dactolisib and Bone-Neoplasms

Ewing sarcomas (ES) are biologically aggressive tumors of bone and soft tissues for which no cure is currently available. Most ES patients do not respond to chemotherapeutic treatments or acquire resistance. Since the PI3K/AKT/mTOR axis is often deregulated in ES, its inhibition offers therapeutic perspective for these aggressive tumors. Herein, by using splicing sensitive arrays, we have uncovered an extensive splicing program activated upon inhibition of the PI3K/AKT/mTOR signaling pathway by BEZ235. Bioinformatics analyses identified hnRNPM as a key factor in this response. HnRNPM motifs were significantly enriched in introns flanking the regulated exons and proximity of binding represented a key determinant for hnRNPM-dependent splicing regulation. Knockdown of hnRNPM expression abolished a subset of BEZ235-induced splicing changes that contained hnRNPM binding sites, enhanced BEZ235 cytotoxicity and limited the clonogenicity of ES cells. Importantly, hnRNPM up-regulation correlates with poor outcome in sarcoma patients. These findings uncover an hnRNPM-dependent alternative splicing program set in motion by inhibition of the mTOR/AKT/PI3K pathway in ES cells that limits therapeutic efficacy of pharmacologic inhibitors, suggesting that combined inhibition of the PI3K/AKT/mTOR pathway and hnRNPM activity may represent a novel approach for ES treatment.

Topics: Alternative Splicing; Antineoplastic Agents; Bone Neoplasms; Cell Line; Exons; Heterogeneous-Nuclear Ribonucleoprotein Group M; Humans; Imidazoles; Nucleotide Motifs; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Sarcoma, Ewing; Signal Transduction; TOR Serine-Threonine Kinases; Transcriptome

Recent studies have identified that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is an important feature of osteosarcoma, where it promotes cell proliferation, survival, and chemo-resistance. Here, we studied the therapeutic potential of NVP-BEZ235, a novel dual PI3K/mTOR dual inhibitor, on osteosarcoma cells in vivo and in vitro. NVP-BEZ235 was cytotoxic and cytostatic to a panel of osteosarcoma lines (MG-63, U2OS and SaOs-2), where it induce apoptosis and cell-cycle arrest. At the molecular level, NVP-BEZ235 inhibited PI3K-AKT-mTORC1 activation and downregulated cyclin D1/cyclin B1 expressions, while increasing MEK/Erk phosphorylation in osteosarcoma cells. MEK/Erk inhibitors PD98059 and MEK-162 increased NVP-BEZ235 activity on osteosarcoma cells. In vivo, oral NVP-BEZ235 inhibited U2OS xenograft in SCID mice, and its anti-tumor efficiency was further enhanced by MEK-162 co-administration. Taken together, our findings indicate that dual inhibition of PI3K and mTOR with NVP-BEZ235, either alone or in combination with MEK/Erk inhibitors, may be an efficient treatment for osteosarcoma.

Topics: Animals; Apoptosis; Bone Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Down-Regulation; Humans; Imidazoles; Male; MAP Kinase Signaling System; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, SCID; Multiprotein Complexes; Osteosarcoma; Phosphatidylinositol 3-Kinase; Phosphorylation; Protein Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and is characterized by the presence of the balanced translocation t(11;22)(q24;q12) in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. This fusion protein is an essential oncogenic component of ES development which is necessary for tumor cell maintenance and represents an attractive therapeutic target. To search for modulators of EWS/FLI1 activity we screened a library of 153 targeted compounds and identified inhibitors of the PI3K pathway to directly modulate EWS/FLI1 transcription. Surprisingly, treatment of four different ES cell lines with BEZ235 resulted in down regulation of EWS/FLI1 mRNA and protein by ~50% with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (-2239/+67) using various deletion constructs identified two 14 bp minimal elements as being important for EWS/FLI1 transcription. We identified SP1 as modulator of EWS/FLI1 gene expression and demonstrated direct binding to one of these regions in the EWS/FLI1 promoter by EMSA and ChIP experiments. These results provide the first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer an additional molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition.

Topics: Antineoplastic Agents; Binding Sites; Bone Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Oncogene Proteins, Fusion; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Promoter Regions, Genetic; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins c-akt; Quinolines; RNA Interference; RNA-Binding Protein EWS; RNA, Messenger; Sarcoma, Ewing; Signal Transduction; Sp1 Transcription Factor; Transcription, Genetic; Transfection

Despite recent improvements in chemotherapy and surgery, the problem of non-response osteosarcoma to chemotherapy remains, and is a parameter that is critical for prognosis. The present work investigated the therapeutic value of NVP-BEZ235, a dual class I PI3K/mTOR inhibitor. NVP-BEZ235 inhibited osteosarcoma cell proliferation by inducing G0/G1 cell cycle arrest with no caspase activation. In murine pre-clinical models, NVP-BEZ235 significantly slowed down tumor progression and ectopic tumor bone formation with decreased numbers of Ki67(+) cells and reduced tumor vasculature. Finally, NVP-BEZ235 considerably improved the survival rate of mice with osteosarcoma. Taken together, the results of the present work show that NVP-BEZ235 exhibits therapeutic interest in osteosarcoma and may be a promising adjuvant drug for bone sarcomas.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Growth Processes; Cell Line, Tumor; Humans; Imidazoles; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Osteosarcoma; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Random Allocation; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Chondrosarcomas are notoriously resistant to cytotoxic chemotherapeutic agents. We sought to identify critical signaling pathways that contribute to their survival and proliferation, and which may provide potential targets for rational therapeutic interventions.. Activation of receptor tyrosine kinases (RTK) was surveyed using phospho-RTK arrays. S6 phosphorylation and NRAS mutational status were examined in chondrosarcoma primary tumor tissues. siRNA or small-molecule inhibitors against RTKs or downstream signaling proteins were applied to chondrosarcoma cells and changes in biochemical signaling, cell cycle, and cell viability were determined. In vivo antitumor activity of BEZ235, a phosphoinositide 3-kinase (PI3K)/mTOR inhibitor, was evaluated in a chondrosarcoma xenograft model.. Several RTKs were identified as critical mediators of cell growth, but the RTK dependencies varied among cell lines. In exploration of downstream signaling pathways, strong S6 phosphorylation was found in 69% of conventional chondrosarcomas and 44% of dedifferentiated chondrosarcomas. Treatment with BEZ235 resulted in dramatic reduction in the growth of all chondrosarcoma cell lines. Tumor growth was similarly inhibited in a xenograft model of chondrosarcoma. In addition, chondrosarcoma cells with an NRAS mutation were sensitive to treatment with a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor. Functional NRAS mutations were found in 12% of conventional central chondrosarcomas.. RTKs are commonly activated in chondrosarcoma, but because of their considerable heterogeneity, targeted inhibition of the PI3K/mTOR pathway represents a rational therapeutic strategy. Chondrosarcomas with NRAS mutations may benefit from treatment with MEK inhibitors.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Chondrosarcoma; Enzyme Activation; Feedback, Physiological; Female; Genetic Association Studies; GTP Phosphohydrolases; Humans; Imidazoles; Inhibitory Concentration 50; MAP Kinase Signaling System; Membrane Proteins; Mice; Mice, Nude; Molecular Targeted Therapy; Mutation, Missense; Phosphatidylinositol 3-Kinases; Phosphorylation; Polymorphism, Single Nucleotide; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Quinolines; Receptor Protein-Tyrosine Kinases; Tissue Array Analysis; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays