Selective Alanine Transporter Utilization Is a Therapeutic Vulnerability in ARID1A-Mutant Ovarian Cancer

Abstract

Subunits of the SWI/SNF chromatin remodeling complex are altered in ∼20% of human cancers. Exemplifying the alterations is the ARID1A mutation that occurs in ∼50% of ovarian clear-cell carcinoma (OCCC), a disease with limited therapeutic options. In this study, we showed that ARID1A mutations create a dependence on alanine by regulating alanine transporters to increase intracellular alanine levels. ARID1A directly repressed the alanine importer SLC38A2 and simultaneously promoted the alanine exporter SLC7A8. ARID1A inactivation increased alanine utilization predominantly in protein synthesis and passively through the tricarboxylic acid cycle. Indeed, ARID1A-mutant OCCCs were hypersensitive to the inhibition of SLC38A2. In addition, SLC38A2 inhibition enhanced chimeric antigen receptor T-cell assault in vitro and synergized with immune checkpoint blockade using an anti-PD-L1 antibody in a genetically engineered mouse model of OCCC driven by conditional Arid1a inactivation in a CD8+ T-cell-dependent manner. These findings suggest that targeting alanine transport alone or in combination with immunotherapy may represent an effective therapeutic strategy for ARID1A-mutant cancers.

Significance: ARID1A mutations regulate expression of alanine transporters to control alanine distribution between cancer cells and the associated tumor microenvironment, which may be exploited therapeutically alone or in combination with immunotherapy.

Figure 1.. ARID1A inactivation increases intracellular alanine leveling by concomitant upregulating importer SLC38A2 and downregulating exporter SLC7A8.

A, Expression of ARID1A in the indicated clear cell ovarian cancer (OCCC) cell lines determined by immunoblot. B, Heatmap of differentially expressed SLC family member genes between control and ARID1A knockout RMG1 cells in a previously published RNA-seq analysis (GEO: GSE120060). Transporters were considered differentially expressed if fold change (FC) ≥ 2.5 and FDR ≤ 0.001. n = 3 biological independent experiments. C, Volcano plot showing changes for metabolites between control and ARID1A knockout RMG1 cells in a previously published metabolomics data (MassIVE: MSV000086347). Plot shows average of 3 biological independent experiments. Metabolites were considered significantly changed if fold change (FC) ≥ 1.5 and P ≤ 0.01. P-values were calculated using two-tailed t-test. D, Peak areas of alanine were determined by LC/MS +/- HILIC in OCCC tissues with wildtype or mutant ARID1A. P-values were calculated using two-tailed t-test. n = 7 independent samples in the ARID1A wildtype group, n = 4 independent samples in the ARID1A mutant group. Error bars represent mean with SD. E and F, Expression of ARID1A and SLC38A2 in the indicated RMG1 cells determined by immunoblot (E). Please note that the multiple bands in SLC38A2 immunoblots represent modified forms of SLC38A2 through glycosylation. The relative levels of intracellular alanine were measured by HRLC/IC-MS in the indicated cells (F). P-values were calculated using two-tailed t-test. n = 3 biological independent experiments. Error bars represent the mean with SD. G and H, Expression of ARID1A and SLC7A8 in the indicated RMG1 cells determined by immunoblot (G). Cells were incubated for 18 h in the presence of 1 mM ¹³C₃-pyruvate and the metabolites in media were collected for analysis. ¹³C labeled alanine secreted from the indicated cell lines were measured by HRLC/IC-MS (H). P-values were calculated using two-tailed t-test. n = 3 biological independent experiments. Error bars represent the mean with SD.

Figure 2.. Alanine regulates the growth of OCCC cells in an ARID1A status dependent manner.

