AAMP is a binding partner of costimulatory human B7-H3

Abstract Background Targeted immunotherapies are of growing interest in the treatment of various cancers. B7 homolog 3 protein (B7-H3), a member of the co-stimulatory/-inhibitory B7-family, exerts immunosuppressive and pro-tumorigenic functions in various cancer types and is under evaluation in ongoing clinical trials. Unfortunately, interaction partner(s) remain unknown which restricts the druggability. Methods Aiming to identify potential binding partner(s) of B7-H3, a yeast two-hybrid and a mass spectrometry screen were performed. Potential candidates were evaluated by bimolecular fluorescence complementation (BiFC) assay, co-immunoprecipitation (co-IP), and functionally in a 3H-thymidine proliferation assay of Jurkat cells, a T-cell lineage cell line. Prognostic value of angio-associated migratory cell protein (AAMP) and B7-H3 expression was evaluated in isocitrate dehydrogenase 1 wildtype (IDH1wt) glioblastoma (GBM) patients from The Cancer Genome Atlas (TCGA)-GBM cohort. Results Of the screening candidates, CD164, AAMP, PTPRA, and SLAMF7 could be substantiated via BiFC. AAMP binding could be further confirmed via co-IP and on a functional level. AAMP was ubiquitously expressed in glioma cells, immune cells, and glioma tissue, but did not correlate with glioma grade. Finally, an interaction between AAMP and B7-H3 could be observed on expression level, hinting toward a combined synergistic effect. Conclusions AAMP was identified as a novel interaction partner of B7-H3, opening new possibilities to create a targeted therapy against the pro-tumorigenic costimulatory protein B7-H3.

Targeted immuno-therapies are of growing interest in the treatment of various cancers. In particular, members of the co-stimulatory/-inhibitory B7-family have come into the focus of research within the last two decades. Immune checkpoint inhibitors have improved the treatment options and prognosis of advanced non-small cell lung cancer, melanoma, and renal cell carcinoma. 1 Although checkpoint inhibitors demonstrated clinical benefit in some cancers, neoplasms such as glioblastoma (GBM) showed diverse responses, with discouraging results in newly diagnosed GBM 2 and beneficial results in recurrent GBM, 3 underlining the need for more individualized treatment options.
In this regard, B7 homolog 3 protein (B7-H3), a member of the co-stimulatory/-inhibitory B7-family, is under evaluation in ongoing clinical trials against various cancer types. Taking advantage of the upregulated expression of B7-H3 in many cancer types, 4 antibody-dependent cell-mediated cytotoxicity (trial NCT01391143) and antibody-drug conjugates against B7-H3 (NCT01099644, NCT01502917, and NCT00089245) are under investigation. 5 Moreover, as B7-H3 has been shown to mediate tumor supporting functions such as invasiveness, metastasis, enhanced therapy resistance, and suppression of the antitumor immune function, other trials (NCT02381314 and NCT02475213) focus on blocking B7-H3 function. 4,[6][7][8][9][10][11][12][13][14][15] Finally, B7-H3 was identified as an interesting target for CAR T-cell therapies in neuroblastoma and GBM. 16,17 Although the importance of B7-H3 in the context of cancer has been well described, the receptor(s) to which B7-H3 binds have still not been comprehensively investigated. This may also be a consequence of B7-H3's two-headed role in immunology as B7-H3 exerts immunestimulating or immune-suppressing properties depending on the tumor type. 18 In 2008, Hashiguchi et al. described TLT-2, triggering receptor expressed on myeloid cells (TREM)-like transcript 2 as the first potential receptor of B7-H3. 19,20 However, the interaction between B7-H3 and TLT-2 which was not confirmed by other authors cannot explain the mostly published pro-tumorigenic function of B7-H3 as the authors describe an immune-stimulating role of the B7-H3/TLT-2 interaction. 21 Recently, IL20-RA was claimed to be the counterreceptor of B7-H3, which is still mostly described as an orphan immune checkpoint member. Therefore, a new platform for extracellular interactome discovery was used but this interaction was not confirmed with further experiments and beyond that, a functional proof of the interaction is missing. 22 Hence, our study aimed to identify novel interaction partners of B7-H3. Therefore, two screening assays were performed. First, a yeast two-hybrid (Y2H) screen was carried out offering the possibility to detect interaction partners beyond the B7-H3 function in the immune system. Next, mass spectrometry was employed for the detection of membrane-bound phospho-proteins of natural killer (NK) cells co-incubated with B7-H3-expressing or B7-H3knockdown GBM cells. The latter was performed after observing that B7-H3-expressing GBM cells can suppress NK cells. 12 Therefore, we aimed to identify proteins with immunoreceptor tyrosine-based inhibitory motif which we postulated to transduce the inhibitory signal of B7-H3 to the NK cells.
The screened candidates were further checked by bimolecular fluorescence complementation (BiFC). Here, CD164, angio-associated migratory cell protein (AAMP), PTPRA, and SLAMF7 were confirmed as potential candidates. Finally, AAMP was substantiated as an interaction partner of B7-H3 by pull-down assay and also by proving interaction on the functional level.
AAMP was described to be involved in angiogenesis and migration of endothelial cells, including cancer cells. 23,24 Forming a WD40 domain and containing immunoglobulinlike domains, AAMP is fitted to interact with a wide plethora of different proteins. Thus, AAMP was until now published to interact with RhoA pathways, 25 thromboxane A2 receptors, [25][26][27] and take part in autophagy 28 among others.

