Abstract

   Immune checkpoint inhibitors (ICIs) have shown promising results in
   bladder cancer (BC). However, only some patients respond to ICIs. DNA
   repair defects (DDR) play an important role in the therapeutic response
   of bladder cancer. Therefore, we aimed to elucidate the association
   between ICIs in bladder cancer and ataxia telangiectasia mutated (ATM),
   a core component of the DNA repair system. From a collected
   immunotherapy cohort (n = 210) and The Cancer Genome Atlas
   (TCGA)-Bladder cancer cohort, which were both retrieved from publicly
   available resources, we performed a series of analyses to evaluate the
   prognostic value and potential mechanism of ATM in bladder cancer
   immunotherapy. We found that ATM-mutant (ATM-MT) bladder cancer
   patients derived greater benefit from ICIs [overall survival (OS),
   hazard ratio (HR) = 0.28, [95% confidence interval (CI), 0.16 to 0.51],
   P = 0.007] and showed a higher mutation load (P < 0.05) and
   immunogenicity (P < 0.05) than ATM-wild-type (ATM-WT) patients. The
   immune inflammatory response to antigenic stimulation, the regulation
   of the IFN pathway and the macrophage activation pathway were
   significantly enriched in the ATM-MT group (NES > 1, P < 0.05), while
   insulin-like growth factor receptor signaling pathways and
   vasculogenesis were significantly downregulated (NES < −1, P < 0.05).
   ATM mutation significantly upregulated the number of DNA damage repair
   pathway gene mutations (P < 0.05). ATM mutations resulted in increased
   bladder cancer sensitivity to 29 drugs (P < 0.05), including cisplatin
   and BMS-536924, an IGF-1R inhibitor. Our results demonstrate the
   importance of ATM as a prognostic signature in bladder cancer and
   reveal that ATM may impact the effects of ICIs by acting on the tumor
   immune microenvironment.

   Keywords: ATM, bladder cancer, immune checkpoint inhibitors, DDR, tumor
   microenvironment

Introduction

   As one of the most common cancers in the world, bladder cancer (BC) is
   more common in men than in women ([33]Antoni et al., 2017).
   Approximately 80–90% of bladder cancers are urothelial carcinomas
   (UCs), and the remainder include adenocarcinomas, squamous cell
   carcinomas and other rare histologies ([34]Willis and Kamat, 2015).
   Currently, the first-line treatment for patients with locally
   progressive or metastatic UC is cisplatin-based chemotherapy ([35]von
   der Maase et al., 2000). However, almost half of patients still show
   recurrence or progression ([36]Oing et al., 2016). Notably, the high
   mutational load of bladder cancer confers high immunogenicity, which
   makes bladder cancer a good candidate for immunotherapy strategies,
   such as immune checkpoint blockade (ICB) ([37]Chabanon et al., 2016).
   In 2016, immune checkpoint inhibitors (ICIs) were approved for the
   treatment of bladder cancer patients with advanced refractory disease
   or who were ineligible platinum chemotherapy, improving patient
   prognosis ([38]Zibelman et al., 2016). Although ICB therapy against the
   checkpoint axis of programmed death ligand-1 (pd-l1)/pd-1 has shown
   some promising results as a UC therapy ([39]Sharma et al., 2017), only
   20–30% of UC patients have a partial or complete response to ICI
   therapy ([40]Mariathasan et al., 2018). Thus, the identification of new
   potential biomarkers to guide targeted clinical therapy and thus
   increase the proportion of patients with bladder cancer responsive to
   immunotherapy is urgently needed.

   DNA repair gene defects play an important role in bladder cancer
   occurrence, development, therapeutic response and prognosis. DNA repair
   gene somatic mutations are often found in bladder cancer ([41]Cancer
   Genome Atlas Research Network, 2014) and are associated with a
   relatively good response to cisplatin-class neoadjuvant chemotherapy
   ([42]Plimack et al., 2015). Liu et al. demonstrated that changes in the
   ERCC2 gene may predict the response of bladder cancer to cisplatin
   neoadjuvant chemotherapy ([43]Freedman and Pfeiffer, 2016). Teo et al.
   showed that DNA damage response (DDR) gene mutations are associated
   with a relatively good prognosis in platinum-treated advanced bladder
   cancer ([44]Teo et al., 2017). These studies confirm the strong link
   between DDR mutations and bladder cancer. In addition, the deficiency
   in the DNA repair machinery has recently been reported to be associated
   with increased response to ICI ([45]Le et al., 2015, [46]2017). [47]Teo
   et al. (2018) found that DDR gene mutation was associated with an
   increased ICI response rate. These results suggest that the DDR pathway
   may play an important role in ICI therapy.