A, Growth of control and ARID1A knockout RMG1 cultured in DMEM containing 10 mM glucose or 1 mM glucose with or without 1 mM alanine supplementation was determined by colony formation assay. P-values were calculated using two-tailed t-test. n = 4 biological independent experiments. Error bars represent mean with SEM. B, Growth of ARID1A-mutated TOV21G cells cultured in RPMI-1640 containing 10 mM glucose or 1 mM glucose with or without 1 mM alanine supplementation was determined by colony formation assay. P-values were calculated using two-tailed t-test. n = 4 biological independent experiments. Error bars represent mean with SEM. C, Growth of control and ARID1A knockout RMG1 cultured in DMEM containing 10 mM glucose or 1 mM, with or without 2 mM glutamine, and at varying concentrations of alanine (1, 10, 100 mM) was determined by alamarBlue cell viability assay. P-values were calculated using two-tailed t-test. n = 5 biological independent experiments. Error bars represent mean with SD. D and E, Expression of ARID1A and SLC38A2 in control and ARID1A knockout RMG1 cells expressing the indicated shSLC38A2s or control determined by immunoblot (D). Growth of the indicated cells was determined by colony formation assay (E). P-values were calculated using two-tailed t-test. n = 3 biological independent experiments. Error bars represent mean with SEM. F and G, Expression of ARID1A and SLC38A2 in the indicated primary OCCC cultures determined by immunoblot (F). Growth of the indicated cells were determined by colony formation assay (G). P-values were calculated using two-tailed t-test. n = 3 biological independent experiments. Error bars represent mean with SEM. H, Growth of RMG1 cells treated with vehicle control, 10 μM BD98 (a ARID1A-specific BAF complexes inhibitor) or 1 μM BRM014 (a BRM/BRG1 ATPase inhibitor) with or without SLC38A2 knockdown was determined by colony formation assay. P-values were calculated using two-tailed t-test. n = 3 biological independent experiments. Error bars represent mean with SD. I and J, Expression of ARID1A and SLC7A8 in control and ARID1A knockout RMG1 cells with or without ectopic FLAG-tagged SLC7A8 expression (I). And growth of the indicated cells was determined by colony formation assay (J). P-values were calculated using two-tailed t-test. n = 4 biological independent experiments. Error bars represent mean with SEM.

Figure 3.. SLC38A2 and SLC7A8 are direct target genes of the SWI/SNF complex.

A and B, Expression of SLC38A2 (A) and SLC7A8 (B) in control and ARID1A knockout RMG1 and OVCAR429 were determined by qRT-PCR. P-values were calculated using two-tailed t-test. n = 4 biological independent experiments. Error bars represent mean with SEM. C, Control and ARID1A knockout RMG1 cells with or without wildtype ARID1A restoration were examined for expression of ARID1A, SLC38A2 and SLC7A8 by immunoblot. D, ARID1A-mutated OVISE cells with or without wildtype ARID1A restoration were examined for expression of ARID1A, SLC38A2 and SLC7A8 by immunoblot. E, SNF5-mutated G401 cells with or without wildtype SNF5 restoration were examined for SNF5, SLC38A2 and SLC7A8 expression by immunoblot. F and G, RMG1 cells with BRG1 knockdown (F) or SNF5 knockdown (G) were examined for SLC38A2 and SLC7A8 expression by immunoblot. H, Expression of SLC38A2 and SLC7A8 were examined by immunoblot in RMG1 cells treated with vehicle control, 10 μM BD98 (a ARID1A-specific BAF complexes inhibitor) or 1 μM BRM014 (a BRM/BRG1 ATPase inhibitor) for 4 days. I and J, The indicated ChIP-seq and input tracks in the SLC38A2 (I) and SLC7A8 (J) gene loci in control and ARID1A knockout RMG1 cells from a previously published GEO dataset (GSE120060). K and L, The association of ARID1A, BRG1, SNF5 and RNA Pol II with the SLC38A2 (K) and SLC7A8 (L) gene promoters in parental and ARID1A knockout RMG1 cells were examined by ChIP–qPCR analysis. An isotype-matched IgG was used as a negative control. P-values were calculated using two-tailed t-test. n = 4 biological independent experiments. Error bars represent mean with SEM. M-O, Representative images of ARID1A, SLC38A2 and SLC7A8 in ARID1A-positive or -negative cases from a OCCC tissue microarray (M). Expression of SLC38A2 and SLC7A8 were scored as high or low based on the median of histological score (H-score) in the indicated ARID1A-positive and -negative OCCCs (N). n = 20 independent ARID1A-positive samples, n = 20 independent ARID1A-negative samples. Correlation between ARID1A and SLC38A2 or SLC7A8 in ARID1A-positive samples (O). n = 23 (20 independent ARID1A-positive OCCC samples, one endometrioid carcinoma and 2 independent endometriosis. The P-values were calculated by Fisher’s exact test in M and N, and by Pearson correlation in O.

