent Syk (30nmol/L) and acceptable PI3K inhibitory properties in vitro. Fig. 5Dshows the chemical structure of SRX3188 and SRX3207.

Pharmacokinetic and pharmacodynamics properties ofSRX3207

Results in Fig. 5E show that SRX3207 is a dual Syk kinase/PI3Kinhibitor that hits both targets in the samemolecule at nmol/L potency

with minimal off-target effects (Fig. 5E, Supplementary Table S1).Supplementary Figure S6A shows that both SRX3188 and SRX3207potently blocks phosphorylation of Syk at 348 site and Y525/526 sitethat are required for complete activation of Syk, described in detail innext section. Moreover, SRX3207, is able to block p-AKT at 10 mmol/Lconcentration (Supplementary Fig. S6A). Finally, we profiled SRX3207against a library of 468 known kinases that demonstrated a selectivityindex (SI) of 0.079 (Supplementary Fig. S6B) documenting a high-levelselectivity of this in silico engineered dual inhibitory compound.SRX3207 has selectivity score of 8 that is defined as number of kinaseshit by the molecule with scores less than 1 [S (1); ref. 36)]. Because ofboth the high potency and excellent kinase selectivity of SRX3207 anoral prototype formulation of SRX3207 was prepared using Pharma-tek's Hot Rod formulations to be used in preliminary in vivo studies.Preliminary pharmacokinetic studies in mice via both the intravenousand oral route indicated a half-life of about 5 hours but with a lowbioavailability of only around 2% (Fig. 5F andG). Despite this being anonoptimized formulation the oral administration of this prototypeformulation of SRX3207 yielded significant antitumor activity (Fig. 6).Additional ADME studies indicate SRX3207 has sufficient solubility inwater (43 mmol/L) relative to its target potencies but suffers from ametabolic liability with a CLint (mL/min/mg protein) of 74. Furtherstudies are in progress to optimize the prototype SRX3207 moleculefrom an ADME standpoint while preserving the desired potencies onthe Syk and PI3K target enzymes. In the meantime, the proof-of-

Figure 5.

In silico design of dual Syk/PI3 kinase inhibitory chemotype. A–C, SRX3188 docked in the catalytic site of Syk kinase (A) or PI3Ka (B) or PI3Kg (C). D, Enzymaticinhibition profile (IC50) of SRX3188 and SRX3207 against different targets. E,Chemical structures of SRX3188 and SRX3207. F andG,Mean concentration time courseof SRX3207 in mouse plasma (E) and pharmacokinetic parameters of SRX3207 in mouse (F) following 5mg/kg IV and 15 mg/kg PO administration.






concept benefits fromhaving a singlemolecule with both Syk and PI3Kinhibition properties are illustrated with the prototype moleculeSRX3207.

SRX3207 blocks tumor immunosuppression and increasesantitumor immunity

We next determined whether SRX3207 blocks immunosuppres-sive M� polarization. Interestingly, we found that SRX3207 potent-ly blocks immunosuppressive gene expression effectively at very lowdose compared with commercially available p110g inhibitor (IPI-549) or Syk inhibitor (R488) alone (Supplementary Fig. S6C andS6D). Moreover, SRX3207 at 10 mg/kg dose blocked tumor growthand increased survival effectively as compared with IPI549 andR788-treated groups at similar doses without toxicity (Fig. 6Aand B; Supplementary S7A). Furthermore, it reduced immunosup-pressive M� polarization and increased infiltration and cytotoxicityof CD8þ T cells in LLC tumors and increased expression of Ifng andGzmb in SRX3207 treated LLC tumors (Fig. 6C–E; SupplementaryS7B). Most notably, SRX3207 did not affect T-cell proliferation(Supplementary Fig. S7C). Moreover, administration of SRX3207did not block CT26 tumor growth in NSG mice but blocked tumorgrowth in Balb/c mice suggesting that SRX3207 blocks tumorgrowth due to its effect on immune compartment (SupplementaryFig. S7D and S7E). Moreover, depleting M�s with anti-CSF1R(CD115) antibody treatment alone significantly blocked LLC tumorgrowth, but the combination with R788 or SRX3207 had no additiveeffects (Supplementary Fig. S7F). Taken together, these resultsvalidate the efficacy of this novel dual Syk/PI3K inhibitor inblocking immunosuppression and activating the adaptive immuneresponse and opened new opportunities to explore it in combina-tion with check point inhibitors.

a4b1–Syk–p110g axis in M�s controls HIF1a stability underhypoxia, immunosuppression, and tumor immunity

In M�s, Syk kinase is known to be activated upon binding tophosphorylated ITAMs of Fc gamma receptor, or by binding to thecytoplasmic domains of integrin adhesion receptors, most notably b1and b3 integrin (9). Our results in Supplementary Fig. S8A show thatonly a4b1 integrin can maximally activate Syk at Y348 site in M�sisolated from the TME (Supplementary Fig. S8A), and BMDMsisolated from a4Y991A mice are defective in phosphorylating Syk atY348 site (Supplementary Fig. S8B). These results suggest that Syk isphosphorylated downstream of a4b1 integrin and mediates tumorgrowth and immunosuppression. Interestingly, Fig. 6F illustrates thatadministration of R788 did not show additive reduction in tumorgrowth in SykMC-KO mice, whereas IPI549 or SRX3207 significantlydecreased tumor growth in the SykMC-KO mice. These results suggestthat Syk and p110g are two separate molecular entities that promotetumor growth and their simultaneous inhibition with singlemolecule is good strategy to reduce tumor growth and immunosup-pression. Our published results (17) and data presented here haveshown that genetic or pharmacologic deletion of p110g and/orSyk results in hypoxic degradation of HIF1/2a and this effect iscompletely blocked by MG132, a proteasome inhibitor (Supplemen-tary Fig. S8C). Figure 6G shows the schematic of Syk and p110gregulation on tumor immunosuppression.

