Protein Kinase STK24 Promotes Tumor Immune Evasion via the AKT‐PD‐L1 Axis

Abstract Immunotherapy targeting PD‐L1 is still ineffective for a wide variety of tumors with high unpredictability. Deploying combined immunotherapy with alternative targeting is practical to overcome this therapeutic resistance. Here, the deficiency of serine‐threonine kinase STK24 is observed in tumor cells causing substantial attenuation of tumor growth in murine syngeneic models, a process relying on cytotoxic CD8+ T and NK cells. Mechanistically, STK24 in tumor cells associates with and directly phosphorylates AKT at Thr21, which promotes AKT activation and subsequent PD‐L1 induction. Deletion or inhibition of STK24, by contrast, blocks IFN‐γ‐mediated PD‐L1 expression. Various murine models indicate that in vivo silencing of STK24 can significantly enhance the efficacy of the anti‐PD‐1 blockade strategy. Elevated STK24 levels are observed in patient specimens in multiple tumor types and inversely correlated with intratumoral infiltration of cytotoxic CD8+ T cells and with patient survival. The study collectively identifies STK24 as a critical modulator of antitumor immunity, which engages in AKT and PD‐L1/PD‐1 signaling and is a promising target for combined immunotherapy.


Human Subjects
We obtained a tissue microarray (TMA) of colorectal cancer tissues and adjacent non-
STK24, AKT1, and PD-L1 (CD274) were knocked out in tumor cell lines using the CRISPR/Cas9 gene-editing system.CRISPR/Cas9 genomic editing for gene deletion was performed as previously described [2].Guide RNA (gRNA) sequences targeting the Akt1 gRNA sequences of knock-in (mAkt1, 5′-CAGGGGAATATATTAAAACC -3′ and 5′-GGTTTTAATATATTCCCCTG-3′) were used to clone the genes into the plasmid gRNA.The AKT1 genomic sequence was cloned into the plasmid pMD18 and T21 site was mutated from ACC to GCC.These constructs were transfected with Cas9-2A-GFP and pMD18 into CT26 or LLC cells.Cells with green fluorescence were then sorted with a flow cytometer (BD FACS Aria II) 48 hours after transfection.Clones were identified by digestion/sequencing identification of genomic PCR products.

Cell proliferation assay
Cells were plated at a density of 3000 cells except for HCT116, LLC and A549 cells at a density of 5000 cells per well in 96-well culture plates and starved overnight.The serum stimulation was a time-decreased course, and plates were read at 96 hours.The number of cells could be represented by the different absorbances measured using the SynergyMx M5 (Molecular Devices).

Generation of the AKT1 T21 phosphorylation antibody.
The AKT1 T21 phosphorylation antibody was produced by Jingyun Biotechnology Co., LTD Shanghai based on the human AKT1 protein sequence.The immunopeptide sequence is CGEYIKp(T)WRPRY and the control peptide is CGEYIKTWRPRY.

Flow cytometry analysis
Tumors were collected after mice sacrifice, weighed, mechanically minced, and incubated in DNase I (100 μg/ml, Solarbio) and collagenase IV (1 mg/ml, Sigma) for 60 min at 37 °C.The dissociated cells were filtered with a 70-μm cell strainer.After blocking with CD16/CD32 (40477) antibody and getting rid of dead cells with Zombie Aqua Fixable Viability Kit (77143), cells were stained with indicated surface antibodies for 20 min on ice.Intracellular antibodies were added after fixation and permeabilization according to the manufacturer's instructions.Stained cells were analyzed by BD Fortessa.Data were further analyzed by Flow Jo 10.0 software.

Pull-down assay
The fusion protein of Flag-STK24-His, HA-EGFP-His, and HA-AKT-His was expressed in HEK293T cells and purified according to standard protocols of the Ni-NTA Column purchased from Sangon Biotech (Shanghai, China) (#C600791).For the HA pull-down assay, approximately 1μg of HA-fusion proteins were mixed with 20μl of pre-cleared anti-HA beads in 500μl of reaction buffer, subsequently, 1μg of Flag-STK24-His protein was added into the mixture and the mixture was incubated at 4°C for 4 hours with gentle rotation.The precipitates were extensively washed three times, and the samples were subjected to SDS-PAGE and immunoblot analysis using indicated antibodies.

