BCL9 Regulates CD226 and CD96 Checkpoints in CD8+ T Cells to Improve PD-1 Response in Cancer

Signal Transduction and Targeted Therapy www.nature.com/sigtrans

ARTICLE OPEN BCL9 regulates CD226 and CD96 checkpoints in CD8+ T cells to improve PD-1 response in cancer

Mei Feng1, Zhongen Wu 2, Yan Zhou3, Zhuang Wei4, Enming Tian2, Shenglin Mei5, Yuanyuan Zhu2, Chenglong Liu2, Fenglian He2, ✉ Huiyu Li2, Cao Xie2, Joy Jin 6, Jibin Dong2, Dehua Yang 3, Ker Yu2, Junbin Qian7, Diether Lambrechts8 , ✉ ✉ Ming-Wei Wang 1,2,8,9 and Di Zhu1,10

To date, the overall response rate of PD-1 blockade remains unsatisfactory, partially due to limited understanding of tumor immune microenvironment (TIME). B-cell lymphoma 9 (BCL9), a key transcription co-activator of the Wnt pathway, is highly expressed in cancers. By genetic depletion and pharmacological inhibition of BCL9 in tumors, we found that BCL9 suppression reduced tumor growth, promoted CD8+ T cell tumor inﬁltration, and enhanced response to anti-PD-1 treatment in mouse colon cancer models. To determine the underlying mechanism of BCL9’s role in TIME regulation, single-cell RNA-seq was applied to reveal cellular landscape and transcription differences in the tumor immune microenvironment upon BCL9 inhibition. CD155-CD226 and CD155-CD96 checkpoints play key roles in cancer cell/CD8+ T cell interaction. BCL9 suppression induces phosphorylation of VAV1 in CD8+ T cells and increases GLI1 and PATCH expression to promote CD155 expression in cancer cells. In The Cancer Genome Atlas database analysis, we found that BCL9 expression is positively associated with CD155 and negatively associated with CD226 expression. BCL9 is also linked to adenomatous polyposis coli (APC) mutation involved in patient survival following anti-PD-1 treatment. This study points to cellular diversity within the tumor immune microenvironment affected by BCL9 inhibition and provides new insights into

1234567890();,: the role of BCL9 in regulating CD226 and CD96 checkpoints

Signal Transduction and Targeted Therapy (2021) 6:313; https://doi.org/10.1038/s41392-021-00730-0

INTRODUCTION polyposis coli (APC) and β-catenin mutations.10 In canonical Wnt Colorectal cancer (CRC) is the third most commonly diagnosed signaling, Frizzled receptor activation regulates the expression/ cancer worldwide.1 In the last few years, significant new insights intracellular localization of β-catenin (β-cat), which binds to co- into the molecular pathways underlying CRC have provided activators such as B-cell lymphoma 9 (BCL9) and its homolog, B-cell several new therapeutic options.2–4 However, despite the lymphoma 9-like (B9L), thereby mediating Wnt transcription.11 We advances in chemotherapeutic and combined targeted treatment previously generated a potent and selective inhibitor targeting the options, most patients with metastatic CRC still exhibit poor interaction between BCL9 and β-cat-hsBCL9CT-24, which sup- survival. As such, there is still an unmet need for more effective presses cancer cell growth and promotes intratumoral infiltration treatments.5,6 Recently, new therapeutic strategies that reinvigo- of cytotoxic T cells by reducing regulatory T cells (Treg).12 rate the immune response directed towards cancer cells were Wnt signaling further contributes to cancer maintenance by developed. Some of these have successfully paved their way to mediating immune evasion and resistance to immunotherapies.13 the clinic, for instance, immune checkpoint inhibitors directed Tumors leverage two main Wnt-mediated mechanisms to subvert against programmed cell death protein 1 (PD-1) or cytotoxic T surveillance and cytotoxicity of the immune response. The first lymphocyte-related protein 4 (CTLA-4) are effective against promotes the differentiation and activity of Treg, while the second microsatellite-unstable CRC.7 Nevertheless, significant challenges minimizes the extent of CD8+ effector T cell (Teff) infiltration into remain, especially for microsatellite-stable CRC, which is char- the tumor microenvironment.14–16 Consequently, activation of acterized by a poor response to these checkpoint inhibitors.7 β-cat results in T cell exclusion, resistance to immunotherapy, and The Wnt signaling pathway is a tightly regulated and receptor- shortened survival of colon cancer patients. Targeting β-cat to mediated transduction pathway that is involved in embryonic block Wnt signaling is a promising strategy to affect immuno- development and adult tissue homeostasis.8,9 It plays a prominent surveillance and prevent tumor initiation and metastasis. Espe- role in CRC with 80% of patient samples displaying adenomatous cially, elevated Wnt signaling has been implicated in

1Department of Pharmacology, School of Basic Medical Sciences and School of Pharmacy, Fudan University, Shanghai, China; 2School of Pharmacy, Fudan University, Shanghai, China; 3Key Laboratory of Systems Biology, Innovation Center for Cell Signaling Network, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; 4Clinical Translational Research Center, Shanghai Pulmonary Hospital, School of Life Science and Technology, Tongji University, Shanghai, China; 5School of Medicine, University of California at San Francisco, San Francisco, CA, USA; 6Department of Gynecologic Oncology, Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China; 7VIB Center for Cancer Biology and Laboratory for Translational Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium; 8School of Life Science and Technology, ShanghaiTech University, Shanghai, China; 9The National Center for Drug Screening and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai, China and 10Shanghai Engineering Research Center of Immune Therapy and Key Laboratory of Smart Drug Delivery, Fudan University, Shanghai, China Correspondence: Diether Lambrechts ([email protected]) or Ming-Wei Wang ([email protected]) or Di Zhu ([email protected]) Received: 3 December 2020 Revised: 18 July 2021 Accepted: 23 July 2021

