Project C: Low-Grade Glioma – Brain tumors
Astrocytomas are the most common tumors of the central nervous system in children and are subdivided according to histologic subtypes, grades I-IV (1). The majority of astrocytomas are grade I and grade II tumors, yet the biological behavior of low-grade astrocytomas reflect a heterogeneous spectrum. Radical surgical resection is the standard therapy; however, whereas most cerebellar pilocytic astrocytomas (WHO I) do not frequently involve the diencephalon, those that do are not resectable due to the tumor’s involvement in the diencepahlon and surrounding eloquent structures. Thus, adjuvant therapy is warranted which includes chemotherapy and/or radiation therapy. The 5-year progression-free survival rate for chemotherapy plus radiotherapy has been reported as 68%, which is superior to chemotherapy alone 38% (2). However, 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 (3, 4). Moreover, the metastatic potential and transformation to a high-grade astrocytoma further contributes to the poor prognosis (5, 6).
In recent years there have been considerable advances in defining subsets of pediatric tumors by genotyping and expression profiling (7-9). Whereas histologically, astrocytomas in children and adults are similar, childhood astrocytomas are distinct clinical entities from those in adults and are not associated with many of the critical genetic alterations found in the adult astrocytomas. With the possible exception of TP53 mutations, frequent genetic alterations detected in adult astrocytomas have been identified at lower frequencies in childhood astrocytomas (10-13). For pediatric astrocytomas, low-grade tumors are associated with activation of BRAF through a tandem duplication that results in the KIAA1549-BRAF fusion (14) or through an activating point mutation of BRAF (predominantly V600E). More recent data suggests that the KIAA1549-BRAF fusion is restricted to grade I pilocytic astrocytoma (100%) whereas BRAFV600E occurs more frequently in grade II-IV gliomas (~23%; although lower frequencies have been reported (15)), and in 60% of xanthoastrocytomas (15, 16). Thus, activating mutation of BRAF appears to be the most common genetic alteration in intermediate grade astrocytoma. Homozygous deletion of the CDKN2A locus is frequent (~70%) in tumors harboring the BRAFV600E mutation (17). Mutations in PIK3CA are reported to be rare in these tumors (18). Findings for BRAF mutation, similar to other tumors with activated BRAF (e.g. melanoma), suggest that activated BRAF may provide a potential drug target (19).
BRAF is a component of the mitogen activated protein kinase (MAPK) signaling pathway that induces multiple proliferative or differentiation signals within tumor cells (20, 21). In many adult carcinomas MAPK activation occurs through activating mutations in RAS or RAF. The frequency of BRAFV600E mutations range from ≥90% in Hairy Cell leukemia, and 60-80% in melanoma to around 10% in colon cancer with other tumors in between (22-28). Cell lines harboring BRAFV600E may be highly sensitive to MEK inhibition, and these agents may have significant utility against melanoma and other tumors with similar mutations (19).
From: Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549:BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma. Jones DT, Kocialkowski S, Liu L, Pearson DM, Ichimura K, Collins VP. Oncogene. 2009 May 21;28(20):2119-23
Initial Testing (Stage 1)
Initial testing (stage 1) of AZD6244 (ARRY-142886) by the Pediatric Preclinical Testing Program.
Kolb EA, Gorlick R, Houghton PJ, Morton CL, Neale G, Keir ST, Carol H, Lock R, Phelps D, Kang MH, Reynolds CP, Maris JM, Billups C, Smith MA. Pediatr Blood Cancer. 2010 Oct;55(4):668-77. PMID: 20806365
BACKGROUND: AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development.
PROCEDURES: AZD6244 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro panel (1 nM-10 microM). In vivo AZD6244 was tested at a dose of 100 mg/kg administered orally twice daily 5 days per week for 6 weeks. Subsequently, AZD6244 was evaluated against two juvenile pilocytic astrocytoma (JPA) xenografts using once and twice daily dosing schedules. Phosphorylation of ERK1/2 was used as a surrogate for in vivo inhibition of MEK1/2 was determined by immunoblotting.
RESULTS: At the highest concentration used in vitro (10 microM) AZD6244 only inhibited growth by 50% in 5 of the 23 cell lines. Against the in vivo tumor panels, AZD6244 induced significant differences in EFS distribution in 10 of 37 (27%) solid tumor models and 0 of 6 acute lymphoblastic leukemia (ALL) models. There were no objective responses. Pharmacodynamic studies indicated at this dose and schedule AZD6244 completely inhibited ERK1/2 phosphorylation. AZD6244 was evaluated against two JPA xenografts, BT-35 (wild-type BRAF) and BT-40 (mutant [V600E] BRAF). BT-40 xenografts were highly sensitive to AZD6244, whereas BT-35 xenografts progressed on AZD6244 treatment.
