bmn-673 and Osteosarcoma

Osteosarcomas are highly aggressive bone tumors that mainly occur in the metaphyses of long bones in children and adolescents. Genetically, they are characterized by complex structural and numerical aberrations with large intra- and interindividual variations which hamper the identification of the initiating and driving events. Sequencing and copy number analyses in a study of 123 pretherapeutic osteosarcoma samples identified mutations in 14 genes as the potential main drivers. Although almost half of all osteosarcomas could be attributed to mutations in TP53 and RB1, no single driver gene could be found that was clearly responsible for the majority of tumors. There were also no unequivocal correlations between single aberrations and clinicopathological parameters; however, when looking at the mutation signatures, a striking resemblance to BRCA-deficient breast cancer was evident in the majority of osteosarcoma profiles. We therefore focused our interest on genes involved in homologous recombination repair and applied different algorithms that have been shown in the literature to be indicators for functional impairment in these signaling cascades. Indeed, >80 % of osteosarcomas showed signatures similar to BRCA-deficient tumors and in osteosarcoma cell lines a response to poly(ADP-ribose) polymerase (PARP) inhibitors could be demonstrated. Our findings thus imply that multiple oncogenic pathways can converge and lead to chromosomal instability during osteosarcoma evolution resulting in the acquisition of BRCA-like traits, which might be of potential therapeutic relevance.

Topics: Adolescent; Algorithms; Bone and Bones; Bone Neoplasms; BRCA1 Protein; Cell Proliferation; Child; Clonal Evolution; DNA Mutational Analysis; Homologous Recombination; Humans; Neoplasm Staging; Osteosarcoma; Phthalazines; Poly(ADP-ribose) Polymerase Inhibitors; Tumor Suppressor Protein p53

Progress in the systemic control of osteosarcoma has been limited over the past decades thus indicating the urgent clinical need for the development of novel treatment strategies. Therefore, we have recently developed new preclinical models to study promising novel agents for the treatment of pediatric osteosarcoma. The checkpoint kinase (chk) inhibitor prexasertib (LY2606368) and its salt form (LSN2940930) have recently been shown to be active in adult and pediatric malignancies, including sarcoma. We have now tested the potency of prexasertib in clonogenic survival assays in two new lines of primary patient-derived osteosarcoma cells and in two established osteosarcoma cell lines as a single agent and in combination with cisplatin and the poly ADP-ribose polymerase (PARP) inhibitor talazoparib. Prexasertib alone results in strongly reduced clonogenic survival at low nanomolar concentrations and acts by affecting cell cycle progression, induction of apoptosis and induction of double-stranded DNA breakage at concentrations that are well below clinically tolerable and safe plasma concentrations. In combination with cisplatin and talazoparib, prexasertib acts in a synergistic fashion. Chk1 inhibition by prexasertib and its combination with the DNA damaging agent cisplatin and the PARP-inhibitor talazoparib thus emerges as a potential new treatment option for pediatric osteosarcoma which will now have to be tested in preclinical primary patient derived in vivo models and clinical studies.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Survival; Cisplatin; Clone Cells; Drug Synergism; Humans; Mice; Osteosarcoma; Phthalazines; Protein Kinase Inhibitors; Pyrazines; Pyrazoles; Xenograft Model Antitumor Assays

We recently discovered mutation signatures reminiscent of BRCA deficiency in the vast majority of a set of primary osteosarcomas (OS). In the current study, we therefore investigated the sensitivity of a panel of OS cell lines to the poly(ADP)-ribose polymerase (PARP) inhibitor talazoparib alone and in combination with several chemotherapeutic drugs (i.e. temozolomide (TMZ), SN-38, doxorubicin, cisplatin, methotrexate (MTX), etoposide/carboplatin). Here, we identified an association between homologous recombination (HR) repair deficiency and the response of OS cell lines to talazoparib. All OS cell lines with molecular features characteristic of BRCA1/2 mutant tumors (so-called "BRCAness"), such as disruptive gains in PTEN or FANCD2 and/or losses of ATM, BAP1, BARD1 or CHEK2, were susceptible to talazoparib-induced reduction of cell viability (i.e. MG63, ZK-58,, SaOS-2 and MNNG-HOS). Consistent with their high sensitivity to talazoparib, MG63 and ZK-58 cells scored positive in a DNA-based measure of genomic instability (i.e. homologous recombination deficiency (HRD)-loss of heterozygosity (LOH) score). In contrast, U2OS cells that carry a heterozygous BRCA2 mutation and therefore most likely have one intact BRCA2 allele left proved to be resistant to talazoparib. Furthermore, we identified TMZ as the most potent chemotherapeutic drug together with talazoparib to synergistically reduce cell viability, as confirmed by calculation of combination index (CI) values, and to suppress long-term clonogenic survival. Mechanistically, talazoparib and TMZ cooperated to induce apoptotic cell death, as demonstrated by activation of BAX and BAK, loss of mitochondrial membrane potential (MMP), caspase activation, DNA fragmentation and caspase-dependent cell death. Genetic silencing of BAX and BAK or pharmacological inhibition of caspases by zVAD.fmk significantly rescued OS cells from talazoparib/TMZ-induced apoptosis. These findings have important implications for the development of novel treatment strategies using PARP inhibitors alone or together with chemotherapy in a subset of OS with features of BRCAness.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bone Neoplasms; Caspases; Cell Line, Tumor; Cell Survival; Cisplatin; Doxorubicin; Drug Synergism; Genes, BRCA1; Genes, BRCA2; Humans; Osteosarcoma; Phthalazines; Poly(ADP-ribose) Polymerase Inhibitors

Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited.

Topics: Bone Neoplasms; Cell Line, Tumor; Exome; Gene Expression Regulation, Neoplastic; Humans; Mutation; Osteosarcoma; Phthalazines; Poly(ADP-ribose) Polymerase Inhibitors