Ethics Statement
All work presented was performed with the approval (application number S310/2019) of the ethics board of the medical faculty of the University of Heidelberg, Germany.

Cells and Cell Culture
All glioma cell lines (U87MG, T98G, LN18, LN319, U138MG,  A172, U231, LN428, LN308, D247, U373, LN229, and Hs683) were maintained in Dulbecco's modified Eagle's medium (DMEM) media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and penicillin (100 IU/ml)/streptomycin (100 mg/ml) (P/S) (Sigma-Aldrich). LN-229 and Hs683 cell lines were kindly provided by Dr N. de Tribolet (Department of Neurosurgery, University Importance of the Study B7 homolog 3 protein (B7-H3), a member of the co-stimulatory/-inhibitory B7-family, was shown to be immunosuppressive and protumorigenic in various cancers. Although evaluated in ongoing clinical trials, a validated binding partner in immune cells has not yet been identified. Using different screening assays, we could identify several probable interaction partners of B7-H3. For one candidate, angio-associated migratory cell protein, we validated this interaction on the functional level in immune cells and could demonstrate a combined synergistic effect on the expression level, offering new possibilities to create a targeted therapy against the pro-tumorigenic costimulatory protein B7-H3.

Primary Cell Culture
Human peripheral blood mononuclear cells (PBMCs) were maintained in RPMI-1640 supplemented with 10% human serum AB (Sigma-Aldrich), P/S, and 2 mM l-glutamine under standard conditions at 37°C and 5% CO 2 . Cell lines were authenticated using Multiplex Cell Authentication by Multiplexion (Heidelberg, Germany) as described. 29 The absence of mycoplasma infection is screened for regularly. 29 Glioma-initiating cells (GICs) cultures were established from freshly dissected tumor tissue. Tumor and neurosphere cultures were cultured as described. Cells were seeded in a neural sphere cell medium containing DMEM F12 enriched with B27 supplement, basic fibroblast growth factor (20 ng/mL) and epidermal growth factor (20 ng/ml). 30

Immunohistochemistry
Formalin-fixed paraffin-embedded tissues of human astrocytoma, oligodendroglioma, and GBM were provided by the Neuropathology Department of University Hospital Heidelberg, Germany. The diagnoses for the current study had been based on WHO classification 2016. Threemicrometer cut sections were processed with Ventana BenchMark XT immunostainer (Ventana Medical Systems). The staining procedure includes the incubation of Ventana Cell Conditioner-treated samples with antihuman AAMP (rabbit anti-AAMP; 1:200, Abcam) primary antibody at 37°C for 32 min, and secondary antibody (P0446; DAKO) at radiotherapy (RT) for 32 min. Further staining procedure is as described before. 12

BiFC Assay
Using the gateway cloning system (Invitrogen), B7-H3 and the extracellular fragments of the screened binding partners bought as codon-optimized GeneArt Strings DNA Fragments from Invitrogen (Supplemental Methods) were cloned into Myc-LC151 and Ha-LN151 containing each a complementary fragment of a fluorescent reporter protein. HEK293 cells were co-transfected with Myc-B7-H3 and Ha-AAMP, vice versa. Upon close proximity of the two proteins tested, the both hemi-fluorescent reporters emitted fluorescence and could be observed under a Leica DM IRB microscope.