immunosuppressive phenotypes, including insufficient tumor Previously, we used the hsBCL9CT-24 inhibitor to block infiltration of immune cells, upon which clinical response to interaction between BCL9 and β-cat, and showed that tumor CTLA-4 and PD-1/programmed cell death-ligand 1 (PD-L1) growth inhibition ratio was higher in immune competent than immune checkpoint inhibitors (e.g., ipilimumab and pembrolizu- immune compromised mice, suggesting that Bcl9 expressed by mab) is dependent.17–20 Recent reports have shown that immune cells also mediates the therapeutic effect of Bcl9 combining Wnt inhibitors with immuno-oncology (IO) therapies knockdown.12 We, therefore, also analyzed MC38 tumor growth may have synergistic effects in preventing cancer progression.12 in WT and Bcl9–/– full knockout mice. MC38 cells continued to CD226 is mainly expressed by monocytes, platelets, T cells, and grow progressively in WT mice, while growth was attenuated in natural killer (NK) cells,21–24 andisregardedasacostimulatory Bcl9–/– mice (TGI of 80.5% on day17; Fig. 1d). Wnt downstream- receptor. It has two extracellular Ig-like domains and an intracellular signaling markers such as Cd44 and Axin2 were reduced in CT26 or kinase domain that phosphorylates downstream signaling effector MC38 tumors infected with Bcl9-shRNA (supplementary Fig. 1c, d), 25 after binding to CD155 or CD112 (nectin-2). CD155 (or poliovirus as well as in tumors treated with hsBCL9CT-24 (compared to receptor (PVR)/nectin-like molecule-5 or necl5) is a cell adhesion vehicle-treated tumors; Fig. 1e), suggesting that BCL9 suppression molecule that is commonly overexpressed in tumors associated with exhibits robust anti-tumor effects by targeting oncogenic Wnt poor outcome.26 Cellular events induced by CD155 include signaling. promotion of cell adhesion,21 increase in cell migration, and reduced To investigate whether Bcl9 depletion affects tumor immune intrinsic cell contacts.27 Recently, CD155 was reported to also infiltration, we characterized immune cells derived from CT26 and regulate the function of tumor infiltrating lymphocytes (TILs) by MC38 tumors implanted in immunocompetent mice by flow directly interacting with both stimulatory and inhibitory signaling cytometry. In Bcl9-depleted CT26 tumors, the ratio of tumor- pathways in T and NK cells. Indeed, CD155 competitively binds to the infiltrated Treg was significantly decreased (Supplementary Fig. costimulatory receptor CD226,26 as well as the inhibitory receptors 1e, while the proportions of cytotoxic granzyme B (GZMB)+CD8+ CD96 and TIGIT. Together, these molecules constitute a pathway that T cells, IFN-γ+ CD8+ T cells, and effector CD8+ T cells were is analogous to that of CD28/CTLA-4.28 Particularly, after costimula- increased (Fig. 1f, g, Supplementary Fig. 1f), implying that Bcl9 tory signaling is established through CD226, NK cell adhesion and depletion inhibits immunosuppressive immune cells. In Bcl9- cytotoxicity as well as cytokine secretion are activated.29 In contrast, depleted MC38 tumors, cytotoxic CD8+ T cells were also interaction of CD155 with TIGIT and CD96 contributes to a “cold significantly increased (Fig. 1h, i), while the ratio of tumor- tumor” phenotype that facilitates tumor escape and metastasis, infiltrated Treg cells were decreased (Supplementary Fig. 1g). leading to poor outcome.25 Overexpression of CD155 in tumor cells Similarly, in Bcl9–/– mice injected s.c. with MC38 cells, the ratio of was also associated with a reduction in TILs and worse treatment tumor-infiltrated Treg cells was significantly decreased (Supple- outcome, presumably due to interactions between CD155 and the mentary Fig. 1h), and the proportion of cytotoxic CD8+ T cells was inhibitory receptors CD96 and TIGIT.30–32 increased (Fig. 1j, k). Overall, it appears that Bcl9 depletion, either In CRC, immune checkpoint inhibitors (ICIs) such as PD-1 and PD- in tumor or stromal cells, not only reduces tumor growth, but also L1 antibodies show poor response, suggesting that other immune promotes infiltration of cytotoxic and effector CD8+ T cells. checkpoints should be targeted to achieve clinical benefits. In a recent study, CD155 was reported to act as an immune checkpoint Bcl9 depletion combined with PD-1 blockade improves the TIME ligand for tumor and tumor-associated myeloid cells, thus represent- We then examined whether Bcl9 depletion has a synergistic effect ing a potential novel ICI target.33 Furthermore, it was reported that with anti-PD-1 on tumor growth. Mice inoculated with Bcl9-shRNA elevated expression of CD155 in human metastatic melanoma is infected in CT26 tumors were treated with a mouse anti-PD-1 associated with decreased sensitivity to anti-PD-1 immunotherapy.34 monoclonal antibody. Tumor growth was significantly reduced in It, however, remains to be established whether a combination of PD- BCL9-depleted tumors treated with anti-PD-1 compared to anti- 1 blockade and downstream activation of CD155-CD226 could create PD-1, with a TGI of 81.8% by day 16 (Fig. 2a, b). Flow cytometry of costimulatory cytotoxic signaling in CD8+ T cells, and whether this CT26 tumors revealed that anti-PD-1 treatment further increased might exert a synergistic therapeutic effect. the abundance of cytotoxic GZMB+CD8+ T cells and IFN-γ+ CD8+ In this study, we explored the involvement of Wnt signaling in T cells, but decreased the Treg cells (Fig. 2c, e). We also examined the tumor immune microenvironment (TIME) by assessing how the combination of Bcl9 depletion and anti-PD-1 in MC38 tumors inactivation of BCL9, which acts as a necessary co-factor of β-cat, compared to NT-shRNA tumors and confirmed that depletion of affects the activity of tumor infiltrating T cells. Specifically, we Bcl9 decreases tumor size in response to anti-PD-1, with a TGI of hypothesized that by inhibiting BCL9 activity, we could shift the 87.1% by day 18 (Fig. 2f). Finally, combination of Bcl9 depletion balance of CD226 and CD96 checkpoints towards more cytotoxi- and PD-1 blockade also improved response and survival rates in city and thereby impede tumor growth. the CT26 mouse model (Fig. 2g). Overall, these results suggest that depletion of Bcl9 combined with anti-PD-1 treatment can further increase T cell cytotoxicity and effector function in the TIME. RESULTS To directly assess the role of T cells in reducing tumor growth in Depletion of Bcl9 inhibits tumor growth by modulating immune Bcl9-shRNA tumors during anti-PD-1 treatment, we depleted CD8+ cell infiltration T cells using a CD8-neutralizing antibody and subsequently To characterize the function of BCL9 (the human gene name) examined the effect of Bcl9-shRNA combined with anti-PD-1 on during CRC growth, we designed a shRNA lentivirus plasmid tumor growth. It was found that in CD8+ T cell-depleted tumor- vector pGIPZ to deplete Bcl9 (the mouse gene name) expression in bearing mice anti-PD-1 no longer exerted an additional inhibitory murine CRC cell lines. Specifically, we depleted Bcl9 in the MC38 effect on tumor growth when combined with Bcl9 depletion (Fig. and CT26 cell lines (supplementary Fig. 1a, b), as these express 2h). The data imply that Bcl9 depletion enhances anti-tumoral high β-catenin levels and are characterized by Wnt/β-cat CD8+ T cell-mediated immune reactions, thereby amplifying the dependent growth.35–37 Tumor growth in mice subcutaneously response to anti-PD-1 in CRC mouse models. bearing CT26 or MC38 cells infected with Bcl9-shRNA was significantly suppressed compared to wild-type (WT) mice: tumor T-cell profiling by scRNA-seq after depletion or pharmacological growth inhibition (TGI) rate of 73.8% by day 16 in the CT26 model inhibition of Bcl9 and 83.9% by day 16 in the MC38 model. Compared to NT-shRNA, Next, we studied the transcriptional changes in the TIME after Bcl9 CT26 or MC38 tumors infected with Bcl9-shRNA exhibited a TGI of depletion and hsBCL9CT-24 treatment using single-cell RNA-seq 74.1% and 85.2% by day 16, respectively (Fig. 1a, b, and c). (scRNA-seq) profiling. CT26 tumors infected with Bcl9-shRNA

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 3 abCT26 MC38 d MC38 2000 500 1500 WT WT Bcl9+/+ -/- ) ) ) NT-shRNA Bcl9 NT-shRNA 3 3 3 400 1500 Bcl9-shRNA Bcl9-shRNA P<0. P<0.0001 P

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size (mm size 1000 s 0 r

1 200 o or 500 mor s um 500 u T T Tum 100

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0.5 Bcl9-shRNA (fold change) Relative expression 0.0 Cd44 Axin2 f CT26 g CT26h MC38 P=0.0187 P=0.0189 P=0.0026 8 80 P=0.0032 40 P=0.0216

6 60 30 cells (%) cells (%) cells (%) + + 4 40 + 20 cells among cells among + + CD8 CD8 cells among CD8 + + + + 2 20 10 GZMB CD45 GZMB CD45 CD45 0 0 0

WT WT NA WT RNA hRNA -shRNA -sh -s T-shR l9 T-shRNAl9 NT-shRNA N N Bcl9 Bc Bc i MC38jk MC38 MC38 P=0.0074 P=0.0381 P=0.0371 50 P=0.0286 15 60

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versus hsBCL9CT-24, or treated with hsBCL9CT-24 versus vehicle, quality expression data for 95,816 cells (Supplementary Fig. 2a, were collected at day 14 and rapidly digested into a single-cell Supplementary Table 2). After graph-based clustering, 8 cell types suspension for scRNA-seq using 10x Genomics (Fig. 3a, Supple- were identiﬁed based on marker gene expression (Supplementary mentary Table 1). Following gene expression normalization for Fig. 2b, Supplementary Table 3), including cancer cells (n = 73,174) read depth and mitochondrial read count, we obtained high- by Wnt10a, NK&T cells (n = 5374) by Cd3e,macrophages(n = 11,823)