CONCLUSIONS: At the dose and schedule of administration used, AZD6244 as a single agent had limited in vitro and in vivo activity against the PPTP tumor panels despite inhibition of MEK1/2 activity. However, AZD6244 was highly active against BT-40 JPA xenografts that harbor constitutively activated BRAF, causing complete regressions.
Figure – AZD6244 activity against pilocytic astrocytoma xenografts. Kaplan-Meier curves for EFS, median relative tumor volume graphs, and individual tumor volume graphs are shown for (A): BT-35 and (B) BT-40. Kaplan-Meier: Controls: black solid line; 100 mg/kg BID × 5/SID × 2 for six consecutive weeks: broken red line; 75 mg/kg BID × 5/SID × 2 for six consecutive weeks: blue broken line: 100 mg/kg SID for six consecutive weeks: broken pink line. Relative Tumor Volume curves: Controls: black solid line; 100 mg/kg BID × 5/SID × 2 for six consecutive weeks: solid brown line; 75 mg/kg BID × 5/SID × 2 for six consecutive weeks: solid blue line: 100 mg/kg SID for six consecutive weeks: solid red line. For individual growth curve plots: Upper left panel: Control; Upper right panel: AZD6244 100 mg/kg BID × 5/SID × 2 for six consecutive weeks; lower left panel: AZD6244 75 mg/kg BID × 5/SID × 2 for six consecutive weeks; Lower right panel100 mg/kg SID for six consecutive weeks.
Development, characterization, and reversal of acquired resistance to the MEK1 inhibitor selumetinib (AZD6244) in an in vivo model of childhood astrocytoma. Bid HK, Kibler A, Phelps DA, Manap S, Xiao L, Lin J, Capper D, Oswald D, Geier B, DeWire M, Smith PD, Kurmasheva RT, Mo X, Fernandez S, Houghton PJ. Clin Cancer Res. 2013 Dec 15;19(24):6716-29. PMID: 24132923
PURPOSE: The BT-40 low-grade childhood astrocytoma xenograft model expresses mutated BRAF(V600E) and is highly sensitive to the MEK inhibitor selumetinib (AZD6244). In this study, we developed and characterized selumetinib resistance and explored approaches to circumventing the mechanisms of acquired resistance.
EXPERIMENTAL DESIGN: BT-40 xenografts were selected in vivo for selumetinib resistance. Resistant tumors were obtained and characterized, as were tumors that reverted to sensitivity. Characterization included expression profiling, assessment of MEK signature and compensatory pathways, MEK inhibition, BRAF expression, and cytokine levels. Combination treatment of BT-40/AZD-resistant tumors with the MEK inhibitor and a STAT3 inhibitor (LLL12) was assessed.
RESULTS: Resistance was unstable, tumors reverting to selumetinib sensitivity when passaged in untreated mice, and MEK was equally inhibited in sensitive and resistant tumors by selumetinib. Drug resistance was associated with an enhanced MEK signature and increased interleukin (IL)-6 and IL-8 expression. Selumetinib treatment induced phosphorylation of STAT3 (Y705) only in resistant xenografts, and similar results were observed in BRAF(V600E) astrocytic cell lines intrinsically resistant to selumetinib. Treatment of BT-40-resistant tumors with selumetinib or LLL12 had no significant effect, whereas combined treatment induced complete regressions of BT-40/AZD-resistant xenografts.
CONCLUSIONS: Resistance to selumetinib selected in vivo in BT-40 tumor xenografts was unstable. In resistant tumors, selumetinib activated STAT3, and combined treatment with selumetinib and LLL12 induced complete responses in resistant BT-40 tumors. These results suggest dual targeting BRAF (V600E) signaling and STAT3 signaling may be effective in selumetinib-resistant tumors or may retard or prevent onset of resistance.