Figure 4.. ARID1A inactivation increases alanine utilization in protein synthesis and the TCA cycle.

A, Oxygen Consumption Rate (OCR) determined by a seahorse mitochondrial stress test in control and ARID1A knockout RMG1 cells cultured in medium with or without 1 mM alanine. Error bars represent mean with SD of 4 biological independent experiments. B, Basal OCR in control and ARID1A knockout RMG1 cells expressing the indicated shSLC38A2s or control was determined by using a seahorse mitochondrial stress test. P-values were calculated using two-tailed t-test. n = 6 biological independent experiments. Error bars represent mean with SEM. C-E, OCR determined by a seahorse mitochondrial stress test in control (C) and ARID1A knockout (D) RMG1 cells expressing shSLC38A2#1 or control with or without acute alanine injection. Basal OCR for the indicated groups were calculated (E). P-values were calculated using two-tailed t-test. n = 5 biological independent experiments. Error bars represent mean with SD. F, Expression of SLC38A2 in control and ARID1A knockout RMG1 cells expressing an inducible shSLC38A2#1 with indicated doxycycline treatments for 72 h determined by immunoblot. G and H, The indicated RMG1 cells were treated with 1 μg/ml doxycycline or vehicle for 72 h, followed by incubating in DMEM containing 1 mM glucose and 1mM ¹³C₃-alanine for 18 h. Intracellular metabolites were extracted to measure the indicated metabolites by LC–MS/MS (G) or secreted metabolites in the media were collected for analysis by HRLC/IC-MS (H). Mass isotopologs (M + X) analysis of the indicated metabolites are shown as percentage of indicated number of carbons labeled with heavy isotype. I, The indicated RMG1 cells were incubated in DMEM containing no glucose and 0.1 μCi/mL ¹⁴C₁-L-alanine for 6 h. Radio signal was detected in total cell lysates or precipitated proteins by liquid scintillation. P-values were calculated using two-tailed t-test. n = 3 biological independent experiments. Error bars represent mean with SD.

Figure 5.. SLC38A2 inhibition suppressed growth of ARID1A-inactivated OCCCs in vivo.

A, Expression of SLC38A2 determined by immunoblot in ARID1A-mutated TOV21G cells expressing an inducible shSLC38A2 with or without the indicated doses doxycycline treatment for 72 h. B, Growth of the indicated ARID1A-mutated TOV21G cells expressing an inducible shSLC38A2 with or without the indicated doses doxycycline treatment was determined by colony formation assay. P-values were calculated using two-tailed t-test. n = 4 biological independent experiments. Error bars represent mean with SEM. C and D, Orthotopic xenografts formed by ARID1A-mutated TOV21G cells expressing an inducible shSLC38A2 treated with vehicle or doxycycline. Shown are images of reproductive tracks with tumors from indicated groups at the end of treatment (C). Tumor weight was measured as a surrogate for tumor burden (D). P-values were calculated using two-tailed t-test. n = 7 mice per group. Error bars represent mean with SEM. E and F, Orthotopic xenografts formed by ARID1A knockout RMG1 cells expressing an inducible shSLC38A2 treated with vehicle or doxycycline. Shown are images of reproductive tracks with tumors from indicated groups at the end of treatment (E). Tumor weight was measured as a surrogate for tumor burden (F). P-values were calculated using two-tailed t-test. n = 7 mice per group. Error bars represent mean with SEM. G and H, Orthotopic xenografts formed by ARID1A wildtype RMG1 cells expressing an inducible shSLC38A2 treated with vehicle or doxycycline. Shown are images of reproductive tracks with tumors from indicated groups at the end of treatment (G). Tumor weight was measured as a surrogate for tumor burden (H). P-values were calculated using two-tailed t-test. n = 7 mice per group. Error bars represent mean with SEM. I and J, Tumors formed by the indicated control or ARID1A knockout RMG1 cells were subjected to H&E staining and immunological staining for SLC28A2, cell proliferation marker Ki67, mitotic marker serine 10 phosphorylated histone H3 (pH3S10) or apoptosis marker cleaved caspase 3 on serial sections (I) and the histological score (H-score) of the indicated markers was quantified from three separate fields from seven tumors from seven individual mice in each of the indicated treatment groups (J). Scale bar = 100 μm. P-values were calculated using two-tailed t-test. Error bars represent mean with SD.