   Ataxia telangiectasia mutated (ATM) is a core component of the DNA
   repair system that activates the ATM-enhanced homologous recombination
   repair pathway upon recognition of a DNA double-strand break ([48]Weber
   and Ryan, 2015). Recently, inhibiting the DDR pathway has become a
   common treatment strategy in many cancers ([49]Cremona and Behrens,
   2014), and the ATM signaling pathway is known to play an important part
   in the development of breast cancer, germ cell carcinoma and other
   cancers ([50]Shiloh and Kastan, 2001; [51]Jin and Oh, 2019).
   [52]Plimack et al. (2015) demonstrated that changes in the ATM, RB1,
   and FANCC genes can predict the efficacy of neocisplatin chemotherapy
   in bladder cancer. Additional studies have reported that ATM gene
   defects are one of the mechanisms of spontaneous tumor-infiltrating
   lymphocyte (TIL) infiltration in breast and ovarian cancer ([53]Rooney
   et al., 2015). In addition, other studies have suggested that
   DDR-deficient cancer cells exhibit IFN response activation and cell
   recruitment via the chemokines CCL5 and CXCL10 ([54]Hartlova et al.,
   2015; [55]Nakad and Schumacher, 2016). These studies may explain some
   of the possible mechanisms underlying the utility of DNA repair gene
   mutations in predicting cancer prognosis, but the significance of DNA
   repair gene mutations as biomarkers for predicting treatment efficacy
   in bladder cancer has not been well defined. At present, there is still
   insufficient research available to be able to stratify patients
   according to treatments and evaluate the effects of specific DDR gene
   mutations on predicting the efficacy of immunotherapy in bladder
   cancer.

   Based on this information, we evaluated the relationship between ATM
   gene mutations and the prognosis of patients receiving ICIs. According
   to the mutation status of the ATM gene, patients who received ICI
   treatment (anti-pd-1/pd-l1 therapies) and had mutation data available
   were divided into ATM-mutant (MT) and ATM-wild-type (WT) groups. We
   found that the ATM-MT bladder cancer patients had better benefits from
   ICI therapy and showed a higher mutation load and immunogenicity. In
   addition, we explored the differences in pathway activation between the
   ATM-MT bladder cancer patients and ATM-WT patients through functional
   enrichment analysis to explain the effect of ATM on the efficacy of
   ICIs and the tumor microenvironment (TME).

Materials and Methods

Relationship Between ATM Gene Mutation and Immunotherapy Prognosis in Bladder
Cancer

   We collected an immunotherapy cohort with clinical and mutational data
   (reported by [56]Samstein et al., 2019), within which 210 bladder
   cancer patients with mutational data received ICI therapy
   (anti-pd-1/pd-l1 therapies). To evaluate the relationship between ATM
   gene mutations and the prognosis of patients receiving ICIs, we divided
   the patients into ATM-MT and ATM-WT groups according to their ATM
   mutational status and performed Kaplan–Meier (KM) analysis. In
   addition, we also collected The Cancer Genome Atlas (TCGA)-Bladder
   cancer cohort with somatic mutation and survival data from the Genomic
   Data Commons^[57]1 using the R package TCGAbiolinks ([58]Colaprico et
   al., 2016) for download. Then, we used cBioportal ([59]Cerami et al.,
   2012) to download the survival data [OS, disease-free survival (DFS),
   and progression-free survival (PFS)] of the TCGA-Bladder cancer cohort
   and conducted similar KM analysis according to the ATM mutational
   status.

Mutation Characteristics and Tumor Immunogenicity

   To identify the gene mutation characteristics and tumor immunogenicity
   of ATM-MT bladder cancer patients, we analyzed the immunotherapy cohort
   ([60]Samstein et al., 2019) somatic mutation data generated by targeted
   next-generation sequencing (NGS; MSK-IMPACT). The neoantigen mutational
   load (neoantigen loading; NAL) data from the TCGA-Bladder cancer cohort
   was also evaluated ([61]Thorsson et al., 2018). Next, we downloaded
   whole-exome sequencing (WES) data for bladder cancer cell lines from
   Genomics of Drug Sensitivity in Cancer (GDSC) ([62]Yang et al., 2013).
   To quantify the tumor mutational burdens (TMBs) of the immunotherapy
   cohort ([63]Samstein et al., 2019), TCGA-Bladder cancer cohort and
   GDSC-Bladder cancer cell line cohort, we referred to other literature
   ([64]Chalmers et al., 2017) and treated the non-synonymous mutations in
   each cohort as raw mutations and divided by 38 Mb. Finally, the R
   package ComplexHeatmap ([65]Gu et al., 2016) was used to visualize the
   mutation rates in the top 20 genes and the clinical features of the
   immunotherapy cohort and TCGA-Bladder cancer cohort. In addition, the R
   package Maftools ([66]Mayakonda et al., 2018) was used to visualize the
   correlation between ATM and the mutation rates in the top 20 genes of
   the TCGA-Bladder cancer cohort. The ATM gene mutation sites in the
   immunotherapy cohort and TCGA-Bladder cancer cohort were visualized
   using the R package Maftools.

Analysis of Immune Characteristics and Drug Sensitivity

   To further compare the relationship between ATM gene mutation status
   and the TME, we compared the mRNA expression levels of immune-related
   genes in the ATM-MT and ATM-WT patients of TCGA-Bladder cancer.
   Immune-related genes and their functional classifications and
   immune-related scores have been reported ([67]Hao et al., 2018). We
   also compared the mRNA expression levels of immune cell-related genes
   in the ATM-MT and ATM-WT patients of TCGA-Bladder cancer ([68]Thorsson
   et al., 2018). The expression levels of all these genes were quantified
   as fragments per kilobase of exon model per million mapped fragments
   (FPKM) and log2-transformed. The cutoff value was set to logFC = 1. In
   addition, TCGAbiolinks was used to download the TCGA-Bladder cancer
   gene expression data (Illumina HiSeq, RNASeq), and CIBERSORT^[69]2
   ([70]Newman et al., 2015) and xCell^[71]3 ([72]Aran et al., 2017) were
   used to compare the immune cell infiltration proportion in the ATM-WT
   and ATM-MT patients of TCGA-Bladder cancer. The R package software MCP
   counter ([73]Becht et al., 2016) was used to quantify the absolute
   abundance of immune and stromal cells. Finally, we downloaded the
   drug-sensitivity data for the bladder cancer cell lines from GDSC and
   analyzed the differences in sensitivity to different drugs according to
   the ATM gene mutation status.