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 4 Fig. 1 BCL9 suppression promotes CD8+ T cells infiltration. a CT26 cells transduced with non-targeting (NT)-shRNA or Bcl9-shRNA were inoculated in BALB/c mice (n = 5 per cohort). b MC38 cells transduced with non-targeting (NT)-shRNA or Bcl9-shRNA were inoculated in C57BL/6 mice (n = 6 per cohort). c Image of tumor tissue from (c). d Tumor growth in Bcl9+/+ (wild-type) and Bcl9–/– (Bcl9 knockout) mice injected subcutaneously (s.c.) with MC38 cells. e qRT-PCR measurement of Cd44 and Axin2 expression in CT26 tumor tissue treated with + + + hsBCL9CT-24 (i.p., 25 mg/kg) or vehicle. f Percentage of GZMB cells among CD45 CD8 T cells in tumors from (a) was analyzed. g Percentage of IFN-γ+ cells among CD45+CD8+ T cells in tumors from (a) was analyzed. h Percentage of GZMB+ cells among CD45+CD8+ T cells in tumors from (b) was analyzed. i Percentage of IFN-γ+ cells among CD45+CD8+ T cells in tumors from (b) was analyzed. j Percentage of GZMB+ cells among CD45+CD8+ T cells in tumors from (c) was analyzed. k Percentage of IFN-γ+ cells among CD45+CD8+ T cells in tumors from (c) was analyzed. Results were denoted as means ± SEM for experiments performed in triplicate. Each experiment was repeated three times, and the statistical significance of differences between groups was determined by Two-way ANOVA or non-parametric test (One-tailed or Two-tailed). P < 0.05 means statistically significant.

by C1qc, while classical (n = 11823) and plasmacytoid dendritic cells found that tumors with low expression of BCL9, CTNNB1,andTCF4 (n = 352) (cDCs and pDCs) were identified based on H2-Aa and (i.e., tumors with low Wnt pathway activation) contained Klk1b27, respectively (Supplementary Fig. 2b, Supplementary Table significantly more infiltrated CD8+ T cells (Supplementary Fig. 3). Compared to NT-shRNA tumors, we observed increased propor- 4e–g). In contrast, tumors expressing high levels of DVL1, AXIN1, tions of T cells, cDCs, and pDCs in Bcl9-shRNA tumors (Supplementary and AXIN2 (i.e., high Wnt pathway activation) contained more Tregs Fig. 2c, d). Similar differences were observed in hsBCL9CT-24-treated (Supplementary Fig. 4h–j). These findings confirm that there is a tumors (Supplementary Fig. 2c, d). correlation between BCL9 and aberrant Wnt pathway activation As infiltration of cytotoxic and effector CD8+ T cells increased and CD8+ T cell suppression in Colon adenocarcinoma (COAD). after genetic depletion and pharmacological inhibition of BCL9,we Finally, we evaluated the correlation between BCL9, CD226, and investigated the cellular landscape associated with this T cell CD8+ T cell infiltration. The fraction of CD8+ T cells was response in more detail using scRNA-seq data. We subclustered significantly lower in COAD when BCL9 was high (Fig. 4i), while T cells using a similar approach as for the cell types (Supplemen- CD226 was negatively correlated with BCL9 (P < 0.05) (Fig. 4j). tary Fig. 3a–d) and observed 6 T cell subclusters in CT26 tumors Additionally, the abundance of CD8+ T cells was increased in treated with hsBCL9CT-24 (Fig. 3b, c, Supplementary Fig. 3e, f). COAD when CD226 was high (P < 0.05; Supplementary Fig. 5a). These included CD8+ T cells (marker Cd8a), Treg cells (marker Since BCL9 was negatively correlated with CD226 and CD8+ T cells Foxp3), and NK&T cells (marker: Klrb1a, Gzma, and Gzmg) (Fig. 3c, in COAD, these findings suggest a correlation between BCL9 and d, Supplementary Fig. 3e–g, k, l, Supplementary Table 3). Cell CD226-positive CD8+ T cell infiltration in CRC. fraction analysis showed that CD8+ T cells and NK&T cells were Since expression of the immune checkpoints PD-1 and PD-L1 is increased after hsBCL9CT-24 treatment (Fig. 3e). In CT26 tumors correlated with infiltrating immune cells and Bcl9 depletion is infected with Bcl9-shRNA and NT-shRNA similar subclusters were synergistic with anti-PD-1 treatment in mouse tumor models, we identified (Fig. 3f, g, Supplementary Fig. 3h, i) including cytotoxic studied the relationship among CD226, PD-1 (or PDCD1), and PD- CD8+ T cells (marker Cd8a), Treg cells (marker Foxp3) and L1 (or CD274) in CRC, lung adenocarcinoma (LUAD), lung NK&T cells (marker Tyrobp and Klrb1a) (Fig. 3g, h, Supplementary squamous cell carcinoma (LUSC) and triple-negative breast cancer Fig. 3h, j, m, n and o). Again, CD8+ T cells and NK&T cells were (TNBC). Both PD-1 and PD-L1 were positively correlated with increased in BCL9-depleted tumors (Fig. 3i). Overall, these result CD226 in LUAD, LUSC, TNBC and COAD (Supplementary Fig. 5b–i). indicate that BCL9 suppression drives a complex remodeling of Triple immunostaining of the murine model of CRC further infiltrating immune cells, including CD8+ T cells and NK&T cells. revealed that CD226 was positive in CD8+ T cells, while CD155 was positive in CT26 cells (Supplementary Fig. 5j, k), consistent with Ligand–receptor interaction identifies the correlation between the immunofluorescence staining data of the human ovarian CD226-CD155 and BCL9 cancer revealing that CD155 was co-stained with P53 for cancer To identify potential interaction between CT26 tumor cells and cells marker (Supplementary Fig. 5i). Overall, it appears that CD226 infiltrating immune cells, we scored ligand–receptor interaction and PD-1/PD-L1 are correlated with multiple cancer types, between cancer cells and CD8+ T cells by calculating average suggesting that CD226 activation may compliment anti-PD-L1/ receptor and average ligand expression in each cell type (Fig. PD-1 immunotherapy by activating distinct T cell subsets with 4a–d). After computing scores for each tumor separately, we synergistic effects. averaged them across each tumor model and treatment condition to identify conserved interactions (Fig. 4e, f). Many of the highest- BCL9 associated APC mutation is negatively correlated with scoring interactions are part of the chemokine family, including immunotherapy outcome TGF-β, IL15, CCL4, and their receptors. Remarkably, CD155/PVR on To further confirm that the interactions of CD155 with CD226 and cancer cells interacted with CD226 expressed in CD8+ T cells in all CD96 play an important immunoregulatory role in CRC, we 4 treatment groups, suggesting that this interaction plays a key quantified cell–cell interaction in a human CRC scRNA-seq dataset. role in immune modulation. Thirteen patients were enrolled with histopathologically con- To further explore this interaction in Bcl9-deficient tumors, RNA- firmed adenocarcinoma diagnosed by colonoscopy. All tumor seq analyses were performed in CT26 tumor-bearing mice treated tissues were obtained during stage I surgical resection and the with hsBCL9CT-24. We found that Cd226 expression increased samples were rapidly digested to a single-cell suspension and significantly compared to vehicle-treated mice (Fig. 4g). In a gene subjected to scRNA-seq. A similar approach as for the mouse CRC set enrichment analysis (GSEA) of differentially-expressed genes, models was used to obtain clusters and identify cell types based hsBCL9CT-24-treated tumors showed positive correlation with on marker genes. When assessing cell type interactions between immune regulation (Fig. 4h), consistent with our scRNA-seq CD8+ T cells (marker gene: CD8A) and tumor cells (marker gene: findings. Interestingly, T cells were activated and cytotoxic, as EpCAM) (Fig. 5a), most of them showed interactions known to be indicated by Prf1 and Gzmb that were expressed in CD8+ T cells mediating immunity, including that of CD155 with CD96 between (Supplementary Fig. 4a–d). CD8+ T cells and tumor cells. Next, we explored the correlation between T cells and the Wnt In order to investigate the clinical significance of BCL9 and signaling pathway using CIBERSORT (https://cibersort.stanford.edu) CD155-CD226 in ICI therapy response, we used a cohort of 110 in CRC tumors profiled by The Cancer Genome Atlas (TCGA). We CRC patients from cBioPortal database to analyze the association

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 5 a CT26 b 2000

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Tumor size (mm Tumor size 500 Bcl9-shRNA

00 +PD-1 Ab Bcl9-shRNA 50 +PD-1 Ab 0 6 8 10 12 14 16 Day c CT26 d CT26 P=0.0909 P=0.0068 0.008 80 P=0.0004 P=0.0868 P=0.0994

cells P=0.0003 P<0.0001 + P=0.0021 0.006 60 cells (%) FOXP3 + + 0.004 40 cells among + CD8 + cells (fold change) CD25 + + 0.002 20 GZMB CD45 0.000 0