Co-immunoprecipitation
To show the interaction between B7-H3 and AAMP, two different co-immunoprecipitation (co-IP) assays were performed.

Identification of Glioma-Derived B7-H3 Binding Partners on NK Cells
To identify potential extracellular binding partners of tumor-derived B7-H3 on NK cells, Y2H (Supplemental Table 1) and a mass spectrometry phosphorylation screen (Supplemental Table 2) were performed. In the mass spectrometry phosphorylation screen, potential binding partners of B7-H3 were identified by analyzing tyrosinephosphorylation sites of NK cells, which were confronted with LN229 GBM cells silenced for B7-H3 or with control-LN229 cells presenting B7-H3. The results of the two screens were narrowed down to 17 genes following a literature review. As we were interested in extracellular binding partners of B7-H3, genes shown to have an extracellular domain or to be secreted into the extracellular space were considered further. We were preferentially interested in candidates proposed to exert an immunologic function. Codon-optimized DNA strings consisting of only the transmembrane and extracellular domain of the selected 17 genes (Supplemental Figure 1) were generated to be analyzed for their interaction with B7-H3. The candidate genes were cloned utilizing the Gateway system. To confirm their interaction with B7-H3, BiFC assays were performed, which revealed four candidate genes: CD164, AAMP, PTPRA, and SLAMF7 as potential interactors of B7-H3 ( Figure 1A). As the BiFC assay was performed by overexpressing recombinant proteins, the interaction with endogenously expressed binding partners was further assessed by co-IP. Since the AAMP candidate has been described to take part in migration and immune response, the co-IP experiments were designed to investigate the interaction between B7-H3 and endogenous AAMP. To this end, recombinant B7-H3 was used as bait for AAMP from Jurkat cells, which express AAMP ( Figure 1C). The co-IP experiments verified an interaction between B7-H3 and AAMP, which was also validated utilizing the human NK-cell line, NKL ( Figure 1B). Since B7-H3 has a role in immune suppression, the expression of AAMP was further examined in glioma cell lines, GICs, immune cell lines, and primary immune cells, which were all positive for this binding partner ( Figure 1C). Finally, as B7-H3 expression in glioma is grade-dependent, 12 it was tested whether AAMP is also expressed depending on glioma grade. Primary glioma tissues were assessed by immunohistochemistry. Although AAMP was clearly expressed by GBM, oligodendroglioma, and grade II astrocytoma, unlike B7-H3, it did not show a grade-dependent expression ( Figure 1D). To get a clearer insight about tumor-and immune-cell expression of AAMP and B7-H3, more detailed in silico analyses were performed (Supplemental Figures 2 and 3). A single cell 10X GBM RNA-sequencing dataset of 28 GBMs 42 allowed us to visualize the distribution of AAMP and B7-H3 expression in different GBM subsets such as mesenchymal-, astrocytic-like, oligodendrocyte progenitor-like-and neural progenitor-like cells. Moreover, we could demonstrate co-expression of AAMP and B7-H3 in immune cells such as CD68-positive macrophages as well as CD4 and CD8A positive lymphocytes. In general, AAMP-expression was more ubiquitous while B7-H3 expression was more distinct in the different subsets as detailed in Supplemental Figure 2. Interestingly, the highest expression of B7-H3 and AAMP was found in the vimentin-positive subset of mesenchymal GBM cells.
Single-cell GBM data and tSNE representation with cell type assignment from four GBMs retrieved from gbmseq.org 40 as well as interactive low-dimensionality data from the brain immune atlas 41 of 7 newly diagnosed GBM and 4 recurrent GBM could further substantiate our expression data (Supplemental Figure 3). The first analysis demonstrated expression of AAMP and B7-H3 in neoplastic cells as well as in myeloid cells (Supplemental Figure 3A and B). The later analysis showed expression of both AAMP and B7-H3 in tumor-associated macrophages while lymphocytes expressed mainly AAMP but not B7-H3 (Supplemental Figure 3C).

