Genome-wide CRISPR/Cas9 library screening identified ATM signaling network genes as critical drivers for resistance to ATR inhibition in soft-tissue sarcomas: synthetic lethality and therapeutic implications.

Soft-tissue sarcoma (STS) are a heterogeneous group of rare tumors with different biological behavior that are fatal in more than 40% of cases, due to their metastatic evolution and inadequate treatment options. ATR inhibition already showed an activity, even if modest, in broad pre-clinical models of STS. By using genome-wide CRISPR/Cas9 library screening, we identified ATM signaling network genes as critical drivers for resistance to the specific ATR inhibitor AZD6738. The role of such genes in resistance to AZD6738 was confirmed by using CRISPR/Cas9 knockout models. More strikingly, the ATM inhibitor AZD0156 works synergistically with AZD6738 in vitro and abolishes STS growth in vivo in our models of most frequent histotypes (such as dedifferentiated liposarcoma, leiomyosarcoma, and undifferentiated pleomorphic sarcoma among others). Moreover, the combination of AZD6738 and AZD0156 induced significantly higher levels of DNA damage than either drug used as single agent alone. In summary, our results demonstrate that targeting ATM is an effective approach to overcome resistance to ATR inhibition in different STS subtypes, including the most frequent histologies.

CHK1 inhibition in soft-tissue sarcomas: biological and clinical implications.

Background: Inhibition of ChK1 appears as a promising strategy for selectively potentiate the efficacy of chemotherapeutic agents in G1 checkpoint-defective tumor cells such as those that lack functional p53 protein. The p53 pathway is commonly dysregulated in soft-tissue sarcomas (STS) through mutations affecting TP53 or MDM2 amplification. GDC-0575 is a selective ATP-competitive inhibitor of CHK1. Methods: We have performed a systematic screening of a panel of 10 STS cell lines by combining the treatment of GDC-0575 with chemotherapy. Cell proliferation, cell death and cell cycle analysis were evaluated with high throughput assay. In vivo experiments were carried out by using TP53-mutated and TP53 wild-type patient-derived xenograft models of STS. Clinical activity of GDC-0575 combined with chemotherapy in patients with TP53-mutated and TP53 wild-type STS was also assessed. Results: We found that GDC-0575 abrogated DNA damage-induced S and G2-M checkpoints, exacerbated DNA double-strand breaks and induced apoptosis in STS cells. Moreover, we observed a synergistic or additive effect of GDC-0575 together with gemcitabine in vitro and in vivo in TP53-proficient but not TP53-deficient sarcoma models. In a phase I study of GDC-0575 in combination with gemcitabine, two patients with metastatic TP53-mutated STS had an exceptional, long-lasting response despite administration of a very low dose of gemcitabine whereas one patient with wild-type TP53 STS had no clinical benefit. Genetic profiling of samples from a patient displaying secondary resistance after 1 year showed loss of one preexisting loss-of-function mutation in the helical domain of DNA2. Conclusion: We provide the first preclinical and clinical evidence that potentiation of chemotherapy activity with a CHK1 inhibitor is a promising strategy in TP53-deficient STS and deserves further investigation in the phase II setting.

BRCA1 haplotype and clinical benefit of trabectedin in soft-tissue sarcoma patients.

BACKGROUND: This study aimed to determine whether the BRCA1 haplotype was associated with trabectedin efficacy in soft-tissue sarcoma (STS) patients. METHODS: We analysed BRCA1 single-nucleotide polymorphisms (SNPs) in tumour specimens from 135 advanced STS patients enrolled in published phase 2 trials or in a compassionate-use programme of trabectedin. Forty-four advanced STS patients treated with doxorubicin and 85 patients with localised STS served as controls. The 6-month nonprogression rate and overall survival (OS) were analysed according to BRCA1 haplotype using log-rank tests. RESULTS: A favourable BRCA1 haplotype (presence of at least one AAAG allele) was significantly associated with an improved 6-month nonprogression rate. It was the only variable significantly associated with OS. No correlations were found between outcomes for patients with localised or advanced STS treated with doxorubicin. CONCLUSIONS: The BRCA1 haplotype represents a potential DNA repair biomarker that can be used for the prediction of response to trabectedin in STS patients.