Immunoprecipitation
The relevant plasmids were transfected in HEK293T cells as indicated groups and cell extracts were lysed by using lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, and 0.5% (vol/vol) Nonidet P-40, 1 mM EDTA) supplemented with a protease inhibitor cocktail (Roche).Next, lysates were incubated with the anti-Flag (M2)-agarose or anti-HA beads (MedChemExpress, HY-K0201) for 4 hours at 4°C.The immunoprecipitants were washed three times with the lysis buffer and subjected to further immunoblot analysis.

Ova-specific cytotoxic CD8 + T-cell killing assay
CD8 + T cells were isolated from spleen of OT-1 mice using the EasySeq mouse CD8 + T-cell isolation kit (STEMCELL, 19853) according to the manufacturer's protocol.
CD8 + T cells were activated with CD3(2μg/ml) and CD28 (2μg/ml) for 48 hours.And Stk24 knockdown MC38-OVA and KPC-OVA cells were first stimulated with mouse recombinant IFN-γ at 10 ng/ml for 12 hours.OVA-specific T cells were then cultured with the stimulated tumor cells at the E:T ratio of 1:1 or 2:1 in T-cell complete growth medium with mouse recombinant interleukin-2 (10ng/ml) for 48 hours.Live tumor cells were counted by flow cytometry.
LNP-siRNA was prepared by mixing the organic lipid components and the aqueous RNA solution with a microfluidic laminal flow rate of 800 μL/min (aqueous:organic flow rate ratio of 3:1) using a NanoAssemblr Benchtop (Precision Nanosystems, Vancouver, Canada).Then, the mixture was dialyzed for 12 hours at 4 °C against PBS (pH 7.4) using a Slide-A-Lyzer Dialysis Cassette 20 K MWCO (Thermo Fisher Scientific, Waltham, MA).Resultant LNP was concentrated with an Amicon Ultra 30 K MWCO (Merk Millipore, Burlington, MA).

Immunoblot analysis of STK24 expression in above cell lines(D).
(E-F) The proliferation of the CT26, MC38, LLC, and KPC cells transfected with STK24-specific (siStk24) or scrambled (siNC) siRNAs(E).Immunoblot analysis of STK24 expression in above cell lines(F).
(G-H) The proliferation of the CT26, MC38, LLC, and KPC cells transfected with shStk24 or scrambled negative control (shCtrl) shRNA(G).Immunoblot analysis of STK24 expression in above cell lines(H).
(I-J) The proliferation of the CTRL and STK24 KO HCT116, NCI-H1299, and A549 cells(I).Immunoblot analysis of STK24 expression in above cell lines(J).
(K-L) The proliferation of the HCT116, NCI-H1299, and A549 cells transfected with siSTK24 or siNC siRNAs(K).Immunoblot analysis of STK24 expression in above cell lines(L).
tumor tissues from the First Affiliated Hospital and Sir Run Shaw Hospital of Zhejiang University (2005DKA21300).The tissue microarray (TMA) of lung adenocarcinoma (SHYJS-CP-1904008) and pancreatic adenocarcinoma (SHYJS-CP-1901009) were obtained from the Shanghai Outdo Biotech Company.Cell culture, plasmid transfection, and siRNA silencing.CT26, MC38, LLC, KPC, NCI-H1299, HCT116, and A549 cell lines were obtained from American Type Culture Collection.Lipofectamine 3000 (Invitrogen) or polyethylenimine (Polysciences) transfection reagents were used for the plasmid transfections.Scramble siRNA and STK24-targeted siRNA were transfected in cells, using INTERFERin@ according to the manufacturer's protocol.The following siRNA

Figure S1 .
Figure S1.STK24 expression is upregulated in multiple cancer types and negatively correlated with the survival rate.(A-C) Representative IHC or IF staining of tumor sections from patients with colorectal