AAMP Is a Functional Interaction Partner of B7-H3
To test whether B7-H3 interacts on a functional level with AAMP, two AAMP knockdown cell lines (AAMP 1 and AAMP 2) were successfully created in an immortalized cell line of human T lymphocyte (Jurkat) cells (Figure 2A). There was a significant effect of the B7-H3 regime (20,30, and 40 μg/ml) on proliferation of Jurkat lines, showing that higher concentrations of B7-H3 significantly inhibited proliferation of both Jurkat control and AAMP-knockdown cells ( Figure 2B) (P < .05). At the lowest dosage of B7-H3 (20 µg/ml), proliferation of the Jurkat control cells was significantly stronger inhibited compared to the AAMP knockdown Jurkat cell lines ( Figure  2C) (P < .05). Taken together, these results suggest that knockdown of AAMP in Jurkat cells inhibits the antiproliferative effect of B7-H3. This demonstrates that B7-H3 exerts its antiproliferative effect in part by interaction with AAMP.

Prognostic value of AAMP and B7-H3 in GBM
The prognostic value of AAMP and B7-H3 expression was evaluated in IDH1wt GBM patients from The Cancer Genome Atlas (TCGA)-GBM cohort (Figure 3). Univariate survival analysis using gene expression as continuous covariates showed a negative prognostic value for B7-H3 but not for AAMP ( Figure 3A). Separation into two groups based on a varying cutoff also revealed a prognostic value for AAMP ( Figure 3B and C). Especially high expression (cutoff values > 12.5) lead to significant separation. Multivariate analysis of both genes showed a trend ( Figure  3D), hinting toward independent latent factors. To further examine this finding, we evaluated genes showing high association with AAMP and B7-H3 expression, using strict filtering criteria: Bonferroni P-value adjustment, cutoff 0.001, only genes with highest and lowest 1% of observed effects/ coefficients. 1d UMAP values were computed from selected genes (AAMP: n = 110 genes; B7-H3: n = 434), hinting toward two distinct populations ( Figure 3E). Single umap values showed high correlation with their respective gene expression values ( Figure 3F). To test for a potential interaction effect, four groups were formed for varying cutoffs ( Figure 3G), and if a significant interaction was observed (P < .05), the respective cutoff was marked (red area in Figure 3G). The lowest P-value is marked with dashed lines and the corresponding Kaplan-Meier survival plot is depicted in Figure 3H. Here, a poor prognosis could be observed for patients with high AAMP and B7-H3 expression (red curve), whereas high B7-H3 expression and low AAMP showed a better prognosis. This separation was not observed in the B7-H3 low group. Therefore, an interaction between AAMP and B7-H3 can be observed on the expression level, hinting toward a combined synergistic effect.

Discussion
We present a new interaction between human B7-H3 and the AAMP. The first evidence of the interaction was given by a Y2H screen. The interaction could be confirmed by BiFC, pull-down assays, and finally on a functional level, showing that the immunosuppressive function of B7-H3 on the proliferation of Jurkat cells is in part conveyed by AAMP. Hence, knockdown of AAMP reduced specifically the anti-proliferative effect of B7-H3 in Jurkat cells. Our results indicate that AAMP is not the only interaction partner of B7-H3 in Jurkat cells, as the anti-proliferative effect of B7-H3 was not completely neutralized. Increasing concentrations of recombinant B7-H3 were still capable to suppress the proliferation of shAAMP-Jurkat cells ( Figure   A B