Copy Number Alteration (CNV) Analysis

   To analyze Copy number alteration (CNV) mutations in the ATM gene in
   the TCGA-Bladder cancer patients, we used Broad GDAC Firehose^[74]4 to
   download Affymetrix 6.0 SNP microarray data (hg19; germline/potential
   false-positive calls were removed). The downloaded CNV segments were
   subjected to GISTIC2.0 analysis by GenePattern^[75]5 ([76]Reich et al.,
   2006). Except for a couple parameters (e.g., confidence level of 0.99;
   X chromosomes not excluded before analysis), the GISTIC2.0 analysis
   used default settings. Finally, the results of the GISTIC2.0 analysis
   were visualized using the R package Maftools ([77]Mayakonda et al.,
   2018).

Pathway Enrichment Analysis and DDR Pathway Mutation Number Analysis

   First, we used the R package edgeR to conduct differential analysis of
   the gene expression data (raw count) of TCGA-Bladder cancer downloaded
   with TCGAbiolink ([78]Robinson et al., 2010). The ClusterProfiler R
   package ([79]Yu et al., 2012) was used to perform gene annotation
   enrichment analysis, and a gene set enrichment analysis (GSEA) dataset
   was obtained from the MSigDB database of Broad Institute
   ([80]Subramanian et al., 2005). P < 0.05 was considered to be
   significant for gene ontology (GO) terms, Kyoto Encyclopedia of Genes
   and Genomes (KEGG) pathways and Reactome pathways. In addition, we
   evaluated and compared the difference in non-synonymous mutation
   numbers in the DDR pathways between the ATM-MT and ATM-WT groups in the
   immunotherapy cohort, TCGA-Bladder cancer cohort, and GDSC-Bladder
   cancer cell line dataset. The gene set of DDR pathways was derived from
   the DDR pathway gene set of the MSigDB database of the Broad Institute
   ([81]Subramanian et al., 2005) (see Additional File: [82]Table S1).

Statistical Analysis

   We used the Mann–Whitney U test to compare the differences in TMB, NAL,
   immune cell abundance, immune-related gene expression, age,
   microsatellite instability (MSI) score and number of mutations in DDR
   pathway components between ATM-MT and ATM-WT groups. In addition, we
   used Fisher’s exact test to determine the relationships between ATM
   gene mutation status and mutation status of the top 20 genes, tumor
   sample type (primary/metastatic), sex, and treatment (single
   drug/combination) in the immunotherapy cohort. We also used Fisher’s
   exact test to determine the relationships between ATM mutations and
   sex, race, ethnicity and clinical stage in the TCGA-Bladder cancer
   cohort. Survival analysis was performed with the KM method and log-rank
   test. P < 0.05 was considered statistically significant, and all
   statistical tests were bilateral. All statistical tests and visualized
   analyses were performed with R software (version 3.6.1). In addition,
   the R package ggpubr was used for visualizing boxplots ([83]Kassambara,
   2018). The false discovery rate (FDR) for CNV visualization was 0.05.

Results

Association Between the ATM Status and Clinical Outcomes

   To evaluate the relationship between ATM gene mutations and the
   prognosis of bladder cancer patients who received ICI treatment, we
   collected an immunotherapy cohort with clinical data and mutational
   data ([84]Samstein et al., 2019). The baseline characteristics of the
   immunotherapy cohort are summarized in [85]Table 1. According to their
   ATM gene mutation status, bladder cancer patients with mutation data
   treated with ICIs (anti-pd-1/pd-l1 therapies) (n = 210) were divided
   into ATM-MT and ATM-WT groups, and then KM analysis was performed. We
   found that the OS time of the ATM-MT bladder cancer patients treated
   with ICIs was longer than that of the ATM-WT patients [[86]Figure 1A,
   hazard ratio (HR) = 0.28, [95% confidence interval (CI), 0.16 to 0.51],
   P = 0.007]. In addition, we downloaded the TCGA-Bladder cancer cohort
   somatic mutation and survival data from the Genomic Data Commons (see
   footnote 1) and similarly performed KM analysis according to the ATM
   mutation status. We found that ATM gene mutation did not predict
   prognosis in the TCGA cohort patients who did not receive ICI treatment
   ([87]Figure 1B: OS, n = 410, HR = 0.76 [95% CI, 0.49 to 1.16], P =
   0.251; [88]Figure 1C: PFS, n = 411, HR = 0.65 [95% CI, 0.43 to 1.00], P
   = 0.09; [89]Figure 1D: DFS, n = 188, HR = 0.64 [95% CI, 0.26 to 1.57],
   P = 0.401). Therefore, among bladder cancer patients treated with ICIs,
   patients with ATM gene mutation exhibited a better prognosis than those
   with ATM-WT.

