Targeting Alpha‐Ketoglutarate Disruption Overcomes Immunoevasion and Improves PD‐1 Blockade Immunotherapy in Renal Cell Carcinoma

Abstract The Warburg effect‐related metabolic dysfunction of the tricarboxylic acid (TCA) cycle has emerged as a hallmark of various solid tumors, particularly renal cell carcinoma (RCC). RCC is characterized by high immune infiltration and thus recommended for immunotherapeutic interventions at an advanced stage in clinical guidelines. Nevertheless, limited benefits of immunotherapy have prompted investigations into underlying mechanisms, leading to the proposal of metabolic dysregulation‐induced immunoevasion as a crucial contributor. In this study, a significant decrease is found in the abundance of alpha‐ketoglutarate (αKG), a crucial intermediate metabolite in the TCA cycle, which is correlated with higher grades and a worse prognosis in clinical RCC samples. Elevated levels of αKG promote major histocompatibility complex‐I (MHC‐I) antigen processing and presentation, as well as the expression of β2‐microglobulin (B2M). While αKG modulates broad‐spectrum demethylation activities of histone, the transcriptional upregulation of B2M is dependent on the demethylation of H3K4me1 in its promoter region. Furthermore, the combination of αKG supplementation and PD‐1 blockade leads to improved therapeutic efficacy and prolongs survival in murine models when compared to monotherapy. Overall, the findings elucidate the mechanisms of immune evasion in anti‐tumor immunotherapies and suggest a potential combinatorial treatment strategy in RCC.

undertook qPCR analysis of these tumor cells.
Primer sequence: qRT-PCR primers seen in Table S2; shRNA primers seen in Table S3.

Immunohistochemistry
To prepare the tissue arrays for analysis, we first deparaffinized them by baking them at 60 °C for 30 minutes, followed by a series of washes with Citrus Clearing Solvent, 100% ethanol, 95% ethanol, and 70% ethanol.To retrieve antigens, we subjected the slides to high temperatures in a pressure cooker with pH 6.0 citrate (Vector Laboratories) for 30 seconds at 125 °C, followed by 10 seconds at 90 °C.Slides were permeabilized with 0.1% TBS-T for 5 minutes, blocked with Bloxall peroxidase block for 10 min and horse serum for 20 minutes.Next, we incubated the arrays overnight with the primary antibody and subsequently incubated them with the secondary antibody for 30 minutes at room temperature.We then applied the Vectastain Elite ABC reagent for 30 minutes and a 1:1 mixture of ImPACT DAB EqV reagent 1 (Chromogen) and reagent 2 (Diluent).After washing the slides with water three times, we applied the bluing reagent once, washed the slides once with water, and dehydrated the tissue by washing them five times with 70% ethanol, followed by ten times with 95% ethanol and twice for 10 minutes with 100% ethanol.Finally, we mounted the slides with Cytoseal 60 (Biosharp) and captured images using a BioTek Cytation 5.

Immunofluorescence analysis
Cells were seeded onto fibronectin-coated glass chamber slides (Biosharp) and then fixed in 4% paraformaldehyde at room temperature for 15 minutes.Following fixation, the samples were blocked with Gelatin Block (10% normal donkey serum, 1% BSA, 0.5% Triton X-100 in PBS) at room temperature.Next, the samples were incubated with the primary antibody overnight at 4 °C or for 1 hour at room temperature, followed by washing with PBS and exposure to the secondary antibody for 1 hour at room temperature.Cells were then stained with DAPI (Servicebio) before imaging.
The antibodies and their corresponding dilutions were used in accordance with the manufacturer's instructions.

CCK-8 assay
Renca cells after corresponding treatment in different groups were respectively seeded in 96-well culture plates (1 × 10 4 /well) and incubated overnight at 37°C in a 5% CO2 incubator.The cells inoculated the day before were washed twice with PBS and then added with 100μl 1640 medium.Discard the old solution at 0, 24, 48 and 96 h after liquid exchange.Add 90μl medium and 10ul CCK-8 to each well of the 96-well test plate and incubate at 37°C for 2 h.A microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) was used to measure OD values at 450nm for each experimental well and to detect changes in cell proliferation ability in each group.