(M- N )
Ctrl and Stk24 KO CT26 cells (5×10 5 , M) or LLC cells (1×10 6 , N) were subcutaneously transplanted into NSG mice.The figures show tumor growth curves (left), tumor weight (middle), and representative images (right).Each dot represents a biological sample.(O-P) Stk24 h/h and wild-type (WT) mice were intraperitoneally injected with urethane dissolved in saline weekly for 10 weeks and sacrificed after 30 weeks from the first urethane injection.Representative images (O) and number (P) of neoplastic lesions of urethane-treated Stk24 h/h and WT mice were shown.Each dot represents a biological sample.Results represent at least two independent experiments and are presented as mean ± SEM. ns, no significant difference.*P<0.05,**P<0.01,***P<0.001,****P<0.0001.P values of tumor weight in M and N and tumor number in P were calculated by unpaired Student's t-tests.P values were calculated by two-way ANOVA in C, E, G, I, K and tumor growth curves of M and N.

Figure S3 .
Figure S3.STK24 deficiency promotes the infiltration and activation of CD8 + T and NK cells.(A-D) Statistical analysis of various tumor-infiltrating immune cells per gram of tumor

Figure S4 .
Figure S4.STK24 downregulation in tumor cells inhibits the expression of PD-L1.(A)Volcano plot from RNA-seq analysis of Ctrl and Stk24 KO CT26 tumor cells.(B) Heat map from RNA-seq analysis of immune checkpoints related gene expression in Ctrl and Stk24 KO CT26 tumor cells.(C) The expression of PD-L1 mRNA levels in Ctrl and Stk24 KO CT26, MC38, KPC and LLC tumor cells were detected by real-time qPCR.n = 3 technically independent samples per group.(D-K) STK24 expression was knockdown in mouse tumor cells by transfection of siRNA (D-G) or shRNA lentiviruses (H-K), and then the protein expression of PD-L1 on the tumor cell surface was detected with or without IFN-γ (20 ng/ml) treatment at

Figure S5 .
Figure S5.STK24 regulates PD-L1 expression via the activation of the AKT pathway.(A-D) Immunoblot analysis of AKT signal pathway with the indicated antibodies in STK24-knockout CT26 (A), LLC (B), MC38 (C), and KPC (D) cell lines generated by CRISPR/Cas9 treated with IFN-γ (100 ng/ml) for 0.5,1 and 3 hours.n = 3 biologically independent samples per group.(E) Pathway analysis (KEGG) of RNA-seq in Ctrl and Stk24 KO LLC tumor cells.(F-G) FACS and quantification analysis of PD-L1 expression levels of Ctrl or Stk24 KO CT26 (F) or LLC (G) cell lines pretreated with or without the AKT inhibitor MK2206 (5 μM) for 12 hours and stimulated with IFN-γ for 12 hours.FACS (left) and MFI (right) of PD-L1 + membrane expression was shown.n = 3 biologically independent samples per group.(H-I) FACS and quantification analysis of PD-L1 expression of IFN-γ treated Stk24 KO CT26 (H) or LLC (I) cells rescued with Flag-GFP, Flag-STK24, or Flag-STK24-KR.FACS (left) and MFI (right) of PD-L1 + membrane expression was shown.(J-K) Immunoblot analysis of AKT signal pathway with the indicated antibodies in Stk24 KO CT26 (J) or LLC (K) cells rescued with Flag-null, Flag-STK24, or Flag-STK24-KR treated with IFN-γ (100 ng/ml) for 0.5,1 and 3 hours.(L)Immunoblot analysis of the interaction between AKT1, AKT2 or AKT3 and STK24 with anti-Flag immunoprecipitated in HEK293T cells.WCL means whole cell lysates.

Figure S7 .
Figure S7.The combination of STK24 deficiency with anti-PD1 therapy boosts the activation of tumor infiltrating lymphocytes (TILs).(A-B) Statistical analysis of various tumor-infiltrating immune cells per gram of tumor tissues Ctrl or Stk24 KO CT26 (A) and MC38 (B) tumor cells subcutaneously inoculated mice models treated with the anti-PD-1 mAb or IgG2ɑ determined by flow

Figure S8 .
Figure S8.STK24 expression is negatively correlated with immune cell infiltration in colorectal, lung, and pancreatic cancer patients.