Atrx deletion impairs CGAS/STING signaling and increases sarcoma response to radiation and oncolytic herpesvirus

ATRX is one of the most frequently altered genes in solid tumors, and mutation is especially frequent in soft tissue sarcomas. However, the role of ATRX in tumor development and response to cancer therapies remains poorly understood. Here, we developed a primary mouse model of soft tissue sarcoma and showed that Atrx-deleted tumors were more sensitive to radiation therapy and to oncolytic herpesvirus. In the absence of Atrx, irradiated sarcomas had increased persistent DNA damage, telomere dysfunction, and mitotic catastrophe. Our work also showed that Atrx deletion resulted in downregulation of the CGAS/STING signaling pathway at multiple points in the pathway and was not driven by mutations or transcriptional downregulation of the CGAS/STING pathway components. We found that both human and mouse models of Atrx-deleted sarcoma had a reduced adaptive immune response, markedly impaired CGAS/STING signaling, and increased sensitivity to TVEC, an oncolytic herpesvirus that is currently FDA approved for the treatment of aggressive melanomas. Translation of these results to patients with ATRX-mutant cancers could enable genomically guided cancer therapy approaches to improve patient outcomes.

Scientific, 155379). Cells were fixed with 4% paraformaldehyde, blocked and washed using the MAXpack Immunostaining Media Kit (Active Motif), then incubated with primary antibody as listed in Supplementary Table 1 overnight at 4°C. Secondary antibody (Supplementary Table 1) was incubated for 1 hour at room temperature. Cells were stained with DAPI and then Prolong Diamond Antifade Mountant. Imaging was performed on a Leica SP5 Inverted Confocal Microscope, with consistent settings to allow for comparison. For each experiment a minimum of 50 cells were imaged then analyzed by a blinded researcher using Leica Suite software (Leica Microsystems). Data from these experiments represent the average of at least three independent experimental technical replicates.

TUNEL Assay
Frozen tissue sections were allowed to thaw at room temperature for 5 minutes, then sections were fixed in cold 4% paraformaldehyde for 10 minutes. Samples were washed twice with ice cold PBS, then incubated with 0.1% Triton X-100 and 0.1% sodium citrate in water for five minutes. Samples were washed 3 times for 5 minutes each in PBS. Then in situ Cell Death Detection Kit, AP (Roche, #11684809910) was used. 100 microliter TUNEL reaction mixture from this kit was added to each sample and incubated for 1 hour at 37°C. Each sample was washed three times with PBS, mounted, and then imaged with a DFC340FX fluorescence microscope using Leica Software.

Mitotic Dysfunction Assays
Micronuclei were defined as regions of DAPI staining located in close proximity to a nucleus with a size between approximately 1/3 and 1/64 the size of a nucleus, which could be determined to be distinct from the nucleus. Chromosome bridges were defined as occurrences of a single thin clear line connecting two otherwise separate nuclei. For telomere dysfunction induced foci (TIF), which are 53BP1 foci that co-localized with telomeres, co-localization was defined by visual inspection in multiple channels and were counted if there was an instance of a 53BP1 focus that was contiguous or overlapping with a telomere focus. Only a single TIF event was called per 53BP1 focus, even if multiple telomere foci were present. Mitotic catastrophe was identified by the presence of multiple micronuclei that were associated with a single nucleus (1-3).

ImmunoFISH
Telomere ImmunoFISH was performed as described in (4)  clinically approved TVEC in that it contains mouse GM-CSF instead of the human GM-CSF) that had a concentration of 1X10^8 PFU/ml was obtained and diluted 1:10 in DMEM. ATRX isogenic 143B human sarcoma cells were plated at 1000 cells per well in a 96 well plate, then treated with 1:2 serial dilutions of the prepared solution. Cells were incubated in a cell culture incubator with 5% CO2 at 37°C for 72 hours before cells were washed twice with PBS then measured via CellTiter-Glo (Promega). IC50 experiments were repeated three times for each cell line shown.

Genotyping
Mice were genotyped using tails collected from mouse pups. Tail genomic DNA was extracted using a KAPA mouse genotyping Kit (KAPA Biosystems KK7352), and PCR was performed using primers as described in Supplementary Table 1 The membranes were washed three times in TBS-T and imaged using an Odyssey CLx (Li-Cor Biosciences). Image analysis for normalization and quantification was performed using Image Studio (version 5.2, Li-Cor Biosciences, P/N 9140-500).

rt-PCR
Cells were lysed with TRIzol reagent (Thermo Fisher, 15596026). RNA was isolated from samples using a Direct-zol RNA MiniPrep kit (Zymo Research, R2051). RNA samples were reverse transcribed to cDNA using an iScript Advanced cDNA Synthesis Kit (Bio-Rad, 1725038). TaqMan probes from Thermo Fisher were used for RT-PCR: mouse probes were IfnB1 (Mm00439552), Ifit3 (Mm00445235), and Rsad2 (Mm00491265). Human probes were IFNB1 (Hs01077958) and ACTB(Hs01060665). Taqman fast advanced master mix was used for the qPCR assay (ThermoFischer Scientific 4444556). Plates were run on a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher), and data were analyzed using the comparative CT method to generate expression fold-change values. Gapdh or Tbp expression was used as an internal control for RNA concentration across samples. Every sample was run in triplicate and results were averaged for each assay.