TABLE 1.

   The baseline characteristics of the immunotherapy cohort.
   Characteristics                   N (%)
   Gender
   Male                             159 (76)
   Female                           51 (24)
   Age decade
   31–50                             12 (6)
   50–60                            43 (21)
   61–70                            66 (31)
   >70                              89 (42)
   Sample Type
   Primary                          121 (58)
   Metastasis                       89 (42)
   Drug class
   PD-1/PD-L1                       187 (89)
   Combo                            23 (11)
   Survival events
   Survival                         92 (44)
   Died                             118 (56)
   ATM Status
   Mutated type                     187 (89)
   Wild type                        23 (11)
   Tumor Mutational Burden (Median)   8.8
   MSI Score (Median)                 0.3
   [90]Open in a new tab

FIGURE 1.

   [91]FIGURE 1
   [92]Open in a new tab

   Association between the ATM status and clinical outcomes in bladder
   cancer. (A) Kaplan–Meier curves comparing the overall survival (OS) of
   patients with ATM-MT with that of patients with ATM-WT in the
   immunotherapy cohort [n = 210, hazard ratio (HR) = 0.28, [95%
   confidence interval (CI), 0.16 to 0.51], P = 0.007]. ATM-MT, ATM mutant
   type; ATM-WT, ATM wild type; BLCA, bladder cancer. (B) Kaplan–Meier
   curves comparing the OS of patients with ATM-MT with that of patients
   with ATM-WT in the TCGA-BLCA cohort (n = 410, HR = 0.76, [95% CI, 0.49
   to 1.16], P = 0.251). (C) Kaplan–Meier curves comparing the
   progression-free survival (PFS) of patients with ATM-MT with that of
   patients with ATM-WT in the TCGA-BLCA cohort (n = 411, HR = 0.65, [95%
   CI, 0.43 to 1.00], P = 0.09). (D) Kaplan–Meier curves comparing the
   disease-free survival (DFS) of patients with ATM-MT with that of
   patients with ATM-WT in the TCGA-BLCA cohort (n = 188, HR = 0.64, [95%
   CI, 0.26 to 1.57], P = 0.401).

Relationships Between the ATM Mutation Status and Clinical Phenotype and
Mutation Load in Bladder Cancer

   To further explore the relationships between the ATM mutation status
   and clinical phenotype and mutation load in bladder cancer, we compared
   the differences in the mutation status in the top 20 genes, tumor
   sample type (primary/metastatic), sex, race, ethnicity, treatment
   (single drug/combination) and clinical stage between the ATM-MT and
   ATM-WT patients in the immunotherapy cohort and TCGA-Bladder cancer
   cohort. The results showed that the ATM-MT group exhibited a better
   prognosis and a higher TMB score and that the mutation frequencies of
   ARID1A, CREBBP, KMT2A, SMARCA4, and EP300T were significantly higher in
   the ATM-MT group than in the ATM-WT group in the immunotherapy cohort
   ([93]Figure 2A, P < 0.05). In the TCGA-Bladder cancer cohort, there was
   no significant difference in prognosis between the ATM-MT group and the
   ATM-WT group. The ATM-MT group had a higher neoantigen load, and the
   mutation frequencies of TTN, KMT20, MUC16, SYNE1, HMCN1, KMT2C, MACF1,
   FAT4, and CSMD3 were significantly higher in the ATM-MT group than in
   the ATM-WT group ([94]Figure 2B, P < 0.05). Correlation analysis showed
   that ATM had co-occurrence with TTN, KMT2C, and CSMD3 genes (P < 0.05),
   and tended to be mutually exclusive with FGFR3 (P < 0.1)
   ([95]Supplementary Figure S1). Additionally, we analyzed the ATM
   mutation sites and CNV. The results showed that the ATM mutation sites
   were evenly distributed among all five important regions ([96]Figure
   2C). Both ATM-MT group and ATM-WT group have a large copy number
   variation regardless of the status of ATM. While comparing to the
   ATM-WT group, we found that there were more CNVs in the ATM-MT group
   ([97]Figure 2D). The significantly amplified regions in ATM-MT patients
   included 1q21.3, 1q23.3, 8q22.3, and 11q13.3, of which 11q13.3 span the
   CCND1 (cyclin D1) gene. A significant deletion in ATM-MT patients
   included 9p21.3, spanning the tumor suppressor genes CDKN2A and CDKN2B
   ([98]Figure 2D).

FIGURE 2.

   [99]FIGURE 2
   [100]Open in a new tab

   Relationship between the ATM status and clinical phenotype. (A) Top 20
   significantly mutated genes in the immunotherapy cohort of bladder
   cancer patients. Samples were ordered based on the somatic
   non-synonymous mutation burden, and genes were ranked by mutation
   frequencies (left panel). The ATM status, sample type
   (metastasis/primary), sex, drug type, OS, OS time (months), TMB score
   and MSI score are annotated in order in the top panel. (B) Top 20
   significantly mutated genes in the TCGA-BLCA cohort. ATM status, age,
   sex, stage, ethnicity, OS, OS time (months), TMB and neoantigen load
   are annotated in order in the top panel. (C) The somatic mutations in
   ATM were evenly distributed in both the immunotherapy cohort and the
   TCGA-BLCA cohort. (D) Maftools was used to visualize the copy number
   alteration (CNV) analysis based on GISTIC2.0 of the TCGA-BLCA cohort.

