Molecular signatures of BRCAness analysis identifies PARP inhibitor Niraparib as a novel targeted therapeutic strategy for soft tissue Sarcomas

Abstract

Background: Patients with advanced soft tissue sarcomas (STS) have a dismal prognosis with few effective therapeutic options. A defect in the homologous recombination repair (HRR) pathway can accumulate DNA repair errors and gene mutations, which can lead to tumorigenesis. BRCAness describes tumors with an HRR deficiency (HRD) in the absence of a germline BRCA1/2 mutation. However, the characteristics of BRCAness in STS remain largely unknown. Thus, this study aimed to explore the genomic and molecular landscape of BRCAness using whole exome sequencing (WES) in STS, aiming to find a potential target for STS treatment. Methods: WES was performed in 22 STS samples from the First Affiliated Hospital of Sun Yat-sen University to reveal the possible genomic and molecular characteristics. The characteristics were then validated using data of 224 STS samples from The Cancer Genome Atlas (TCGA) database and in vitro data. The analysis of the potential biomarker for BRCAness was performed. Targeted drug susceptibility and combination therapy screening of chemotherapeutics for STS were evaluated in STS cell lines, cell-line-derived xenografts (CDX), and patient-derived xenografts (PDX). Results: Compared with 30 somatic mutation signatures of cancers, high cosine-similarity (0.75) was identified for HRD signatures in the 22 STS samples using nonnegative matrix factorization. Single nucleotide polymorphism indicated a low mutation rate of BRCA1/2 in the 22 STS samples (11.76% and 5.88%, respectively). However, copy number variation analyses demonstrated widespread chromosomal instability; furthermore, 54.55% of STS samples (12/22) carried BRCAness traits. Subsequently, similar genomic and molecular characteristics were also detected in the 224 STS samples from TCGA and in vitro. Poly (ADP-ribose) polymerases (PARP)-1 could be a promising reflection of HRD and therapeutic response. Furthermore, the level of PAR formation was found to be correlated with PARP-1. Subsequently, STS cell lines were determined to be sensitive to PARP inhibitor (PARPi), niraparib. Moreover, based on the screening test of the five common PARPis and combination test among doxorubicin, ifosfamide, dacarbazine, and temozolomide (TMZ), niraparib and TMZ were the most synergistic in STS cell lines. The synergistic effect and safety of niraparib and TMZ combination were also shown in CDX and PDX. Conclusions: BRCAness might be the common genomic and molecular characteristics of majority of STS cases. PARP-1 and PAR could be potential proper and feasible theranostic biomarkers for assessing HRD in patients. STSs were sensitive to PARPi. Moreover, the combination of niraparib and TMZ showed synergistic effect. Niraparib and TMZ could be a promising targeted therapeutic strategy for patients with STS.

Keywords: BRCAness; Genomic characteristic; PARP inhibitor; PDX models; Soft tissue sarcomas.

Figure 1

Genomic and molecular characteristics of BRCAness were observed in 22 STS samples. A. Flow chart of genomic instability analysis from whole exome sequencing (WES) of 22 STS samples from the First Affiliated Hospital of Sun Yat-sen University. B. Four signatures out of 30 somatic mutation signatures of cancers were identified in 22 STS samples. The four most similar signatures were signature 1 (cosine-similarity: 0.94, etiology: spontaneous deamination of 5-methylcytosine), signature 3 (cosine-similarity: 0.86, etiology: defects in DNA-DSB repair by homologous recombination), signature 4 (cosine-similarity: 0.76, etiology: smoking), and signature 7 (cosine-similarity: 0.97, etiology: UV exposure). C. Distribution of BRCAness-associated gene (BAP1, FANCC, BRIP1, CHEK2, WRN, ATM, BRCA1, ATR, CDK12, PALB2, RAD51C, BLM, BRCA2, FANCA, FANCE, NBN, RAD51B, RAD51D) and the significant mutated gene (LOR, UBA52, MARCH7, TP53, LTBP3, PSMD11, CMTM8, ELP2, IFNGR2, LMTK2, MMP19, P2RX2, RB1, LRRIQ1, MSH3, NUMBL, PTEN) single-nucleotide variants (SNVs) with predicted pathogenic effects and indels across 17 patients with STS. Blue, homozygous deletion; red, amplification; purple, truncation; green, substitution or indel; gray, no alteration. D. Copy-number profile of genomes from 22 STS samples. Light red, gain (1 ≤ copy-number variation [CNV] < 2); dark red, high copy-number gain (CNV > 2); wathet, heterozygous deletion (-2 < CNV < -1); navy blue, homozygous deletion (CNV < 2). E. The number of CNV meeting the specific criterion of a BRCAness characteristic for each STS tumor (threshold line in red). F. Homologous recombination deficiency (HRD) score for each STS tumor (HRD score > 35, threshold line in red). G. Circos plot demonstrating the integral mutation in chromosomes, SNVs, CNVs, and gene rearrangements from 22 samples (from outer to inner side); italic font text denotes BRCAness-associated genes.

