Low-grade astrocytomas (LGA) of childhood present a significant clinical challenge. While 5-year progression-free survival rate for chemotherapy plus radiotherapy is ~ 68%, significant morbidity is associated with the presence of residual tumor and the current therapy that includes neuroendocrine- cognitive deficits, visual deficits, vasculopathy and secondary tumors. Moreover, the metastatic potential and malignant transformation to a high-grade astrocytoma further contributes to the poor prognosis. Recent data show that BRAF is mutated in ~23% of LGA's, and 60% of xanthoastrocytomas. We have established two of the only childhood LGA models as direct patient- xenografts in mice. These tumors retain expression profiles and genomic alterations characteristic of the original patient's tumor, thus present unique models to develop alternative, less debilitating, curative therapy. BT-40 xenografts are heterozygous for mutated BRAF (V600E) whereas BT-35 xenografts have wild type BRAF. BT-40 tumors are exquisitely sensitive to the MEK inhibitor AZD6244, whereas BT-35 tumors are unresponsive, typical of other childhood tumor xenografts with wild type BRAF. Resistance to AZD6244 is characterized by an increase in the MEK-dependent gene signature, enhanced IL-6 transcription and secretion, and activation of STAT3 signaling. Resistance to AZD6244 is unstable, as tumors passaged in untreated mice revert to drug sensitivity, and correlates with decreased IL-6 and STAT3 activation. Further, two BRAF (V600E) mutant astrocytic cell lines intrinsically resistant to AZD6244 induce STAT3 activation as MEK is inhibited. The role of STAT3 activation in resistance will be studied in Aim 1. For BT-40 tumors sensitivity to AZD6244 is characterized by complete inhibition of TORC1 signaling, suggesting that mutant BRAF controls the PI3K/TORC1 signaling axis. The role of Akt and STAT3 signaling in MEK regulation of TORC1, and the simultaneous inhibition of MEK and STAT3 to prevent development of resistance, or reverse resistance to AZD6244, will be studied in Aim 2. We plan to exploit the control of mutant BRAF over TORC1 signaling therapeutically. In BT-40 xenografts, MEK inhibition leads to rapid and complete loss of the DNA repair protein FANCD2, downstream of TORC1, thus potentially selectively sensitizing tumor cells to cisplatin, etoposide and ionizing radiation. Further, we will test the concept that combination of cisplatin o etoposide with AZD6244 will prevent the emergence of AZD6244 resistant cells, as mutant BRAF is required to maintain Akt signaling associated with survival in cells exposed to cisplatin or etoposide. We will establish additional models of mutant BRAF LGA's, and determine whether results from BT-40 xenografts are generally applicable. These data will form the basis for rapid translation of novel non-genotoxic and also more conventional therapies that are both effective and less debilitating compared to current therapeutic approaches, for children with recurrent or surgically non-resectable LGA.
Using unique models of childhood low-grade astrocytomas in mice, we have discovered that in BRAF-mutant tumors this oncogene has a gain of function that can be exploited to selectively kill astrocytoma cells. The studies proposed will identify how tumors become resistant to inhibitors of BRAF signaling, and exploit mutant BRAF to prevent development of resistance and synergize with drugs and ionizing radiation therapy. We anticipate that the studies proposed will lead to both increased cures for children with non-resectable astrocytomas, and reduce the toxicity of radiation and cytotoxic drug therapies, thus reducing the debilitating sequellae of current therapies.
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