bmn-673 and Bone-Neoplasms

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

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

BMN 673 is a potent inhibitor of poly-ADP ribose polymerase (PARP) that is in clinical testing with a primary focus on BRCA-mutated cancers. BMN 673 is active both through inhibiting PARP catalytic activity and by tightly trapping PARP to DNA at sites of single strand breaks.. BMN 673 was tested in vitro at concentrations ranging from 0.1 nM to 1 μM and in vivo at a daily dose of 0.33 mg/kg administered orally twice daily (Mon-Fri) and once daily on weekends (solid tumors) for 28 days.. The median relative IC50 (rIC50 ) concentration against the PPTP cell lines was 25.8 nM. The median rIC50 for the Ewing cell lines was lower than for the remaining cell lines (6.4 vs. 31.1 nM, respectively). In vivo BMN 673 induced statistically significant differences in EFS distribution in 17/43 (39.5%) xenograft models. Three objective regressions were observed: a complete response (CR) in a medulloblastoma line (BT-45), a maintained CR in a Wilms tumor line (KT-10), and a maintained CR in an ependymoma line (BT-41). BMN 673 maintained its high level of activity against KT-10 with a threefold reduction in dose. KT-10 possesses a truncating mutation in PALB2 analogous to PALB2 mutations associated with hereditary breast and ovarian cancer that abrogate homologous recombination (HR) repair.. The PPTP results suggest that single agent BMN 673 may have limited clinical activity against pediatric cancers. Single agent activity is more likely for patients whose tumors have defects in HR repair.

Topics: Animals; Bone Neoplasms; Drug Evaluation, Preclinical; Enzyme Inhibitors; Fanconi Anemia Complementation Group N Protein; Female; Glioblastoma; Humans; Mice; Mutation; Neuroblastoma; Nuclear Proteins; Phthalazines; Poly(ADP-ribose) Polymerase Inhibitors; Rhabdomyosarcoma; Sarcoma, Ewing; Tumor Suppressor Proteins

Inhibitors of PARP, an enzyme involved in base excision repair, have demonstrated single-agent activity against tumors deficient in homologous repair processes. Ewing sarcoma cells are also sensitive to PARP inhibitors, although the mechanism is not understood. Here, we evaluated the stereo-selective PARP inhibitor, talazoparib (BMN 673), combined with temozolomide or topotecan.. Talazoparib was tested in vitro in combination with temozolomide (0.3-1,000 μmol/L) or topotecan (0.03-100 nmol/L) and in vivo at a dose of 0.1 mg/kg administered twice daily for 5 days combined with temozolomide (30 mg/kg/daily x 5; combination A) or 0.25 mg/kg administered twice daily for 5 days combined with temozolomide (12 mg/kg/daily x 5; combination B). Pharmacodynamic studies were undertaken after 1 or 5 days of treatment.. In vitro talazoparib potentiated the toxicity of temozolomide up to 85-fold, with marked potentiation in Ewing sarcoma and leukemia lines (30-50-fold). There was less potentiation for topotecan. In vivo, talazoparib potentiated the toxicity of temozolomide, and combination A and combination B represent the MTDs when combined with low-dose or high-dose talazoparib, respectively. Both combinations demonstrated significant synergism against 5 of 10 Ewing sarcoma xenografts. The combination demonstrated modest activity against most other xenograft models. Pharmacodynamic studies showed a treatment-induced complete loss of PARP only in tumor models sensitive to either talazoparib alone or talazoparib plus temozolomide.. The high level of activity observed for talazoparib plus temozolomide in Ewing sarcoma xenografts makes this an interesting combination to consider for pediatric evaluation.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Bone Neoplasms; Cell Line, Tumor; Dacarbazine; Drug Synergism; Female; Humans; Immunoblotting; Mice; Mice, SCID; Phthalazines; Poly(ADP-ribose) Polymerase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Sarcoma, Ewing; Temozolomide; Xenograft Model Antitumor Assays

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