Figure 2

Genomic and molecular characteristics of BRCAness validated using The Cancer Genome Atlas (TCGA) database. A. Four signatures out of 30 somatic mutation signatures of cancers were identified in 224 STS samples from TCGA. The four most similar signatures in the TCGA samples were signature 1 (cosine-similarity: 0.92, etiology: spontaneous deamination of 5-methylcytosine), signature 3 (cosine-similarity: 0.87, etiology: defects in DNA-DSB repair by homologous repair), signature 7 (cosine-similarity: 0.97, etiology: UV exposure), and signature 24 (cosine-similarity: 0.57, etiology: exposure to aflatoxin). B. Distribution of BRCAness-associated gene (FANCD2, ATM, BLM, BRCA2, BRIP1, NBN, RAD51B, WRN, FANCC, FANCE, BAP1, CHEK1, CDK12, CHEK2, PALB2, FANCA, FANCF, RAD51C, RAD51D, BRCA1) and significant mutated genes single-nucleotide variants with predicted pathogenic effects and indels across 224 STS samples. Blue, homozygous deletion; red, amplification; purple, truncation; green, substitution or indel; gray, no alteration. C. Copy-number profile of 224 STS genomes. Light red, gain (1 ≤ copy number variation [CNV] < 2); dark red, high copy-number gain (CNV > 2); wathet, heterozygous deletion (-2 < CNV < -1); navy blue, homozygous deletion (CNV < 2). D. The number of CNVs meeting the specific criterion of a BRCAness characteristic for each STS tumor. The threshold for considering a tumor to be BRCA deficient was set to 15 MB CNA (red dashed line). E. Receiver operating characteristic (ROC) curve and Youden index analysis were performed to determine the optimal cut-off point for homologous recombination deficiency (HRD) scores in 224 samples from TCGA. According to the ROC curve, the maximal Youden index (sensitivity + specificity - 1 = 0.171) corresponded to HRD scores 34.50; therefore, HRD score 35 was defined to be threshold of BRCAness and could predict the therapeutic response and prognosis of 224 samples from TCGA. The HRD score for each STS tumor (HRD score > 35, threshold line in red). F. Comparison of the overall survival rate between the high HRD and low HRD score groups examined using the log-rank test (cutoff: HRD score 35, p = 0.0257).

Figure 3

Impaired DSB repair ability in STS cell lines. A. Fibroblasts and HT-1080 cells were treated with 20 µM etoposide or DMSO (vehicle group) for 2, 4, 8, 16, 24 and 36 h. qRT-PCR detected that among these timepoints, the relative expression of RAD51 (a marker of homologous recombination repair) was highest at 4 h. B. Fibroblasts and HT-1080 cells were treated with 10 µM MK4827 or DMSO (vehicle group) for 2, 4, 8, 16, 24 and 36 h. Again, qRT-PCR detected that the relative expression of RAD51 was highest at 4 h. C. When cells were treated with 20 µM etoposide (a topoisomerase II inhibitor) or DMSO (vehicle group) for 4 h, western blots (WB) showed elevated expression of γH2AX, a marker of double-strand breaks (DSBs), in the etoposide group. WB indicated that no significant DSBs developed in the vehicle group, wherein the expression of RAD51 was negligible. However, RAD51 expression was lower in treated STS cells than in treated fibroblasts. D. WB demonstrated similar results when STS cell lines were treated with MK4827. E. When cells were treated with the same etoposide concentrations described above, an obvious increased green focus of RAD51 was observed in the treated fibroblasts using immunofluorescence (IF) staining. F. IF findings were similar when cells were treated with MK4827. G. After etoposide treatment, the RAD51 foci in fibroblasts were more than those in STS cell lines. H. After MK4827 treatment, the RAD51 foci in fibroblasts were more than those in STS cell lines. HT-1080, fibrosarcoma; SW982, synovial sarcoma; RD, rhabdomyosarcoma; SW872, liposarcoma; SK-LMS-1, leiomyosarcoma; VA-ES-BJ, epithelioid sarcoma; MK4827, niraparib. Scale bar, 5 µm.