ATM Mutation Enhances Immunogenicity and Activates Antitumor Immunity

   To determine how ATM mutations enhance the immunogenicity of bladder
   cancer and activate antitumor immunity, we compared the expression
   differences in 74 immune-related genes (antigen
   presentation/stimulation/inhibition) and immune cell-related genes
   between the ATM-MT group and AMT-WT group. The results showed that in
   the ATM-MT group, the expression of the immune-related genes CCL5 and
   AGE1 was significantly increased (logFC > 1, adjusted P < 0.05). Among
   the immune cell-related genes, the expression of mast cell-related CLC
   and NTRK1, T helper (Th) 17 cell-related SH2D6, and regulatory T (Treg)
   cell-related IL1RL1 was significantly upregulated (logFC > 1, adjusted
   P < 0.05) ([101]Figures 3A,B). Moreover, the ATM-MT group showed a
   higher mutation load and stronger antigen-specific immunity than the
   ATM-WT group in the immunotherapy cohort, TCGA-Bladder cancer cohort
   and GDSC-Bladder cancer cell line dataset ([102]Figures 3C–F, P <
   0.05). Next, we used CIBERSORT to evaluate the data and then compared
   the levels of 22 immune cells between the ATM-MT and ATM-WT groups. The
   results showed that the proportion of activated dendritic cells was
   significantly upregulated in the ATM-MT group ([103]Figure 3G, P <
   0.05). In addition, xCell analyses showed that the proportion of CD4 +
   Tem cells was upregulated in the ATM-MT group ([104]Supplementary
   Figure S2A, P < 0.05), while there were no significance in MCP counter
   analyses ([105]Supplementary Figure S2B).

FIGURE 3.

   [106]FIGURE 3
   [107]Open in a new tab

   ATM mutation was associated with enhanced tumor immunogenicity and
   activated antitumor immunity. (A) Heatmap showing average changes in
   the expression levels of immune-related gene between the ATM-MT and
   ATM-WT patients in the TCGA-BLCA cohort. The genes corresponding to the
   same lymphocyte or function are identified by the same color on the
   left side of the squares, and each square with an exact number
   represents the logFC of a gene, filled with different back colors,
   i.e., from red to gray. The logFC values marked in black font indicate
   that the absolute value of logFC is ≥1 with statistical significance (P
   < 0.05), while the logFC values in white font are non-significant (P >
   0.05). Using Mann–Whitney U test tested the differences in
   tumor-infiltrating lymphocytes and immune-related gene expression
   between ATM-MT and ATM-WT samples. (B) Heatmap showing average changes
   in the expression levels of immune-cell-related gene between the ATM-MT
   and ATM-WT patients in the TCGA-BLCA cohort. The genes corresponding to
   the different cell types are identified by the different colors on the
   left side of the squares, and each square with an exact number
   represents the logFC of a gene, filled with different back colors,
   i.e., from red to blue. The logFC values marked in black font indicate
   that the absolute value of logFC is ≥1 with statistical significance (P
   < 0.05). Using Mann–Whitney U test tested the differences in
   immune-cell-related gene expression between ATM-MT and ATM-WT samples.
   (C) Comparison of the tumor mutational burden between the ATM-MT and
   ATM-WT tumors in the immunotherapy cohort. (D) Comparison of the tumor
   mutational burden between the ATM-MT and ATM-WT tumors in the TCGA-BLCA
   cohort. (E) Comparison of the neoantigen load between the ATM-MT and
   ATM-WT tumors in the TCGA-BLCA cohort. (F) Comparison of the tumor
   mutational burden between the ATM-MT and ATM-WT tumors in the
   GDSC-Bladder cancer cell line dataset. (G) CIBERSORT analyses
   quantifying the proportion of 22 immune cells in the ATM-MT and ATM-WT
   tumors in the TCGA-BLCA cohort. (C–G), *p < 0.05; ****p < 0.0001.

Functional Enrichment Pathways Associated With ATM Mutations

   Next, we analyzed the functional enrichment pathways of the ATM-MT and
   ATM-WT groups in the TCGA-Bladder cancer cohort and GDSC dataset by
   GSEA. The results showed that the immune inflammatory response to
   antigenic stimulation, the regulation of the IFN pathway and the
   macrophage activation pathway were significantly enriched in the ATM-MT
   group (NES > 1, P < 0.05) ([108]Figures 4A–C). Insulin-like growth
   factor receptor signaling pathways and vasculogenesis were
   significantly downregulated in the ATM-MT group (NES < −1, P < 0.05)
   ([109]Figures 4D–F). Additionally, we analyzed the effect of ATM
   mutation on the sensitivity of bladder cancer cells to drug therapy in
   the GDSC cohort. We found that ATM mutation resulted in increased
   bladder cancer sensitivity to 29 drugs, including cisplatin (P < 0.05),
   BMS-536924, an IGF-1R inhibitor (P < 0.05), motesanib, a VEGFR
   inhibitor (P < 0.05), and WHI-P97, a JAK inhibitor (P < 0.05)
   ([110]Figure 5). In combination with the significant downregulation of
   insulin-like growth factor receptor expression in the ATM-MT group
   shown in the GSEA analysis described above, BMS-536924 may have a
   synergistic effect with ATM mutation in inhibiting IGF-1R.