Mouse Whole Exome Sequencing
Mouse whole exome sequencing was performed by Novogene using their standard protocol for sample preparation and data analysis. Each mouse tumor was submitted along with a portion of normal muscle from the same mouse, then WES was performed by Novogene. Briefly The genomic DNA was randomly sheared into short fragments with the size of 180-280 bp. The obtained fragments were end repaired, A-tailed, and further ligated with Illumina adapters. The fragments with adapters were PCR amplified, size selected, and purified. Hybridization capture of libraries was proceeded according to the following procedures. Briefly, the prepped libraries were hybridized in the buffer with biotin-labeled probes, and magnetic beads with streptavidin were used to capture the exons of genes. Subsequently, non-hybridized fragments were washed out and probes were digested. The captured libraries were enriched by PCR amplification. The original fluorescence images obtained from high throughput sequencing platforms were transformed to short reads by base calling. These short reads (Raw data) are recorded in FASTQ format, which contains sequence information (reads) and corresponding sequencing quality information.For quality control, read pairs were discarded if either one read contained adapter contamination, if more than 10% of bases in either read were uncertain, or if the the proportion of low quality bases was over 50% in one read. Q30 was required to above 80% for the samples.
Burrows-Wheeler Aligner (BWA) was utilized to map the paired-end clean reads to the mouse reference genome (mm10). After sorting with Samtools and marking duplicates with Picard, the results of read alignment was finally stored in the format of BAM. The coverage and depth was then computed based on the final BAM file. Somatic SNP/InDel detection and filtering was performed using GATK, as well as MuTect/Strelka. Somatic CNV detection was performed using Control-FREEC, and annotation was performed using ANNOVAR.

RNA-seq Bioinformatic Analysis
Raw counts from Novogene's processing pipeline were obtained from the company. For cell line RNA seq analysis, genes were first filtered if they did not have at least 10 reads in a single sample from each of the 3 replicates. Normalization and differential expression were carried out using the DESeq2 Bioconductor package with the R statistical programming environment. In each analysis, 'replicate' was used as a cofactor in the model. The false discovery rate was calculated to control for multiple hypothesis testing. Gene set enrichment analysis (5) was performed to identify gene ontology terms and pathways associated with altered gene expression for each of the comparisons performed.

Cancer Genomic Database Analysis
For determination of the frequency of ATRX mutation in human cancers, the TCGA database was queried via cBioportal. For overall prevalence of ATRX in human cancer, "Curated set of nonredundant studies" was selected, and was queried for genomic profile of mutations and copy number alterations in ATRX. For data on frequency and position of ATRX mutations in soft tissue sarcoma, "Sarcoma (TCGA, PanCancer Atlas)" study was selected, UPS, LMS and MFS sarcoma subtypes were selected, then were queried for genomic profile of mutations and copy number alterations in ATRX. Disease free survival data was obtained via CBioportal. Whole genome sequencing mutation and phenotype data of undifferentiated pleomorphic sarcomas was accessed from (2). Samples harboring frameshift or missense mutations in ATRX exons, as well as samples with copy number deletion, fusions or inversions were classified as "ATRX altered".

cGAMP ELISA Assay
The ELISA assay was performed for cGAMP according to the protocol provided (Cayman Chemical no. 501700). Briefly, 96-well plates were washed, ELISA standards and samples were added, and 2'3'-cGAMP-HRP Tracer and then 2'3'-cGAMP Polyclonal Antiserum were added to the wells. Plates were incubated overnight at 4C, HRP Stop Solution was added, and plate was read at 450 nm (Molecular Devices SpectraMax i3).

Cell Growth Assay
Mouse sarcoma primary isogenic cell lines were cultured in 96-well plates. After 60 hours, plates were equilibrated at room temperature for 30 minutes before addition of 100μl Cell Titer Glo Reagent (Promega CellTiter-Glo Luminescent Cell Viability Assay, 77573).
Reagents were mixed on a shaker for 2 minutes and incubated for 10 minutes at room temperature. Relative fluorescence was recorded using a luminometer (Molecular Devices SpectraMax i3).          Table showing most frequent recurrent mutations (each row is a different mutation) in sarcomas from P7 KPA and P7 KP mice (each column is a different mouse sarcoma). Mutations were identified via whole exome sequencing as described in the methods section. Specific mutation types are color coded as shown in the legend