Figure 4

PARP-1 expression is correlated with HRD and the therapeutic response. A. According to the ROC curve, the maximal Youden index (sensitivity + specificity - 1 = 0.168) corresponded to PARP-1 RPKM 3694.49815; therefore, PARP-1 RPKM 3695 was defined to be the cut-off for the 224 samples. The overall survival rate was compared between the high (n = 55) and low PARP-1 expression (n = 169) groups for the 224 STS patients from TCGA using the log-rank test (cutoff: PARP-1 RPKM 3695, p = 0.009). Western blots (WB) indicated higher expression of PARP-1 in six STS cell lines than in fibroblasts. B. Pearson correlation analysis showed that the expression level of PARP-1 (RPKM) had a significantly positive correlation with HRD scores in 224 STS cases from the TCGA database (r = 0.4593, p < 0.0001). C. Western blots (WB) indicated higher expression of PARP-1 in six STS cell lines than in fibroblasts. D. WB showed higher expression of PARP-1 in STS tumor tissue than in paired adjacent normal tissue. Case 1, spindle cell sarcoma; case 2, rhabdomyosarcoma; case 3, undifferentiated pleomorphic sarcoma; N, normal tissue; T, tumor tissue. E. IC₅₀ (µM) of cell lines for three time points (24 h, 48 h, 72 h), denoted as mean ± standard deviation (SD). F. Spearman correlation analysis showed that the expression level of PARP-1 (gray value of the protein band on WB) had a moderate negative correlation with the PARPi IC₅₀ of cell lines (ρ = -0.608, p = 0.003). G. WB indicated similar expression patterns between PARP-1 and PAR protein in six STS cell lines and fibroblasts. H. Bar chart demonstrating that after 10 µM MK4827 treatment for 24 h, cells arrested in S and the number of cells in G2/M increased. I. Plot showing that after 10 µM MK4827 treatment for 48 hours, apoptosis increased. HT-1080, fibrosarcoma; SW982, synovial sarcoma; RD, rhabdomyosarcoma; SW872, liposarcoma; SK-LMS-1, leiomyosarcoma; VA-ES-BJ, epithelioid sarcoma; MK4827, niraparib.

Figure 5

The investigation for combination effect of MK4827 and temozolomide (TMZ) against STS cell lines in vitro. A. A normalized isobologram for the drug combination was constructed to demonstrate the treatment combination index (CI) of MK4827 and four chemotherapeutic agents for all six cell lines, which follows the median effect principle. CIs < 1, = 1, and > 1 indicate synergistic, additive, and antagonistic effects, respectively. The normalized isobologram showed the synergistic effect of the combination of MK4827 and TMZ against STS cell lines, with CIs < 0.5 for most combinations tested. B. Combination therapy had a superior inhibitory effect on colony formation than each reagent alone (treatment for 14 days). C. The bar charts reveal the synergistic effects of this combination in inhibiting proliferation, as assessed by colony formation assays. D. Measurement of PARP-1 activity during apoptosis indicated that compared with each regimen alone, the MK4827 and TMZ combination augmented DNA damage through a synergistic effect. E. Western blots (WB) showed higher expression of γH2AX and RAD51 in HT-1080 cell lines treated with combination therapy than in those treated with MK4827 or TMZ alone. F. WB demonstrated higher expression of γH2AX and RAD51 in SK-LMS-1 cell lines treated with combination therapy than in those treated with MK4827 or TMZ alone. G. MTT results showing that HT-1080 (left) and SK-LMS-1 (right) were less sensitive to niraparib under PARP-1 knockdown; H. MTT results showing that HT-1080 (left) and SK-LMS-1 (right) were more sensitive to niraparib under PARP-1 overexpression. CFN: colony formation number; HT-1080, fibrosarcoma; SW982, synovial sarcoma; RD, rhabdomyosarcoma; SW872, liposarcoma; SK-LMS-1, leiomyosarcoma; VA-ES-BJ, epithelioid sarcoma; MK: MK4827 (niraparib); ADM, doxorubicin; IFO, ifosfamide; DTIC, dacarbazine; PARPi: PARP inhibitor; OE: overexpression.