WT IgG WT IgG Ratio of CD4 among CD45 -shRNA PD-1 Ab-shRNA -shRNA PD-1 Ab-shRNA NT-shRNA NT-shRNA Bcl9 Bcl9 +PD-1 Ab Bcl9 Bcl9 +PD-1 Ab e CT26 f MC38 250 100 P=0.0078

P=0.0031 P=0.0396 ) P 3

200 <0 80 WT .0 P<0 P P<0

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0 0 0 04010 20 30 50 6 8 101214161820 22 Day Day between gene mutation and overall survival (OS) after treat- TCGA (Fig. 5b–d).39 However, expressions of CTLA-4, PD-L1, and ment.38 To further explore the relationship between APC mutation PD-1 were signiﬁcantly reduced in APC mutant compared to APC and marker genes, we employed the TCGA dataset, and found WT tumors (Fig. 5e–g). that APC mutation was positively correlated with BCL9 and CD155/ Given the adverse role of mutated APC in the immune PVR expression, but negatively correlated with CD226 in COAD in microenvironment and its effect on PD-1 expression, patients

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 6 Fig. 2 Inhibition of BCL9 enhances response to anti-PD-1 antibody in CRC models. a Combination treatment of Bcl9-shRNA and anti-PD-1 antibody resulted in almost complete regression of the CT26 tumor. BALB/c mice were either inoculated with CT26 cells via single flank implantation and treated with immunoglobulin G (IgG), anti-PD-1 antibody [twice-weekly (BIW)], or with CT26 cells that transduced with non- targeting (NT)-shRNA or Bcl9-shRNA. The mice were subsequently treated with anti-PD-1 antibody as indicated after tumor volume reached 40–60 mm3 (n = 5 per cohort). b Image of tumor tissue from (a). c Ratio of CD4+CD25+FOXP3+ cells among CD45+ cell populations in the tumors from (a). d Percentage of GZMB+ cells among CD45+CD8+ T cells in the tumor from (a) was analyzed. e Percentage of IFN-γ+ T cells among CD45+CD8+ T cells in the tumor from (a) was analyzed. f Mice inoculated with Bcl9-shRNA-transduced-MC38 cells and treated with anti-PD-1 antibody resulted in almost complete regression of the MC38 tumor. C57BL/6 mice were inoculated with MC38 cells via single flank implantation and treated with immunoglobulin G (IgG) or anti-PD-1 antibody (BIW), or with Bcl9-shRNA-transduced-MC38 cells and treated with anti-PD-1 antibody as indicated after tumor volume reached 40–60 mm3 (n = 5 per cohort). Ab, antibody. Results were denoted as means ± SEM for experiments performed in triplicate. g Kaplan–Meier analysis of survival end point (tumor size >2000 mm3, n = 8 per cohort). h Mice were inoculated with Bcl9-shRNA-transduced-CT26 cells used anti-PD-1 antibody resulted in almost complete regression of the CT26 tumor. BALB/c mice were inoculated with CT26 cells via single flank implantation and treated with anti-PD-1 antibody (BIW), the mice were inoculated with Bcl9-shRNA-transduced-CT26 cells used anti-CD8 antibody (intraperitoneal injection, i.p., 300 μg per mouse at days 12, 15, and 19 after tumor cell inoculation) plus anti-PD-1 antibody as indicated after tumor volume reached 40–60 mm3 (n = 5 per cohort). Each experiment was repeated three times, and the statistical significance of differences between groups was determined by Two-way ANOVA or non-parametric test (Two-tailed). P < 0.05 means statistically significant.

with APC mutation may not benefit from immunotherapy. We environment–infiltrating myeloid cells restrained anti-tumor analyzed the association between APC mutation and immunother- immunity by impairing anti-tumor T lymphocyte and NK cell 43 apy outcome using a cohort of CRC patients treated with ICIs function. hsBCL9CT-24 treatment increased mRNA expression of showing that they had significantly shorter OS time after CD155/Pvr in CT26 cells (Fig. 6j), while Bcl9 depletion in immunotherapy than that of APC WT patients (Fig. 5h). CT26 similarly increased expression of CD155/Pvr (Fig. 6k). It was reported that sonic hedgehog activates CD155 gene expression.44 Depletion of BCL9 modulates CD226-CD155 signaling via We thus examined whether key components of sonic hedgehog phosphorylation of VAV1 Gli1 (transcription factor) and Patch1 (receptor) were changed. CD226 is a transmembrane receptor present on NK and CD8+ Increased mRNA levels of Gli1 and Ptch1 were seen in hsBCL9CT-24 T cells. CD155 competitively binds to CD226 and CD96.26 The or Bcl9-shRNA treated CT26 tumor cells (Fig. 6l, m, Supplementary guanine nucleotide exchange factor VAV1 is required for the Fig. 6e, f), suggesting that BCL9 suppression activates CD155 activation of T cells. Interestingly, upon T-cell activation, the expression via sonic hedgehog signaling. costimulatory molecule CD226 was reported to be one of the most enriched VAV1 interactors.40 Significantly less CD96+CD8+ T cell Depletion of BCL9 inhibits Treg migration infiltration was observed in Bcl9–/– mice bearing CT26 tumor cells The role of BCL9 in Treg cells was investigated using the Transwell suggesting that Bcl9 deficiency may promote T cell activation and migration method to simulate tumor infiltration by immune cells. potentially enhance responsiveness to PD-1 checkpoint blockade In vitro migration of Treg cells was reduced in Bcl9-depleted CT26 (Fig. 6a). Decreased mRNA expression of Cd96 is also found in Bcl9- cells compared with the NT-shRNA group (supplementary Fig. 7a). shRNA treated CD8+ T cells compared with NT-shRNA (Fig. 6b), Likewise, Treg cells migration in Bcl9-depleted MC38 cells was also hsBCL9CT-24 and vehicle control (Supplementary Fig. 6a). In reduced (Supplementary Fig. 7b). In MC38 cells treated with contrast, depletion of Bcl9 in CT26 tumor cells increased hsBCL9CT-24 decreased migration of Tregs was observed (Supple- CD226+CD8+ T cell tumor infiltration accompanied by increased mentary Fig. 7c). When Ctnnb1 was depleted via shRNA in MC38 expression of Pvr/CD155 (Fig. 6c). In Bcl9-depleted MC38 tumors, cells, Treg cell migration was also reduced, as expected CD226+CD8+ T cell tumor infiltration was also increased (Fig. 6d). (Supplementary Fig. 7d). These results suggest that Bcl9 suppression promotes Next, we explored the molecular mechanisms underlying Treg cell CD226+CD8+ T cell tumor infiltration, but decreases that of migration by examining expression levels of known regulators, CD96+CD8+ T cells. CD226 was reported to impact CD8+ T cell including Ccl4, Ccl22,andTgf-β using real-time quantitative PCR. Bcl9 activity and function.41 In MC38 tumors, Bcl9 depletion and depletion significantly increased Ccl4 expression and significantly knockout both increased T cell proliferation indicted by staining of reduced Tgf-β expression levels in CT26 cells (Supplementary Fig. 7e, 12 Ki67 (Fig. 6e, Supplementary Fig. 6b). Interestingly, Bcl9-shRNA or f). hsBCL9CT-24 and CCR4 antagonist C021 were employed to + hsBCL9CT-24 treated CT26 tumor cells co-cultured with CD8 investigate changes in the ability of Tregs to infiltrate tumors. Both T cells promoted the proliferation of CD8+ T cells in vitro (Fig. 6f). inhibitors significantly reduced the tumor infiltration ratio of Treg We then used CD226 and CD155 neutralizing antibodies in a CD8+ cells, but interestingly, it did not change the proportion of Treg cells T cell proliferation assay. The proliferation of CD8+ T cells induced in the spleen (supplementary Fig. 7g, h). This result suggests that by Bcl9-shRNA was abolished by neutralizing antibodies against Treg cell infiltration is mediated via CCR4 and BCL9/β-cat pathways. CD155 and CD226 (Fig. 6g, Supplementary Fig. 6c), suggesting that CD8+ T cell proliferation is dependent on CD226-CD155 BCL9 depletion induces transcriptional differences in CD8+ T cells signaling. Engagement of CD226 is known to elicit tyrosine GSEA was performed to compare T cells in hsBCL9CT-24-treated vs. phosphorylation of VAV1.40 Neutralizing antibodies against CD226 vehicle-exposed CT26 tumors. This revealed that pathways related indeed decreased the activation of VAV1, as shown by its to IL-2, CD4+ T cell memory formation and stimulation of CD8+ T phosphorylation on Tyr174 (Fig. 6h), implying that engagement were enriched in tumor-infiltrating T cells treated with hsBCL9CT- of CD226 induces tyrosine phosphorylation of VAV1. Treatment of 24 (Supplementary Fig. 8a, c). Tumor-infiltrating T cells in Bcl9- + hsBCL9CT-24 or Bcl9-shRNA in CD8 T cells increased the level of shRNA compared to NT-shRNA bearing tumors exhibited path- tyrosine phosphorylation of VAV1, AKT, and ERK, while stimulation ways enriched for TLR, FOXP3, and IL-2 (Supplementary Fig. 8b, d). of CD8+ T cells increased the global quantities of tyrosine We found a couple of pathways related to CD8+ T cells that are phosphorylation (Fig. 6i, Supplementary Fig. 6d). Our results enriched in hsBCL9CT-24 and Bcl9-shRNA groups (Supplementary indicate that suppression of Bcl9 promotes VAV1-AKT-ERK signal- Fig. 9a–f). ing cascades in CD8+ T cells. Next, transcription factor activity underlying specific mouse CD155, expressed on the surface of cancer cells, was shown to CD8+ T-cell clusters was analyzed in the scRNA-seq data using promote tumor invasiveness.42 Its upregulation in tumor SCENIC.45 This identified MYC, JUN, STAT1, JUNB, and FL1 as