Figure 6.. SLC38A2 inhibition and anti-PD-L1 are synergistic in suppressing ARID1A-inactivated OCCCs.

A and B, Kaplan–Meier analysis of overall survival (OS) based on SLC38A2 (A) or SLC7A8 (B) mRNA levels in cancer patients receiving immunotherapy. n = 933 independent samples. P-values were calculated by Log-rank test. C, Killing of control and ARID1A knockout RMG1 cells expressing CD19 with or without SLC38A2 knockdown was measured after coculture with anti-CD19 CAR T cells for 48 h at the 1:3 E:T ratio. P-values were calculated using two-tailed t-test. n = 3 biological independent experiments. Error bars represent mean with SD. D, Expression of SLC38A2 and loading controls Na+/K+ ATPase in anti-CD19 CAR T cells with or without SLC38A2 expression was determined by immunoblot. E, Killing of control and ARID1A knockout RMG1 cells expressing CD19 was measured after coculture with anti-CD19 CAR T cells with or without SLC38A2 overexpression for 48 h at the 1:3 E:T ratio. P-values were calculated using two-tailed t-test. n = 3 biological independent experiments. Error bars represent mean with SD. F, Expression of Slc38a2 in Arid1a^−/−;Pik3ca^H1047R tumors developed from mice injected with a lentivirus encoding both Cre-recombinase and a sgRNA targeting mouse Slc38a2 or control was determined by immunoblot. G-J, Mice bearing Arid1a^−/−;Pik3ca^H1047R OCCCs were randomized for the four indicated treatment groups. Images of reproductive tracts with tumors from the indicated groups at the end of treatment are shown (G). Tumor weight was measured as surrogate for tumor burden (H). Ascites produced in the indicated treatment groups (I) was quantified (J). Statistical co-efficiency of drug interaction (CDI) analysis revealed that the CDI for the combination were 0.88 for tumor weight and 0.89 for ascites volume (<1, indicative of a synergistic effect). P-values were calculated using two-tailed t-test. n = 5 mice per group. Error bars represent mean with SD.

Figure 7.. CD8 T cells contribute to the anti-tumor effects of SLC38A2 inhibition and anti-PD-L1 combination.

A, Arid1a^−/−;Pik3ca^H1047R tumors dissected the indicated treatment groups were subjected to H&E staining, sequential immunofluorescence staining (seqIF) and mass spectrometry imaging (MSI). Representative images showing H&E images were analyzed by Visiopharm image analysis software and used for tissue segmentation to separate tumor and non-tumor areas. SeqIF images were analyzed by Visiopharm image analysis software and used to visualize the spatial distribution of immune cells (e.g. CD4⁺ and CD8⁺ T cells), along with their exhaustion (PD-1) and proliferation (Ki67) markers. MSI images were analyzed by SCiLS Lab 2024a Pro and used to map spatial distribution of metabolites (e.g. alanine). The analyzed seqIF, H&E and MSI images were aligned using Visiopharm image analysis software. B, The ratio of alanine mean intensity in the tumor region of interest (ROI) area to that in the non-tumor ROI area. The alanine mean intensity in each ROI area were measured by Visiopharm for the indicated treatment groups. P-values were calculated using two-tailed t-test. n = 5 mice per group. Error bars represent mean with SD. C, Mean intensity of alanine in CD45⁺ cells were measured by Visiopharm for the indicated treatment groups. P-values were calculated using two-tailed t-test. n = 5 mice per group. Error bars represent mean with SD. D, Numbers of infiltrating CD8⁺ T cells were counted by Visiopharm and normalized to the whole tissue ROI area in the indicated treatment groups. CD8⁺ T cells were identified by the overlapping immunofluorescence signals of CD45 and CD8a. P-values were calculated using two-tailed t-test. n = 5 mice per group. Error bars represent mean with SEM. E, Mice bearing Arid1a^−/−;Pik3ca^H1047R OCCCs were randomized for the three indicated treatment groups. After completing treatment, mice were followed for survival and the Kaplan–Meier survival curves for each of the indicated groups are shown (n = 5 mice per group), P-values were calculated by Log-rank test.