DiscussionTAMs are reported as the most abundant immune cells in the TME

of solid tumors, where they release immunosuppressive factors thatinhibit T-cell–mediated antitumor immune response (37). Therapeu-tic approaches that are aimed at blocking tumor immunosuppression

Figure 6.

SRX3207 increases antitumor immune response. A and B, Show tumor volume (A) and Kaplan–Meier survival data (B) of LLC inoculated subcutaneously in WTmice and treated with 10 mg/Kg of R788, or IPI549 or SRX3207 (n ¼ 5, �� , P < 0.001, one-way ANOVA with Tukey post hoc test). C, RTPCR analysisof cDNAs reflecting TAMs isolated from LLC tumors grown inWTanimals and treatedwith SRX3207. �� ,P <0.001; ��, P <0.01; and � ,P <0.05, t test.D, Quantificationof CD4þ and CD8þ T cells in the LLC tumors treated with SRX3207 (n ¼ 3, � , P < 0.05, t test). E, In vitro tumor cell cytotoxicity induced by T cells isolated fromSRX3207-treated tumors (n¼ 3), two-way ANOVA using Bonferroni test. F, Tumor volume of LLC tumors implanted in SykMC-KO mice and treated with R788, IPI549or SRX3207 data was analyzed by one-way ANOVA using Tukey multiple comparison tests (n ¼ 5). G, Schematic representation of Syk, PI3Kg , HIF1/2a axis in thecontrol over tumor immunosuppression.

Joshi et al.





by inhibiting M� polarization have shown great efficacy in murinecancer models (38, 39) and open new and effective strategies to treatcancer.

In this report, we focused on two major immuno-oncology targets,namely Syk kinase (reported in this article) and PI3Kg (6, 17). Weprovided several lines of evidence to prove that inhibition of Syk kinaseis associated with reduction of tumor growth, M�-mediated immu-nosuppression and activation of adaptive immune responses in TME(Figs. 1–4). Although recent study has shown the efficacy of Sykinhibitor in solid tumors (40), but the role of M�s in activatingadaptive immune responses has never been reported. Our results haveshown that Syk kinase is minimally expressed in T cells and inhibitionof this kinase increases infiltration and activation of CD8þ T cells withreduction in percentage of CD4þT cells, which can be explained byreduction in the number of immunosuppressive CD4þ regulatory Tcells (Treg). Our initial preliminary results have shown decrease innumber of Tregs in R788-treated tumors but the results did not reachto significance (Fig. 4D). Recent studies have shown that similar todendritic cells, M�s can also prime CD8þ T cells to generate cytotoxiceffector cells in vivo (41). Our results in Figs. 2 and 3 clearly provideevidence that Syk kinase inhibition in M�s indirectly promotescytotoxic adaptive immune responses and represents a majorimmune-oncology target.

Herein, we describe the in silico design and synthesis of a novel dualSyk–PI3K inhibitor, SRX3207 that potently inhibited Syk and PI3Ksignaling and augmented the antitumor immune response in lungcarcinoma tumor model with no toxicity (Fig. 6). Taken in con-text (16, 17, 42), the results presented here elucidate the mechanisticinterconnection of integrin a4b1, p110g , Syk, HIF axis in M�s. Inaddition, data provided in Fig. 6F provide the rationale to target bothSyk and p110g with single molecule for effective antitumor immunityand to explore SRX3207 in combination with checkpoint inhibitors incancers driven by M�-mediated immunosuppression.

Disclosure of Potential Conflicts of InterestA.B. Sharabi is an advisory board member for Astrazeneca, Jounce Therapeutics

and reports receiving commercial research grant from Varian Medical Systems andPfizer, and has ownership interest (including patents) in Toragen Inc. G.A.Morales isa director R&D and has ownership interest (including patents) in SignalRx Pharma-ceuticals. J.R. Garlich is a chief scientific officer and has ownership interest (includingpatents) in Signalrx Pharmaceuticals Inc. D.L. Durden has ownership interest(including patents) in SignalRx Pharmaceuticals Inc. No potential conflicts of interestwere disclosed by the other authors.

Authors’ ContributionsConception and design: S. Joshi, G.A. Morales, J.R. Garlich, D.L. DurdenDevelopment ofmethodology: S. Joshi, K.X. Liu, T.V. Pham,D. Shiang, D.L.Durden,P.D. SandersAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): K.X. Liu, M. Zulcic, A.R. SinghAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Joshi, K.X. Liu, D. Skola, C.K. Glass, A.B. Sharabi,T.V. Pham, P. Tamayo, H.Q. Dinh, C.C. Hedrick, D.L. DurdenWriting, review, and/or revision of the manuscript: S. Joshi, K.X. Liu, A.B. Sharabi,C.C. Hedrick, D.L. DurdenAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): S. Joshi, A.B. Sharabi, G.A. Morales, J.R. Garlich,D.L. DurdenStudy supervision: S. Joshi, D.L. DurdenOther (study design of mass cytometry experiments): C.C. Hedrick

AcknowledgmentsThis work was supported by R01 CA215651 (to D.L. Durden).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 2, 2019; revised December 5, 2019; accepted January 9, 2020;published first January 23, 2020.

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Joshi et al.




2020;19:755-764. Published OnlineFirst January 23, 2020.Mol Cancer Ther Shweta Joshi, Kevin X. Liu, Muamera Zulcic, et al. Immunosuppression

PI3K Inhibitory Chemotype Relieves Tumor−Novel Dual Syk Inhibits Antitumor Immunity: SRX3207, a γPI3K−Macrophage Syk

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