FIGURE 4.

   [111]FIGURE 4
   [112]Open in a new tab

   GSEA of the functional enrichment pathways of the ATM-MT and ATM-WT
   groups in the TCGA-BLCA cohort and GDSC-Bladder cancer cell line
   dataset. (A) The inflammatory response in the antigen pathway was
   significantly enriched in the ATM-MT group. (B) Interferon-gamma
   production and regulation pathways were significantly enriched in the
   ATM-MT group. (C) Macrophage activation involved in the immune response
   pathway was significantly enriched in the ATM-MT group. (D) The
   insulin-like growth factor receptor signaling pathway was significantly
   downregulated in the ATM-MT group. (E) The insulin-like growth factor
   receptor signaling pathway was significantly downregulated in the
   ATM-MT group. (F) Angiogenesis-related pathways were significantly
   downregulated in the ATM-MT group.

FIGURE 5.

   [113]FIGURE 5
   [114]Open in a new tab

   Comparison of drug sensitivity between ATM-MT and ATM-WT in the
   GDSC-Bladder cancer cell line cohort. We observed that the sensitivity
   to 29 drugs were increased in the ATM-MT group (P < 0.05), including
   Alectinib, AZD7762, Bleomycin, Carmustine, CCT_018159, Cisplatin,
   CX_5461, Cytarabine, Genentech_Cpd_10, YK_4_279, GW_2580, IOX2,
   KIN001_270, KU_55933, Lenalidomide, Motesanib, OSI_930, Pevonedistat,
   PFI_3, RO_3306, rTRAIL, Selisistat, Sepantronium_bromide, SN_38,
   STF_62247, Talazoparlb, WHI_P97, XMD14_99, and XMD15_27. IC50s reported
   in GDSC were log-transformed. Using the Mann–Whitney U test tested the
   differences in the ln(IC50) values of different drugs between the
   ATM-MT and ATM-WT cell lines. *P < 0.05; **P < 0.01.

ATM Mutation and Mutated DDR Pathway Gene Number Variation

   Since DNA repair gene defects play an important role in the occurrence,
   development, therapeutic response and prognosis of bladder cancer, we
   further explored the role of ATM mutations in DDR pathways. The eight
   gene sets related to DNA repair gene defects (DDR) came from MSigDB
   ([115]Supplementary Table S1). We analyzed the relationship between ATM
   mutations and gene mutations in the eight DDR-related pathways in the
   immunotherapy cohort, TCGA-Bladder cancer cohort and GDSC-Bladder
   cancer cell line dataset. We found that patients with ATM alterations
   in homologous recombination (HR) (P < 0.0001), MMR (P < 0.001), DSB (P
   < 0.0001), and FA (P < 0.0001) showed comparably high mutation counts
   than ATM intact patients in the immunotherapy cohort. However, SSB
   failed to show the significant difference in all cohorts ([116]Figure
   6).

FIGURE 6.

   [117]FIGURE 6
   [118]Open in a new tab

   Association between ATM mutation and mutated DNA damage repair pathway
   gene number variation. Mutated DNA damage repair pathway gene number
   variation increased in the ATM-MT group in the immunotherapy, TCGA-BLCA
   and GDSC-Bladder cancer cell line cohorts. The x axis indicates eight
   DDR-related pathways. BER, base excision repair; HR, homologous
   recombination; MMR, mismatch repair; SSB, single-stranded DNA binding;
   DSB, double-strand break repair; NER, nucleotide excision repair; NHEJ,
   non-homologous end-joining; FA, Fanconi anemia. *P < 0.05; **P < 0.01;
   ***P < 0.001; ****P < 0.0001.

Discussion

   Recently, the high mutational load of bladder cancer confers high
   immunogenicity, which makes bladder cancer a good candidate for
   immunotherapy strategies. [119]Fan et al. (2019) conducted a
   meta-analysis of 14 clinical trials and showed that PD-1/PD-L1
   treatment was effective with acceptable incidence of treatment-related
   adverse events. Unfortunately, no effective biomarker has been found to
   predict the efficacy of immunotherapy in bladder cancer patients.
   Therefore, we collected an immunotherapy cohort and the TCGA-Bladder
   cancer cohort containing clinical data and mutation data. Through
   survival analysis, we found that the patients in the ATM-MT group in
   the immunotherapy cohort had a significantly better immunotherapy
   prognosis than those in the ATM-WT group. There was no significant
   difference in the TCGA-Bladder cancer cohort, which may be related to
   the lack of sufficient information to stratify patients according to
   treatment. These results suggest that anti-pd-1/pd-l1 ICI therapy is
   more effective in ATM-MT bladder cancer patients. Through further
   analysis, we found that ATM is likely to impact the efficacy of ICIs by
   affecting the tumor immune microenvironment.