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 7 a TIME NT-shRNA

Tumor Bcl9-shRNA + Cells Oil CD8

(PVR) Vehicle TAM

DC CD96 Primer/barcod hsBCL9 CT containin beads + -

bdceC Gzma Vehicle l u

st hsBCL9CT er 6 C Foxp3 l u st e r T

+ CD8 Cl Cd8a uster

tSNE_ + Vehicle CD8 C Gzmg

hsBCL9 l +

CT u ster CD8 5 6 Activ Prolifer

f gih NT-shRNA

Tyrobp Bcl9-shRNA

+ CD8 Cluster5 Foxp3

T Cd8a

NT-shRNA CD8+ Bcl9-shRNA + Proliferating CD8 Memor Prolifer Fig. 3 Depletion of BCL9 changes T-cell cellular landscapes. a The workﬂow of scRNA sequencing. b tSNE plot of the tumor sample following treatment with vehicle or hsBCL9CT-24 (two groups), color-coded by their associated clusters. c Dot plot of the six clusters of T cells from (b). d tSNE plot of color-coded expression (gray to orange) of marker genes for the clusters from (c). e The proportion of CD8+ T and NK&T cells from 6 samples (vehicle and hsBCL9CT-24). f tSNE plot of the tumor sample that CT26 cells transduced with non-targeting (NT)-shRNA or Bcl9-shRNA (two groups), color-coded by their associated clusters. g Dot plot of the seven clusters of T cells from (f). h tSNE plot of color-coded expression (gray to orange) of marker genes for the clusters from (g). i The proportion of CD8+ T and NK&T cells from 6 samples (NT-shRNA and Bcl9- shRNA)

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 8 adb c g 01 Vehicle hsBCL9CT-24 NT-shRNA Bcl9-shRNA CD8-CT26 CD8-CT26 CD8-CT26 CD8-CT26 PVR_CD96 PVR_CD96 PVR_CD96 PVR_CD96 Ifng PVR_CD226 PVR_CD226 PVR_CD226 PVR_TIGIT Cd200r1 PVR_TIGIT PVR_TIGIT PVR_TIGIT SPP1_CD44 TIGIT_NECTIN2 Tlr7 TIGIT_NECTIN2 CRTAM_CADM1 SPP1_CCR8 CD226_NECTIN2 Ctsk CD226_NECTIN2 TIGIT_NECTIN2 PDCD1_FAM3C SPP1_CD44 TNFRSF1B_GRN SPP1_CD44 CD226_NECTIN2 C2 SPP1_CCR8 CD74_COPA SPP1_CCR8 SPP1_CD44 Gli1 PDCD1_FAM3C CD44_HBEGF SPP1_CCR8 TNFRSF1A_GRN TNFRSF1B_GRN CD74_APP Cxcl13 TNFRSF1B_GRN TGFB1_TGFBR3 CD44_HBEGF CCL4_SLC7A1 Cfh CD44_HBEGF LAMP1_FAM3C CCL4_SLC7A1 FGFR1_FGF7 C6 ICAM1_SPN PDCD1_FAM3C CSF1_SLC7A1 BMP2_SMO FAM3C_CLEC2D Rora ADORA2A_NAMPT TNFRSF1B_GRN PLXNB2_SEMA4D PLXNB2_SEMA4D NRP1_SEMA3A CD74_COPA LRP6_CKLF Cd226 CD44_HBEGF TNFSF9_TNFRSF9 Mmp2 CCL4_SLC7A1 LTBR_LTB TNFSF9_TNFRSF9 TNFSF10_RIPK1 ADORA2A_NAMPT IL15 receptor_IL15 SELL_CD34 Vsig4 NPPB_DPP4 CCL4_SLC7A1 NKG2DIIR_ULBP1 SELP_CD34 Btla SELE_GLG1 PLXNB2_SEMA4D CCL3_IDE IFNG_Type II IFNR Havcr2 TNFSF9_TNFRSF9 TNFSF10_RIPK1 LTBR_LTB Cd300lg AXL_IL15RA PLXNB2_SEMA4D IL15 receptor_IL15 NKG2DIIR_ULBP1 Gli2 T cell IL15_IL15RA TNFSF9_TNFRSF9 T cell + + NRG1_a6b4complex LTBR_LTB LTBR_LTB Myh2 IL15 receptor_IL15 IFNG_Type II IFNR

IL15 receptor_IL15 CT26|CT26 Hpx T cell|CT26 CRLF2_TSLPR NKG2DIIR_ULBP1 NKG2DIIR_ULBP1 + Vav1 TGFB1_TGFBR2 TGFB1_TGFBR1 CT26|CD8 CD8

+ Il20rb CT26|CT26 T cell|CT26 CT26|CT26 T cell|CT26 + Cd33 + CD8 Dpp4 F CD8 CT26|CD8 T cell|CD8 CD8 CT26|CD8 T cell|CD8 + Pdcd1 + Gfi1 CD8 CD8 Hist1h3c -log10 Log2 mean -log10 Log2 mean -log10 Log2 mean -log10 Log2 mean Gpr68 (P value)(Molecule 1, (P value)(Molecule 1, (P value)(Molecule 1, (P value)(Molecule 1, Molecule 2) Molecule 2) Molecule 2) Molecule 2) Gli3 Scin 0 0 150 0 0 150 60 Prf1 100 1 1 100 1 1 40 100 Lbp 2 2 2 50 2 20 Nr1h4 50 50 3 3 3 3 0 Zbtb16 0 0 0 Mertk Gprc5b ef hsBCL9 V Vehicle- hsBCL9CT-24 NT-shRNA-Bcl9-shRNA ehicle T Type II IFNR

CD226 ype II IFNR ADGRE5

C AREG CCR5 ULBP1 CCR2 CD34 AREG ULBP1 4

TNFRSF9 TNFRSF9 D TSLPR 3 TSLPR CT 4 -24

TGFBR3 C CD96 CD44 TIGIT CD96 CLEC2D CLEC2D TIGIT COPA TGFBR2 FAM3C GRN h GLG1 TGFBR1 GRN SLC7A1SMO HBEGF HBEGF SEMA4D IL15 IL15 ITGAL LTB NECTIN2 LTB GO_REGULATION_OF SLC7A1SPN MIF NK MIF SEMA4D NK NAMPT NAMPT NECTIN2 NECTIN2 SEMA3A SELPLG IMMUNE_SYSTEM_PROCESS SEMA4D LTB SELPLG RIPK1 SLC7A1 IL15 SEMA4D NECTIN2 SPN HBEGF SLC7A1 NAMPT TGFBR1 SPN 0.25 TGFBR2 GRN GLG1 TGFBR3 LTB TGFBR3 TIGIT 0.20 IL15RA CD8 TIGIT FGF7 CD8 TNFRSF9 TNFRSF9 IL15 FAM3C ULBP1 0.15 IDE ULBP1 ADORA2A COPA ADORA2A 0.10 HBEGF AXL BMP2 GRN CCL3 CLEC2D CCL2 0.05 CCL4 CKLF CCL4 FAM3C CCL5 0.00 DPP4PA CD226 CD96 CD226 CO CD44 CD44 CD44 CD55 CD74 -0.05 CD96 CD72 CD34 CRLF2 CD44 CD74