Identification of SNPs associated with response of breast cancer patients to neoadjuvant chemotherapy in the EORTC-10994 randomized phase III trial.

Using cell line panels we identified associations between single-nucleotide polymorphisms (SNPs) and chemosensitivity. To validate these findings in clinics, we genotyped a subset of patients included in a neoadjuvant breast cancer trial to explore the relationship between genotypes and clinical outcome according to treatment received and p53 status. We genotyped 384 selected SNPs in the germline DNA extracted from formalin-fixed paraffin-embedded non-invaded lymph nodes of 243 patients. The polymorphisms of five selected genes were first studied, and then all 384 SNPs were considered. Correction for multiple testing was applied. CYP1B1 polymorphism was significantly associated with pathological complete response (pCR) in patients who had received DNA-damaging agents. MDM2, MDM4 and TP53BP1 polymorphisms were significantly associated with pCR in patients harboring a p53-positive tumor. In the complete SNP panel, there was a significant association between overall survival (OS) and a SNP of ADH1C, R272Q (P=0.0023). By multivariate analysis, only ADH1C genotype and p53 status were significantly associated with OS.

The association between the T309G polymorphism of the MDM2 gene and sensitivity to anticancer drug is dependent on the p53 mutational status in cellular models.

BACKGROUND: We investigated, in the panel of 60 human tumour cell lines of the National Cancer Institute (NCI-60), whether the R72P polymorphism of TP53 and the T309G polymorphism of MDM2 were associated to the in vitro cytotoxicity of anticancer agents, extracted from the NCI database. For validation, the same study was performed independently on a second panel of tumour cell lines, JFCR-45. METHODS: Both SNPs were identified in cell DNA using PCR-RFLP techniques confirmed by direct sequencing and by pyrosequencing. For the analysis of the results, the mutational status of p53 was taken into account. RESULTS: In the NCI-60 panel, the TP53 rare-allele frequency was 32% and the MDM2 rare-allele frequency 39%. The MDM2 alleles were distributed according to Hardy-Weinberg equilibrium whereas this was only found, for the TP53 alleles, in p53 non-mutated cell lines. Comparable results were obtained in the JFCR-45 validation set. The TP53 SNP had low impact on anticancer drug cytotoxicity in either panel. In contrast, the MDM2 gene polymorphism had a major impact on anticancer drug cytotoxicity, essentially in p53 non-mutated cell lines. Presence of the rare allele was associated to significantly higher MDM2 protein expression and to increased sensitivity to DNA-interfering drugs. In the JFCR-45 panel, a similar effect of the MDM2 gene polymorphism was observed, but was less dependent on the p53 mutational status. CONCLUSIONS: We hypothesised that cell lines harbouring the MDM2 G allele presented a lower availability of p53 for DNA repair, translating into higher sensitivity to DNA-damaging agents.

Down-regulation of bcr-abl and bcl-x(L) expression in a leukemia cell line and its doxorubicin-resistant variant by topoisomerase II inhibitors.

K562 cells are usually resistant to apoptosis induction, probably because of the expression of bcr-abl, the hybrid gene characteristic of the Philadelphia chromosome (t 9;22). However, we have previously shown that amsacrine and, to a lesser extent, doxorubicin, could induce apoptosis in the doxorubicin-resistant variant of this cell line. In order to elucidate the role of bcr-abl in triggering apoptosis, we investigated the effect of the topoisomerase II inhibitors doxorubicin, amsacrine, and etoposide on the expression of several genes that may be related to apoptosis induction in both cell lines. This was done using a technique of reverse transcription-polymerase chain reaction coupled with HPLC of the amplified fragments to obtain semiquantitative evaluations. We showed that amsacrine, at pharmacologically relevant concentrations, was able to decrease the expression of bcr-abl down to 20% of the basal value in the doxorubicin-resistant variant only, whereas doxorubicin and etoposide were unable to do so. No effect of these drugs was seen on the expression of the normal abl gene. In addition, there was an effect of amsacrine on the expression of bcl-x(L) in the resistant cell line only, but at concentrations higher than the IC(50) of this drug. Our results emphasize the role of bcr-abl in protecting cells from apoptosis and the possible involvement of specific topoisomerase II inhibitors in overcoming resistance to apoptosis.