Figure. – Mice bearing BT-40, BT-40/AZD or BT-40/REV tumors were either not treated (0 hr) or treated with selumetinib (75 mg/kg BID). Tumors were harvested (n=3) at the time points shown. STAT3 and pSTAT3(Y705) were determined. Actin was used as the loading control B. Quantitation of STAT3 and pSTAT3(Y705). C. LLL12 reverses selumetinib resistance in BT-40/AZD xenografts. Mice (n=10 per group) were treated with vehicle (DMSO, control); LLL12 (5 mg/kg/day); selumetinib (75 mg/kg BID); selumetinib and LLL12 combined for up to 6 weeks. D. At termination tumors from each control or treatment group were harvested. FFPE sections were stained to assess proliferation (Ki67), apoptosis (TUNEL) or histology (hematoxylin and eosin). Right panels show quantitation of Ki67 and TUNEL staining.
Inhibition of MEK
Inhibition of MEK confers hypersensitivity to X-radiation in the context of BRAF mutation in a model of childhood astrocytoma. Studebaker A, Bondra K, Seum S, Shen C, Phelps DA, Chronowski C, Leasure J, Smith PD, Kurmasheva RT, Mo X, Fouladi M, Houghton PJ. Pediatr Blood Cancer. 2015 May 15. PMID: 25981859
PURPOSE: Curative therapy for childhood glioma presents challenges when complete resection is not possible. Patients with recurrent low-grade tumors or anaplastic astrocytoma may receive radiation treatment; however, the long-term sequellae from radiation treatment can be severe. As many childhood gliomas are associated with activation of BRAF, we have explored the combination of ionizing radiation with MEK inhibition in a model of BRAF-mutant anaplastic astrocytoma.
EXPERIMENTAL DESIGN: The regulation of TORC1 signaling by BRAF was examined in BT-40 (BRAF mutant) and BT-35 (BRAF wild type) xenografts, in a cell line derived from the BT-40 xenograft and two adult BRAF mutant glioblastoma cell lines. The effect of MEK inhibition (selumetinib), XRT (total dose 10 Gy as 2 Gy daily fractions), or the combination of selumetinib and XRT was evaluated in subcutaneous BT-40 xenografts.
RESULTS: Inhibition of MEK signaling by selumetinib suppressed TORC1 signaling only in the context of the BRAF-mutant both in vitro and in vivo. Inhibition of MEK signaling in BT-40 cells or in xenografts lead to a complete suppression of FANCD2 and conferred hypersensitivity to XRT in BT-40 xenografts without increasing local skin toxicity.
CONCLUSIONS: Selumetinib suppressed TORC1 signaling in the context of BRAF mutation. Selumetinib caused a rapid downregulation of FANCD2 and markedly potentiated the effect of XRT. These data suggest the possibility of potentiating the effect of XRT selectively in tumor cells by MEK inhibition in the context of mutant BRAF or maintaining tumor control at lower doses of XRT that would decrease long-term sequelae.
Figure. Mice bearing BT-40, BT-40/AZD or BT-40/REV tumors were either not treated (0 hr) or treated with selumetinib (75 mg/kg BID). Tumors were harvested (n=3) at the time points shown. STAT3 and pSTAT3(Y705) were determined. Actin was used as the loading control. B. Quantitation of STAT3 and pSTAT3(Y705). C. LLL12 reverses selumetinib resistance in BT-40/AZD xenografts. Mice (n=10 per group) were treated with vehicle (DMSO, control); LLL12 (5 mg/kg/day); selumetinib (75 mg/kg BID); selumetinib and LLL12 combined for up to 6 weeks. D. At termination tumors from each control or treatment group were harvested. FFPE sections were stained to assess proliferation (Ki67), apoptosis (TUNEL) or histology (hematoxylin and eosin). Right panels show quantitation of Ki67 and TUNEL staining.
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- Inhibition of MEK confers hypersensitivity to X-radiation in the context of BRAF mutation in a model of childhood astrocytoma. Studebaker A, Bondra K, Seum S, Shen C, Phelps DA, Chronowski C, Leasure J, Smith PD, Kurmasheva RT, Mo X, Fouladi M, Houghton PJ. Pediatr Blood Cancer. 2015 Oct;62(10):1768-74. doi: 10.1002/pbc.25579. Epub 2015 May 15.PMID:25981859
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- Development, characterization, and reversal of acquired resistance to the MEK1 inhibitor selumetinib (AZD6244) in an in vivo model of childhood astrocytoma. Bid HK, Kibler A, Phelps DA, Manap S, Xiao L, Lin J, Capper D, Oswald D, Geier B, DeWire M, Smith PD, Kurmasheva RT, Mo X, Fernandez S, Houghton PJ. Clin Cancer Res. 2013 Dec 15;19(24):6716-29. doi: 10.1158/1078-0432.CCR-13-0842. Epub 2013 Oct 16.PMID:24132923
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