Enrichment score -0.10

CRLF2 CSF1

CD226 CT26 FAM3C CD226 CT26 C CCR8 x CCR8 F R APP FGFR1 ICAM1AM3CT ICAM1 CADM1 IFNG AM IFNG IL15 IL15 R IL15R LRP6 TNFSF9 LAMP1 L L NKG2D II R NKG2D II R NRG1 NPPB PDCD1 TBR NRP1 TNFSF10 PDCD1 TBR TNFSF9 TIGIT a6b4 comple TIGIT 2

B TNFRSF1B TGFB1SPP1 TGFB1SPP1 SELP SELP N SELL TNFRSF1B SELL TNFRSF1A SELE SELE PVR PVR High Low

PLXNB2 i j P=0.0116 Level High Low P=0.0017 Level High Low 10 0.4 12 R=-0.13 10 9 0.3 11 P=0.0058 8 8 0.2 10 7 expression expression 6 CD226 6

T cell infiltration 9

0.1 Normalized + 5 BCL9

8 CD226 CD8 0.0 4 4 High Low 0.0 0.1 0.2 0.3 0.4 High Low 8 9 10 11 12 BCL9 expression level CD8+ T cell cibersort score BCL9 expression level BCL9

potential transcription factors of CD8+ T cells in Bcl9-shRNA but gene promoters (Supplementary Fig. 10a). We then used SCENIC not NT-shRNA tumors (Supplementary Fig. 10a). It was found that to analyze co-expression of transcription factors and their putative MYC and STAT1 expression were linked to CD155-related signaling, target genes. This identiﬁed Irf1, Junb, Jun, Stat1, and Elf1 as while downregulation of Jun, Junb, and Fli1 were linked to CD226- candidate transcription factors underlying gene expression related signaling, in which Jun and Junb were identiﬁed as CD226 differences in T cells (Supplementary Fig. 10b–f).

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 9 Fig. 4 BCL9 is associated with CD155-CD226 checkpoint in CT26-CD8 T+ cell interaction. a Interaction analysis between CD8+ T and CT26 cells + + in vehicle group. b Interaction analysis between CD8 T and CT26 cells in hsBCL9CT-24 treated group. c Interaction analysis between CD8 T and CT26 cells in NT-shRNA group. d Interaction analysis between CD8+ T and CT26 cells in Bcl9-shRNA group.eCircos plots of all of the putative ligand–receptor interactions in vehicle and hsBCL9CT-24 treated groups. The color of the line represents the average expression of the ligand–receptor pair in the pair of cells, and the thickness of the line represents the significance of the ligand–receptor enrichment in the pair of cells. The solid line represents the interaction of the hsbcl9CT-24 group, and the dotted line represents the interaction of the vehicle group. f Circos plots of all of the putative ligand–receptor interactions in NT-shRNA and Bcl9-shRNA groups. The solid line represents the interaction of Bcl9-shRNA group, and the dotted line represents the interaction of the NT-shRNA group. g Heatmap analysis of CT26 tumor pretreated with vehicle or hsBCL9CT-24 (i.p., 25 mg/kg). h GSEA analysis of CT26 tumor pretreated with vehicle or hsBCL9CT-24 (i.p., 25 mg/kg). i Scatter and boxplot analyses of CD8+ T cell infiltration associated with BCL9 expression level in TCGA. j Scatter and boxplot analyses of CD226 expression associated with BCL9 expression level in TCGA. P < 0.05 means statistically significant.

DISCUSSION BCL9 modulation and CD155-CD226 as a potential checkpoint, our In this paper, we uncovered that cytotoxic CD8+ T cell tumor data will certainly help CRC diagnosis and therapy. infiltration was increased by pharmacological inhibition and BCL9 may be important mainly in APC mutated tumors.51–53 genetic depletion of BCL9. We analyzed the TIME associated with CRC patients with APCmut showed significantly shorter overall this intervention at single-cell resolution. RNA sequencing survival after ICI immunotherapy than that of APC wild-type technique was applied to characterize CD8+ T and NK&T cells patients. BCL9 may be negatively correlated with ICI treatment, and to study their roles in the TIME models. Cellular landscapes suggesting inhibition of BCL9 may enhance the outcome of ICI and transcriptional features were presented with T, NK, and tumor immunotherapy in patients with APCmut. cells. Key cell clusters and signaling properties among CD8+ T and There are a number of reports about BCL9 role in non-immune NK&T cells were described and CD155-CD226 was found to be a regulation in cancer development. Loss of BCL9/9l was reported to potential checkpoint regulated by BCL9. We found that CD155/ block colonic tumorigenesis and mutations.51 Bcl9 and Pygo were PVR-CD226 signaling occurs through VAV1 phosphorylation and found to synergize downstream of Apc to effect intestinal BCL9 inhibition promotes antibody-mediated PD-1 blockade via neoplasia in Apcmin/+ mouse models.53 BCL9/9L-β-catenin signal- promoting cytotoxic CD8+ T cell tumor infiltration in mouse ing is associated with poor outcome and affects stemness models. BCL9 suppression blocks VAV1 phosphorylation in CD8+ T maintenance in colorectal CRC.54 Deregulation of BCL9 is an cell as well as increases GLI1 and PATCH expression to promote important contributing factor to tumor progression in CRC.55 But CD155 expression in cancer cells (Supplementary Fig. 12). TCGA there is no reports about the role of BCL9 in immune- analysis revealed that BCL9 upregulation is associated with APC oncology yet. mutation. CD226 expression is not only associated with PD-1/PD-L1 in We for the first time reveal BCL9 suppression results in altered CRC, but also in triple-negative breast cancer, lung adenocarci- immune infiltration. Enrichment of CD8+ T and NK&T cells in noma and lung carcinoma suggested by TCGA. It is positively tumor is upon BCL9 genetic depletion and pharmacological correlated with APC mutation, which is an indicator of OS of CRC inhibition. BCL9 suppression leads to the engagement of CD226 patients treated with ICIs.56 It appears that high infiltration of which in turn activates of VAV1 and promotes CD155 expression CD226+CD8+ T cells might be a survival marker associated with possibly via increasing mRNA levels of Gli1 and Patch. BCL9 the outcome of ICI treatment in patients of different cancer types. depletion also significantly decreases CCL22 expression in tumor In the mouse scRNA-seq, we identified that both CD155-CD226 cells. Pharmacological inhibition of BCL9 reduces tumor infiltration and CD155-CD96 interactions were significantly enriched between by Treg cells due to inhibition of CCR4 expression. Inhibition of CD8+ T and CT26 cells while in the human scRNA-seq dataset, only Wnt signaling may function through the CCL22-CCR4 axis in Treg CD155-CD96 was identified. This may be due to the difference of cells tumor infiltration.46–48 two immune systems and CD226 may plays different roles in the This study is the first demonstration of using scRNA-seq to human vs. the mouse. Another possibility is that missing CD155- identify cell clusters (e.g., T cells, macrophages, fibroblasts, CD226 in human samples is an isolated case because the scRNA- endothelial cells, dendritic cells, and granulocytes) in mouse seq data were generated among CRC patients with liver models with Bcl9 genetic depletion and pharmacological inhibi- metastasis. tion. We present comprehensive cell distribution map in tumors PVR, the ligand for TIGIT, is shared with CD226. TIGIT exerts with genetic depletion of BCL9 and pharmacological inhibition. immunosuppressive action by competing with CD226 for the Alterations in the proportions of T-cell subpopulations, including same CD155 ligand. Blockage of CD226 completely abrogated the decreased percentages of CD8+ T cells, tumor-associated macro- effect of TIGIT/PD-L1 in the tumor but did not impact IFN-γ- phages, cancer-associated fibroblasts, tumor endothelial cells, producing CD8+ T cell frequency in the tumor-draining lymph tumor-associated neutrophils, and dendritic cells, indicate a node,57 suggesting a unique interplay among CD226, TIGIT and complex TIME in tumors regulated by BCL9. These findings PD-1 in the tumor microenvironment. However, the primary focus introduce a new way to investigate the role of BCL9 in TIME. of this paper was to study co-stimulatory role of CD226 and co- Clearly manipulation of cellular microenvironment is of value to inhibitory role CD96 in TIME of CRC, respectively. dissect functional roles of immune cells of different type in the To investigate the role of Wnt signaling in cancer cells, immune context of cancer therapeutic outcome evaluation and prognosis cells, and TIME, receptively, we used three different approaches, prediction. including genetic depletion, knockout and pharmacological Immune checkpoint blockade is effective in only a subset of inhibition. First, in the CT26/MC38 genetic depletion model, CRC patients with microsatellite instability-high (MSI-H) tumors.49 CT26 or MC38 cell was Bcl9 depleted, while the BALB/c and C57BL/ In these patients, the TIME is dynamic during malignant 6 mice are wild-type. This model shows how Bcl9 depleted cancer progression. Recent studies have emphasized the role of the cell impacts on the wild-type CD8+ T cell tumor infiltration. TIME in therapeutic resistance in cancer.50 Our scRNA-seq study Second, in the model of Bcl9 knockout model, the mice were sheds light on the composition of the TIME, which may facilitate subcutaneously implanted with wild-type MC38 cell. This model the development of novel immunotherapy and enhance under- demonstrates how Bcl9 knockout immune cells were changed in standing about drug resistance in CRC. By identifying differences wild-type TIME and how BCL9 driven Wnt signaling alters + + in immune cell subtypes (e.g., CD8 T and NK&T cells) following transcriptional profile of CD8 T cells. Third, in the hsBCL9CT-24