   We first explored the role of ATM mutations in bladder cancer by
   evaluating the associations between the gene mutation status and
   clinical phenotypes in the immunotherapy cohort and TCGA-Bladder cancer
   cohort. We found that the ATM-MT group showed a higher mutation load
   and immunogenicity in both cohorts. Compared to the ATM-WT group,
   ARID1A and TTN mutations were significantly upregulated in the ATM-MT
   group in the immunotherapy cohort and the TCGA-Bladder cancer cohort,
   respectively. The different results between two cohorts may be related
   to the lack of sufficient treatment information to stratify patients in
   the TCGA-Bladder cancer cohort. Zhu et al. showed that ARID1A and TTN
   genes were among the 11 genes with frequent mutations in bladder cancer
   ([120]Zhu et al., 2020), indicating their importance in predicting
   immune response in bladder cancer, and suggesting that co-mutations of
   ARID1A or TTN with ATM may be potential molecular biomarkers for
   predicting the efficacy of immunotherapy for bladder cancer. We further
   compared the expression differences in immune-related genes (antigen
   presentation/stimulation/inhibition) and immune cell-related genes
   between the ATM-MT and ATM-WT groups. The results showed that the
   expression of the chemokines CCL5 and AGE1 was significantly
   upregulated in the ATM-MT group. The chemokine CCL5 plays important
   roles in the chemotaxis and activation of various immune cells, such as
   monocytes/macrophages, mast cells and T lymphocytes ([121]Lapteva and
   Huang, 2010; [122]Marques et al., 2013), and has potential clinical
   application value as an adjuvant to enhance antitumor immunity
   ([123]Yang et al., 2017; [124]Dugas et al., 2019). Additionally, we
   found that cytotoxic cells, memory CD8 cells, mast cells, Th17 cells,
   Treg cells and other immune cell marker molecules were significantly
   upregulated. Baharlou R et al. showed that Th17 cells might play an
   important roles in the antitumor immune response in early stage bladder
   cancer ([125]Baharlou et al., 2015). Next, we used CIBERSORT to further
   analyze the proportion of 22 immune cell subtypes in the ATM-MT group
   and found that the proportion of activated dendritic cells was
   significantly upregulated in the ATM-MT group. Hartana et al. noted
   that large numbers of T cells and other cytotoxic cells infiltrated a
   tumor and that the tumor stage of patients was relatively low,
   suggesting the possibility of using cytotoxic cells as a new
   immunotherapy regimen. Additionally, bladder cancer cells may reduce
   the infiltration of CD8 T cells by inhibiting the ICAM-1/TGFβ2 pathway
   ([126]Hartana et al., 2018a, [127]b). Several studies have shown that
   various methods of activating the immune activity of dendritic cells in
   bladder cancer can induce the dendritic cells to initiate an antitumor
   immune response ([128]Xiu et al., 2018; [129]Domingos-Pereira et al.,
   2019). Above all, ATM-MT bladder cancer cells are likely to promote the
   infiltration of cytotoxic cells and Th17 cells into tumor tissues by
   secreting relatively large amounts of chemokines, such as CCL5, to
   activate dendritic cells and promote the therapeutic effect of immune
   checkpoint inhibition.

   In addition to the chemokine CCL5, another significantly upregulated
   gene in the ATM-MT group identified by differential analysis of immune
   cell-related gene expression was AGE1. The AGE1 gene is a core
   component of the insulin signaling pathway. Age-1 inactivates the FOXO
   transcription factor daf-16 by activating downstream AKT, thereby
   blocking the insulin/insulin-like growth factor receptor signaling
   pathway ([130]Paradis and Ruvkun, 1998; [131]Kenyon, 2010). Next, we
   conducted functional pathway enrichment analysis of the TCGA-Bladder
   cancer cohort by GSEA. The results showed that the insulin-like growth
   factor receptor pathway was significantly downregulated in the ATM-MT
   group. Previous studies have demonstrated that high expression of
   insulin-like growth factor receptor significantly promotes bladder
   cancer invasion and metastasis through pathways such as the AKT and
   mitogen-activated protein kinase (MAPK) pathways ([132]Metalli et al.,
   2010; [133]Safarinejad et al., 2011) and plays an important role in
   lung cancer, breast cancer and other tumor proliferation ([134]Selinski
   et al., 2012; [135]Szarvas et al., 2012). Therefore, ATM-MT bladder
   cancer cells are likely to downregulate the activity of the
   insulin-like growth factor receptor pathway by upregulating AGE1 gene
   expression, thereby inhibiting the invasion and metastasis of bladder
   cancer and improving survival. To confirm our hypothesis, we further
   analyzed the effect of ATM mutations on bladder cancer by evaluating
   drug sensitivity data from the GDSC database. The sensitivity to
   twenty-nine drugs, including cisplatin, BMS-536924, an IGF-1R
   inhibitor, motesanib, a VEGFR inhibitor, and WHI-P97, a JAK inhibitor,
   was found to be increased in the ATM-MT group. IGF-1R was reported to
   be capable of transmitting mitogenic signals to the neoplastic cells
   and may be a possible target of immunotherapy in the head and neck
   carcinomas ([136]Friedrich et al., 2010). Martina et al. showed that
   tamoxifen combined with an IGF-1R inhibitors produced a enhanced
   response in HER2 positive breast cancer ([137]McDermott et al., 2017).
   BMS-536924 is an ATP-competitive IGF-1R inhibitor, and the sensitivity
   to it was enhanced in the ATM-MT group, suggesting that ATM mutation
   has a synergistic effect with the IGF-1R inhibitor. We inferred that
   ATM mutation is likely to improve the prognosis of bladder cancer
   patients by downregulating the activity of the insulin-like growth
   factor receptor pathway and ATM-MT bladder cancer patients may benefit
   from the combined application of ICIs and IGF-1R inhibitors, which
   opens a new approach for clinical treatment ([138]Figure 7). In
   addition, the sensitivity to VEGFR inhibitor and JAK inhibitor were
   enhanced in the ATM-MT group, which consistent with the results of
   GSEA, samely suggesting the synergistic effect of ATM mutation with
   VEGFR inhibitor and JAK inhibitor. However, our attempt to explain the
   mechanism of ATM mutation promoting immunotherapy for bladder cancer
   was based on a hypothetical model, which need to be further verified
   ([139]Figure 7).