Cell apoptosis assay
Apoptosis was analyzed using a flow cytometry assay (Becton Dickinson).For co-cultivation experiments, we first collected lymphocytes from Balb/c spleens, and then collected CD8 + T cells purified by positive selection with magnetic beads from lymphocytes isolated from Balb/c mouse spleen and stimulated them with anti-CD3, anti-CD28 antibody and β-mercaptoethanol for 48 hours.

Cell Transfection
Plasmids V101-3flag and PCDH-H1 were gifts from the Laboratory of Biliary and Pancreatic Disease in Tongji hospital.Neofect was used to transfect the plasmids.
For RNA interference, gene-specific shRNA in combination with PSPAX and PMD2G were transfected into 293T cells.Twenty-four hours post transfection, the culture medium was replaced with fresh culture medium with 10% FBS.After 24 h, the medium without 293T cells was harvested, filtered and mixed with the medium containing cultured tumor cells.3 days later, puromycin (10 µg mL −1 ) was used to select positive renal cancer cells infected with shRNAs.

Chromatin immunoprecipitation and Chip-seq
Following 1% formaldehyde cross-linking, chromatin was isolated and sonicated using a Diagenode Bioruptor Pico sonicator to generate fragments with a size range of 300-700 base pairs.For immunoprecipitation, 250 μl of the fragmented chromatin was incubated overnight at 4°C with 5 μg of anti-H3K4me1 antibody (Sigma-Aldrich 17-10489) or normal rabbit IgG antibody (12-370) as a control, bound to protein A/G coated magnetic beads with rotation.The DNA was then reverse-crosslinked and eluted from the protein-DNA complex using an elution buffer (1% SDS, 100mM NaHCO3) while shaking at 65 °C for 1 hour.The purified DNA was extracted using the Maxtract tube (QIAGEN) with a phenol-chloroform extraction method, followed by overnight settling of ethanol.The immunoprecipitated DNA was further processed for sequencing using Illumina technology with modified TruSeq adapters and NEBNext ChIP-Seq Library Prep Master Mix Set.

Figure
Figure S1.GDH1 is positively correlated with the prognosis and B2M expression.(A) Overall survival and (B) tumor stages of TCGA-KIRC patients with high or low GDH1 expression.(C-E) Gsea results and B2M expression in the control group and ShGDH1 group in human 786O cells

Figure S2 .
Figure S2.High expression of B2M predicts better survival and correlates with CD8 + T cell infiltration.(A) Overall survival of TCGA-SKCM patients with high or low B2M expression.(B) Correlation of B2M expression and tumor-infiltrating CD8 + T cell proportions in multiple tumors of TCGA.(C-D) Correlation of B2M expression and tumor-infiltrating DCs and CD4 + T cell proportions in TCGA-KIRC.Immune data were analyzed using Timer database.

Figure S3 .
Figure S3.AKG promoted B2M expression in Renca cells.(A) pH values and (B) acetate levels of cell media after αKG/PBS (NC) supplementation within 24 hours.(C) PKM2 expression in clinical RCC tumor sanples and adjacent normal tissues using IHC analysis.(D) Expression of B2M in Renca cells after αKG supplementation at different time.(E) Expression of B2M in Renca cells after αKG supplementation with different doses.(F) Expression of B2M and H3K4me1 in Renca cells after CPI455 supplementation.(G) Expression of B2M and Sirt2 in Renca cells after αKG Nicotinamide supplementation.(H) Expression of B2M and AcH3K9 in Renca cells after trichostatin A supplementation.(I) Expression of B2M in Renca cells after αKG, Nicotinamide and/or trichostatin A supplementation with/without KMT2D overexpression (KMT2D ++ ).

Figure S4 .
Figure S4.AKG supplementation augmented CTL cytotoxicity to tumor cells and improved tumoral infiltration of DCs.(A) Representative flow plots and quantification of Renca cell proportions in the αKG group treated with 5mM αKG and control group after co-cultivation with CD8+