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 10

a CEACAM1_CEACAM6 LGALS9_SLC1A5 KLRB1_CLEC2D COL17A1_a2b1 complex COL17A1_a1b1 complex PVR_CD96 EGFR_GRN CD8 receptor_LCK HLA-C_FAM3C -log10 Log2 mean (P value)(Molecule 1, HLA-E_KLRC2 Molecule 2) CD74_MIF 0 4 LGALS9_DAG1 1 0 2 EGFR_COPA -4 TNFRSF1B_GRN 3 MIF_TNFRSF14 CCL4L2_VSIR EPHB2_EFNB2 LRP5_FAM3B EREG_EGFR EGFR_MIF CD55_ADGRE5 CD47_SIRPG MDK_LRP1 MDK_SORL1 EPHA1_EFNA4 + +

+ |Tumor Tumor|Tumor + |Cytotoxic CD8 Cytotoxic CD8 Tumor|Cytotoxic CD8

Cytotoxic CD8 b P=0.0080 cdP=0.1231 P<0.0001 eP<0.0001 6 12.0 on i 11.5 68 45 11.0 express expression xpression 10.5 e expression 10.0

891011 9.5 123 PVR BCL9 CTLA-4 CD226 9.0 0 024

WT WT WT WT mutant mutant mutant mutant APC APC APC APC APC APC APC APC h f P=0.0001 g P<0.0001 100 APC mutant group 90 APC WT group

n 80 70 ssio 60 pre 50 ex expression 40 2468 30 Overall survival (%) CD274 02468 PDCD1

0 20 10 P=0.0297 WT WT 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 mutant mutant APC APC Month APC APC Fig. 5 BCL9 and CD155-CD226 are associated with APC mutation, which is correlated with patient survival after immune checkpoint inhibitor treatment. a Cell–cell interaction in scRNA-seq database for human CRC samples. b Boxplot analyses of BCL9 expression associated with APC mutation level in TCGA. c Boxplot analyses of CD226 expression associated with APC mutation level in TCGA. d Boxplot analyses of CD155/PVR expression associated with APC mutation level in TCGA. e Box plots showing expression differences of CTLA-4 between APC non-mutation and APC mutation tumors. f Box plots showing expression differences of PDCD1 (PD-L1) between APC non-mutation and APC mutation tumors. g Box plots showing expression differences of CD274 (PD-L1) between APC non-mutation and APC mutation tumors. h Correlation of APC mutation with immunotherapy outcome in CRC specimens. OS, overall survival. P < 0.05 means statistically signiﬁcant.

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 11 CT26 MC38 a b cdP=0.0168 P=0.0491 6 P=0.0311 1.5 P<0.0001 50 P=0.0133 30 P=0.0317 40 4 1.0 20 Cells (%) cells (%)

cells (%) 30 + + + cells among cells among + cells among + 20 + CD8 CD8 CD8 mRNA expression 10 + +

+ 2 0.5

(Fold change) 10 CD96 CD226 CD226 Cd96 Cd96 CD45 CD45 CD45 0 0.0 0 0 +/+ -/- A NA WT WT NA RN R Bcl9 Bcl9 -shRNA -sh -shRNA -shRNA

Relative T-shR T l9 N N c NT-sh Bcl9 + B Bcl9 e MC38 f CD8 T cell P=0.0130 P=0.0167 P=0.0259 P=0.0243 80 ) 4000

P=0.0407 U

P=0.0276 A A

i N

R h

60 3000 R

T c

T cells (%) 40 2000

B N + cells among + 20 1000 CD8

Ki67 p-VAV1 0 Luminescence (RL 0 WT WT -24 RNA VAV1 h CT -shRNA -shRNA l9 l9 NT-shRNAc NT-s B Bc hsBCL9 P-AKT g CD8+ T cell h P=0.0481 1.0 anti-CD3 - + + AKT 0.8 anti-CD226 - - + 0.6 P-ERK1/2 P-VAV1 0.4

0.2 VAV1 ERK1/2 Relativeproliferation 0.0 NT-shRNA + ++- Actin GAPDH Bcl9-shRNA --+- anti-CD226 - +-- anti-CD155 --+- j Pvr k Pvr lmGli1 Ptch1 P=0.0009 P=0.0013 P<0.0001 P<0.0001 2.5 2.5 5 5 2.0 2.0 4 4 1.5 1.5 3 3 1.0 1.0 2 2 0.5 0.5 1 1 (fold change) (fold change) (fold change) (fold change) 0.0 0.0 0 0 A A Relative mRNA expression Relative mRNA Expression -24 Relative mRNA Expression Relative mRNA Expression N NA CT R Vehicle -shRN -shRNA -shR -shRNA -sh -shRNA T l9 l9 T l9 N c NT c N c hsBCL9 B B B

treated CT26/MC38 model, hsBCL9CT-24, which is a BCL9 and β-cat signiﬁcantly reduced in Bcl9-depleted tumors treated with anti- inhibitor, suppressed transcription of Wnt signaling both in PD-1 antibody, compared to NT-shRNA tumors receiving anti-PD-1 immune cells and cancer cells, without depleting or knockout alone, with a TGI of 81.8% by day 16 (Fig. 2a, b), while in the MC38 BCL9 and β-cat protein. model with MSI, anti-PD-1 therapy displayed a TGI of 56.72%. We In the CT26 microsatellite stable (MSS) tumor model, anti-PD-1 also observed in the same model that depletion of Bcl9 markedly treatment showed a TGI of 33.88%. Tumor growth was decreased the tumor size in response to PD-1 antibody, with a TGI