Figure 6

The combination of MK4827 and temozolomide showed synergistic effects against STS in vivo. A. A subcutaneous tumor model was utilized to assess the effect of the MK4827 and TMZ combination on HT-1080 cells in vivo. The tumors were harvested 4 weeks after injection. The tumor volume was measured by electronic calipers twice a week, and the volume was calculated according to the equation V = L × W²/2. The growth curve of HT-1080 CDX tumors was shown (left). A subcutaneous tumor model was also used to assess the effect of combination treatment on SK-LMS-1 cells in vivo. The tumors were collected at 3 weeks after injection, the growth curve of SK-LMS-1 CDX tumors was shown (middle). A patient-derived xenograft model built using a spindle cell sarcoma was utilized to confirm the synergistic effects. The tumors were collected 5 weeks after surgery. The growth curve of spindle cell sarcoma PDX tumors was shown (right). B. Bar charts demonstrated the tumor volumes in the four groups before harvest (HT-1080, n = 7 per group, left; SK-LMS-1, n = 7 per group, middle; PDX, n = 7 per group, right). C. The chart displayed the tumor weights before harvest (HT-1080, left; SK-LMS-1, middle; PDX, right). D. Representative images of immunohistochemical (IHC) staining against Ki67, γH2AX, and RAD51 in PDX tumor specimens. Scale bar, 100 µm (Positive: Vector Brown). E. Plot charts showing the IHC staining scores for Ki67, γH2AX, and RAD51 in PDX tumors. Significant differences were indicated as *: p < 0.05, **: p < 0.01, ***: p < 0.001, and ns: no significance. HT-1080, fibrosarcoma; SK-LMS-1, leiomyosarcoma; MK4827, niraparib.

Figure 7

Expression of HR-associated proteins, including PARP-1, γH2AX and RAD51, significantly correlated with the prognosis of 123 patients with STS. A. Representative images of immunohistochemistry (IHC) staining (positive and negative expression) for PARP-1, γH2AX, RAD51, and RPA32 in samples from patients with fibrosarcoma (FS), synovium sarcoma (SS), leiomyosarcoma (LMS), liposarcoma (LS), and rhabdomyosarcoma (RMS), respectively. (400×, positive: brown). B. Bar chart showing the number of patients with positive and negative staining for PARP-1, γH2AX, RAD51, and RPA32 IHC. C-F. Log-rank tests showed that the patients in the high RAD51 expression group had better event-free survival (EFS; events were defined as local recurrence, distant metastasis, lung metastasis, and death; p < 0.0001), while patients in the high PARP-1 and γH2AX expression groups had worse EFS (p < 0.001); RPA32 expression was not associated with the prognosis of the STS patients (p = 0.9584). The IHC scores for staining extent were evaluated as follows: 0: 0% of cells showing staining, 1: < 5% of cells showing staining, 2: 5- 50% of cells showing staining, and 3: over 50% of cells showing staining. Staining intensity was scored as 0: negative, 1: weak, 2: intermediate, and 3: strong. The final score was determined as the sum of both the extent and intensity scores. Based on the final score, patients were divided into low (scores 0 and 2) and high (3- 6) expression groups. EFS: event-free survival; FS: fibrosarcoma; SS: synovial sarcoma; LMS: leiomyosarcoma; LS: liposarcoma; RMS: rhabdomyosarcoma.