Neuro-Oncology Advances
2B). Furthermore, a difference between the proliferation of shAAMP-and shControl-Jurkat cells could only be recorded at the lowest concentration of recombinant B7-H3 treatment arguing for additional interaction partners of B7-H3 that compensate for the immunosuppressive function of AAMP with mounting doses of B7-H3. This might also be an indication of a cooperating action of B7-H3 and AAMP on a common receptor which can be substituted by higher doses of B7-H3.
To substantiate the finding that AAMP and B7-H3 are interaction partners, the prognostic value of AAMP and B7-H3 expression was evaluated in IDH1wt GBM patients from the TCGA-GBM cohort ( Figure 3). Multivariate analysis of expression of both genes showed a trend ( Figure  3D), hinting toward independent latent factors. Of note, a poor prognosis could only be observed for patients with high AAMP and B7-H3 expression, supporting the interaction between AAMP and B7-H3 on expression level and toward a combined synergistic effect.
Unfortunately, other potential binding partners such as SLAMF7, PTPRA, and CD163 which were identified in the screening assay, and partly verified by BiFC could not be further substantiated by pull-down assays. This might be due to a weaker or shorter interaction, technical issues such as poorly evaluated antibodies against these less known proteins, and the necessity for different culture models. Furthermore, Y2H systems are known to produce false-positive hits. In our Split-Ubiquitine system, there is a known bias toward membrane and cytosolic proteins, due to the detection system that utilizes Ubiquitinase, which is not very common in the nucleus. The interaction partners from our screen that could not be validated may be detected in Y2H due to unphysiological high expression in this system, incorrect folding of the proteins due to missing chaperons, or incorrect post-translational processing of the proteins. These restraints might also explain, why other published interaction partners such as IL-20-RA could not be identified with our screens.
Interestingly, tumors are capable of specifically modulate the glycosylation pattern of B7-H3 as shown for oral cancer which also has an impact on the interaction with their receptors. 43 In this regard, it is entirely possible, that recombinant B7-H3 produced in HEK-cells does not mimic the necessary glycosylation pattern required to successfully perform a pull-down assay in our models. Moreover, it might be necessary to perform further pull-down assays with different subsets of immune cells to confirm other potential candidates.
Interestingly, it has been shown that AAMP is upregulated in some cancers and correlated with a worse prognosis. 25,26 Moreover, AAMP has the capacity to take part in the activation/inhibition of the innate and adapted immune system by its interaction with nucleotide-binding oligomerization domain-containing protein 2, an intracellular pattern recognition receptor having an important role in recognizing bacterial peptidoglycans and stimulating immune reactions. 44,45 Noncanonical activation of NOD1/2 via AAMP can modulate the Nf-B-pathway, receptorinteracting serine/threonine-protein kinase 2 involved in programmed cell death, autophagy, and mitogen-activated protein kinase pathway. [44][45][46][47] These pathways are suitable to explain the pro-tumorigenic and immunosuppressive action of B7-H3 via AAMP.
To conclude, we have identified new potential binding partners of the costimulatory B7-H3 by using different screening assays. While SLAMF7, PTPRA, and CD163 could be confirmed only by BiFC assays, further validation experiments are required. AAMP was further substantiated by pull-down assays and on the functional level. We demonstrate in detail the ubiquitous expression of AAMP in the tumor and immune compartment, proving that AAMP is a rational candidate and can explain the immunosuppressive,

Neuro-Oncology Advances
pro-tumorigenic function of B7-H3. AAMP is the first potential binding partner of B7-H3 that could be confirmed on the functional level and proved synergism with B7-H3 on the expression level. In view of ongoing trials attempting to target B7-H3 to overcome its immunosuppressive nature and to target various cancers due to their specific upregulation of B7-H3, our results offer an alternative possibility by blocking the interaction of B7-H3 and AAMP.

Supplementary Material
Supplementary material is available at Neuro-Oncology Advances online. Keywords brain tumor | glioblastoma | immune therapy | precision medicine | targeted therapy Funding This work was supported by SFB 1389 UNITE, subproject A03 and C05. UNDERSTANDING AND TARGETING RESISTANCE in Glioblastoma (UNITE Glioblastoma) is a unique research initiative funded within the frame of the program for Collaborative Research Centers (Sonderforschungsbereiche, SFB) of the German Research Foundation (Deutsche Forschungsgemeischaft).