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 12 Fig. 6 Inhibition of BCL9 promotes CD155-CD226 checkpoint, which signals via VAV1 phosphorylation. a Percentage of CD96+ cells among CD45+CD8+ T cells from MC38 tumor tissue in BCL9+/+ and BCL9−/− mice was analyzed. b qRT-PCR measurement of Cd96 in CD8+ T cells treated with NT-shRNA or Bcl9-shRNA. c Percentage of CD226+ cells among CD45+CD8+ T cells from CT26 tumor tissue in BALB/c mice inoculated with wild-type (WT), NT-shRNA or Bcl9-shRNA-transduced-CT26 cells was analyzed. d Percentage of CD226+ cells among CD45+CD8+ T cells from MC38 tumor tissue in C57BL/6 mice inoculated with WT, NT-shRNA or Bcl9-shRNA-transduced-MC38 cells was analyzed. e Percentage of Ki67+ cells among CD45+CD8+ T cells from MC38 tumor tissue in C57BL/6 mice inoculated WT, NT-shRNA or Bcl9- + shRNA-transduced-MC38 cells was analyzed. f Proliferation of CD8 T cell co-cultured with CT26 were treated with Bcl9-shRNA or hsBCL9CT-24 and analyzed by Cell titer-Glo® Luminescent cell viability assay. g Mouse CD8+ T cells proliferation was measured after treatment with NT- shRNA or Bcl9-shRNA in the presence of anti-CD155 and anti-CD226 antibodies. h Immunoblots showing VAV1 and VAV1 phosphorylation on Tyr174 (p-VAV1) in equal amounts of total lysates from CD8+ T cells that were treated with or without CD3 antibody (Thermo, 16-0032-81, 0.25 μg) and/or anti-CD226 antibody (Thermo, 0.25 μg) for 5 min. i Immunoblots showing phosphorylated VAV1 (p-VAV1), phosphorylated ERK1/2 (p-ERK1/2), phosphorylated AKT (p-AKT) as well as total VAV1, AKT, and ERK1/2 in equal amounts of total lysates from CD8+ T cells treated with NT-shRNA or Bcl9-shRNA. j qRT-PCR measurement of CD155/Pvr in CT26 cells treated with hsBCL9CT-24 (5 μM) or vehicle for 24 h. k qRT-PCR measurement of CD155/Pvr expression in CT26 cells transduced with NT-shRNA or Bcl9-shRNA. l qRT-PCR measurement of Gli1 expression in CT26 cells treated with NT-shRNA or Bcl9-shRNA. m qRT-PCR measurement of Ptch1 expression in CT26 cells treated with NT- shRNA or Bcl9-shRNA. Results were denoted as means ± SEM for experiments performed in triplicate. Each experiment was repeated three times, and the statistical signiﬁcance of differences between groups was determined by non-parametric test. P < 0.05 means statistically signiﬁcant.

of 87.06% by day 18 (Fig. 2f). Our results show that Bcl9 depletion via i.p. injection. Tumor size of >2000 mm3 was set as the enhanced the therapeutic effect of anti-PD-1 antibody in both endpoint. models irrespective of MSS or MSI. In the CT26 combination experiment (n = 5) and MC38 (n = 8) Although our results suggest that targeting the BCL9-CD155- model, mice were grouped into six randomized cohorts and given CD226 cascade might have therapeutic implication for CRC, IgG control, anti-PD-1 antibody (10 mg/kg, i.p., BIW), or a additional preclinical studies are required to validate this combination arm (Bcl9-shRNA + anti-PD-1 antibody) via i.p. hypothesis. injection. In summary, we found that BCL9 suppression reduced tumor For CD8 depletion, anti-CD8α (YTS169.4, BioXcell BE0117) was growth, promoted CD8+ T cell tumor infiltration, and enhanced injected i.p. (300 μg per mouse) at days 12, 15, and 19 after tumor anti-PD-1 response. Single-cell RNA-seq revealed that CD226 and cell inoculation. CD96 immune checkpoints plays an important role in TIME. Our analyses confirmed many important observations that were 10x library preparation and sequencing previously made by bulk sequencing, either in vitro or in animal The single cell suspensions concentration was 700–1200 cells/μlto models, and highlighted key areas for further studies of BCL9 in input and barcode with a 10x Chromium Controller (10x the tumor immune microenvironment. These findings would Genomics). Each barcoded cell sample’s RNA was reverse- extend our understanding of BCL9 involvement in the tumorigen- transcribed to generate libraries with the reagents from 10x esis of CRC. genomics Single Cell 5’ Gel Bead Kit according to the manufacturer’s instructions. Finally sequencing was performed with an Illumina Hiseq 3000 according to the manufacturer’s instructions MATERIALS AND METHODS (Illumina). Mouse xenograft model CT26 Bcl9/NT-shRNA cancer cells were cultured as above and RNA isolation and RNA-Seq analysis 5 harvested for subcutaneously (s.c.) inoculation (4 × 10 cells in We obtained a total of 6 tumor samples of vehicle and hsBCL9CT- PBS) in the right flank region of BALB/c female mice (purchased 24 groups, from which we extracted RNA using the Tirol regent from Charles River) at 6–8 weeks of age. MC38 Bcl9/NT-shRNA above. Samples were sequenced using the BGISEQ-500 platform cancer cells were cultured as above and harvested for s.c. at The Beijing Genomics Institute (BGI) for Genomics and inoculation (1 × 106 cells in PBS) in the right flank region of Bioinformatics. Raw counts were then normalized to fragments C57BL/6 female mice (purchased from Charles River) at 6–8 weeks per kilobase of transcript per million mapped reads (FPKM). of age. BCL9–/– mice, on a C57BL/6 background and were obtained Differential gene expression was performed based on the negative from Konrad Basler’s laboratory, Switzerland. MC38 cancer cells binomial distribution with the DEGSeq package using the default were cultured as above and harvested for s.c. inoculation (1 × 106 settings (Wald significance test). To identify enriched signaling cells in PBS) in the right flank region of BCL9–/– or WT C57BL/6 pathways, we utilized GSEA and GO analysis. female mice. Tumor volume was measured every other day (V = 0.5 ab,2 where a and b are the long and short diameters of the Quantification and statistical analysis tumor, respectively). Tumor growth inhibition rate (TGI %) per PCA, t-SNE, and UMAP analysis. All cells that had the number of dosing group was calculated according to the following formula: UMI sequences of low-quality were removed. From the remaining TGI% =[1 − (TVi − TV0)/(TVvi − TVv0)] × 100%, in which TVi repre- cells, the similarity and variability of cells were summarized by sents the mean tumor volume of a dosing group on a specific day, principle component analysis (PCA). As a result, the similarity TV0 is the mean tumor volume of a dosing group on day 0, TVvi is between cells was observed by the aid of PCA. The expression the mean tumor volume of vehicle group on a specific day, and trend of cell genes is proportional to the sample distance. For TVv0 is the mean tumor volume of vehicle group on day 0. All the visualization, the dimensionality of each dataset was further animal experiments and care protocols were approved by the reduced using either the Barnes-Hut t-Distributed Stochastic Animal Care Committee of Fudan University and conformed to the Neighbor embedding (t-SNE) or Uniform Manifold Approximation Animal Management Rules of the National Health Commission of and Projection (UMAP) with Seurat functions Run-TSNE and Run- the People’s Republic of China. UMAP. The 7 principle components were summarized and In the CT26 model survival experiment, mice were grouped into visualized by tSNE (t-Distributed Stochastic Neighbor Embedding) six randomized cohorts (n = 8) and given IgG control, anti-PD-1 using the default settings of the RunTSNE function. We reanalyzed antibody [10 mg/kg, intraperitoneal (i.p.) injection, twice-weekly cells from each of these seven cell types separately in order to (BIW)], or a combination arm [Bcl9-shRNA + anti-PD-1 antibody] identify subclusters. The cells were contrasted using the Seurat

Signal Transduction and Targeted Therapy (2021) 6:313 BCL9 regulates CD226 and CD96 checkpoints in. . . Feng et al. 13 Find Markers function to identify marker genes for these and J.B.Q. edited the manuscript. M.-W.W. and D.L. advised the project, analyzed the subclusters. data, and wrote the manuscript jointly with D.Z.

TMB analysis. TCGA COAD patient mutation information and expression data was downloaded from BROAD GDAC Firehose ADDITIONAL INFORMATION (http://firebrowse.org/). We kept only the primary solid tumor Supplementary information The online version contains supplementary material patient samples for the following analysis. TMB score was available at https://doi.org/10.1038/s41392-021-00730-0. calculated as follows: total number of truncating mutations * 2.0 + total number of non-truncating mutations * 1.0 Truncating Competing interests: The authors declare no competing interests. mutations include nonsense, frame-shift deletion or insertion, and splice-site mutations. Non-truncating mutations include missense, in-frame deletion or insertion, and nonstop mutations.58 REFERENCES 1. Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2018. CA Cancer J. Clin. 68, GSVA analysis. 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