FIGURE 7.

   [140]FIGURE 7
   [141]Open in a new tab

   Mechanism of ATM mutation enhancing the efficacy of immune checkpoint
   inhibitors in bladder cancer based on a hypothetical model.

   Additionally, in the GSEA results, we also found that the immune
   inflammatory response to antigens and macrophage activation pathways
   were significantly enriched in the ATM-MT group, which verified that
   ATM mutation can promote the antitumor immune response. The IFN
   signaling pathway was also significantly enriched in the ATM-MT group.
   A number of studies have demonstrated the antiproliferative effect of
   IFNγ on bladder cancer cells, and inhibition of IFNγ leads to increased
   proliferation and invasion ([142]Li et al., 2002; [143]Riemensberger et
   al., 2002). In addition, the significant downregulation of
   vasculogenesis pathways in the ATM-MT group suggested that ATM
   mutations may affect blood perfusion of bladder cancer tumor cells. All
   these results indicate that ATM mutations affect the immune
   microenvironment and thus affect the efficacy of ICIs. Moreover, as a
   core component of the DNA repair system, ATM plays an important role in
   DNA repair. We found that patients with ATM alteration in DDR-related
   pathways showed comparably high mutation counts than ATM intact
   patients, suggesting that ATM mutation patients may show a higher
   immunogenicity and better efficacy of ICI therapy.

   However, there are some limitations to our research. First, although we
   included two independent cohort studies with clinical data and
   mutational data, the immunotherapy cohort was relatively small in size,
   and the analysis results may have statistical errors. Second, while we
   verified that ATM-MT bladder cancer cells are likely to downregulate
   the activity of the insulin-like growth factor receptor pathway by
   upregulating AGE1 gene expression, the functional pathway analysis of
   ATM mutations needs to be further verified in vitro and in vivo. Third,
   we propose that ATM-MT bladder cancer patients may benefit from the
   combined application of ICIs and IGF-1R inhibitors, which opens a new
   approach for clinical treatment, but further experimental confirmation
   is still needed. Finally, this study focused on the prognostic value
   rather than the predictive value of ATM mutations in patients with
   bladder cancer who received ICI therapy.

Conclusion

   In conclusion, we found that ATM-MT bladder cancer patients showed
   greater benefit from ICI therapy than ATM-WT patients in the
   immunotherapy cohort and TCGA-Bladder cancer cohort. We further found
   that the ATM-MT group showed a higher mutation load and immunogenicity
   in both cohorts. By comparing the ATM-MT group immune-related gene and
   immune cell-related gene expression with that of the ATM-WT group, we
   found that ATM-MT bladder cancer cells might promote the infiltration
   of cytotoxic cells and Th17 cells into tumor tissues by secreting
   increased amounts of the chemokine CCL5 and activating dendritic cells
   to enhance ICI therapy. Additionally, we found that ATM-MT bladder
   cancer cells were likely to downregulate the activity of the
   insulin-like growth factor receptor pathway by upregulating AGE1 gene
   expression, thus promoting the efficacy of ICIs. Through functional
   enrichment analysis, we also found that the immune inflammatory
   response to antigens, the regulation of the IFN signaling pathway and
   macrophage activation pathways were significantly enriched in ATM-MT
   bladder cancer patients, while vasculogenesis and insulin-like growth
   factor receptor signaling pathways were significantly downregulated. We
   also demonstrated that ATM mutation patients showed comparably high
   mutation counts than ATM intact patients in DDR-related pathways, thus
   improving the efficacy of ICI therapy. Finally, by analyzing the GDSC
   database, we found that ATM mutations resulted in increased bladder
   cancer sensitivity to 29 drugs, including cisplatin, the first-line
   treatment for bladder cancer, and BMS-536924, an IGF-1R inhibitor.

Data Availability Statement

   We collected an immunotherapy cohort with clinical and mutational data
   (reported by [144]Samstein et al., 2019). In addition, we also
   collected The Cancer Genome Atlas (TCGA)-Bladder cancer cohort with
   somatic mutation and survival data from the Genomic Data Commons
   ([145]https://portal.gdc.cancer.gov/). Then, we used cBioportal to
   download the survival data [overall survival (OS), disease-free
   survival (DFS), and progression-free survival (PFS)] of the
   TCGA-Bladder cancer cohort.

Author Contributions

   RY analyzed the data and wrote the manuscript. AL was involved in
   performing computational coding. MC, AX, PL, and JZ collected the data
   and designed the study. JZ and PL were involved in the manuscript
   editing and the supervision of the entire work. All authors have
   approved the final manuscript.

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Acknowledgments