Aim 1. To investigate biologic consequences of TrkB/PI-3Kinase/AKT activation on NB tumor biology We have continued to collaborate with Dr. Zhijie Li our former fellow who is now Research Director at China Medical University in Shenyang China, to study on how activated PI3K and MAPK signal transduction pathways affect NB metastatic potential of TrkB expressing tumor cells. Previously we had identified that the BDNF/TrkB pathway attenuated the effects of cytotoxic drugs on the survival of neuroblastoma tumor cells. In a recent study the contribution of the TP53 and BCL2 family members was evaluated in this model. It was identified that etoposide induced TP53 and PUMA expression mediated NB cell death and the etoposide induced TP53 and PUMA expression was attenuated when TrkB signaling was activated. This study was recently published in Apoptosis 23:408-19, 2018 Aim 3. To investigate STAT3 activation and NB tumor biology Our a previous published study evaluated the involvement of aberrantly activated IL-6/JAK/STAT pathway in Neuroblastoma, Rhabdomyosarcoma and Ewings Sarcoma and showed that inhibition of the JAK1/2 using AZD1480 decreased the viability of 7/7NB, 7/7RMS and 2/2 EWS cell lines tested (median EC50 is 1.5uM, ranging from 0.36-5.37uM). This was the first study showing as proof of principle that targeting the JAK/STAT3 pathway was efficacious in pediatric solid tumors. Off-target effects of AZD1480 and toxicity in the Phase I study led to the discontinuation of drug development. However, we continued pre-clinical studies evaluating genetic and pharmacogenomic inhibition of STAT3. We evaluated the biologic consequences of specific targeting of STAT3 in NB, by assessing the effects of genetic inhibition of STAT3 using engineered NB cell lines containing shRNAs targeting STAT3 and we evaluated r a generation 2.5 antisense oligonucleotide, AZD9150, which targets STAT3. Recent clinical studies showed activity in lymphomas using this first ever in cancer pharmacogenomic antisense to target a transcription factor. To date the direct drugging of transcription factors has been difficult. We found that tumor targeting of STAT3 had only a minor effect on in vitro growth on plastic but did remarkably decrease anchorage independent growth. Both genetic and AZD9150 had little effect on primary tumor growth. This result contrasted with our previous studies using AZD1480 and another study using STATTIC which showed significant inhibition of tumor growth in vivo. Unlike the systemic administration of AZD1480 and STATTIC which would affect both STAT3 levels in the human tumor as well as levels in murine tissues, AZD9150 only targets human STAT3. This raises the point that tumor microenvironment levels of STAT3 contribute may significantly contribute to xenograft tumor growth. Although primary tumor growth was not significantly affected by AZD9150, xenografts from AZD9150 treated mice did have a significant reduction ability to form secondary NB tumors. The frequency of tumor initiating cells (TIC) in NGP is 1/2,000 but in AZD9150 treated xenografts in which STAT3 expression was silenced, the TIC precursor frequency dropped a 1000-fold to 1/180,000. Additionally, we found that although inhibition of STAT3 did not affect primary tumor growth, the addition of cisplatin decreased tumor growth and increased survival of tumor-bearing mice. This study supports the development of strategies to target STAT3 when used in combination with conventional chemotherapy for patients with high-risk NB. These studies also indicate that targeting STAT3 decreases the frequency of TIC or cancer stem cells in NB xenografts. These studies were published in Clin. Cancer Res. 23:1771-1784,2017 Aim 3. To develop relevant in vivo NB models we are currently developing PDX models from NB patients seen in the POB and St. Judes hospital. Primary patients frozen tissues has been passaged in NSG mice in an orthotopic location for 3 passages and samples cryopreserved for future experiments. Each passage representative tumors are being analyzed for histology, DNA whole exome sequencing and RNAseq.
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