Project A: Childhood Glioma

In recent years there have been considerable advances in defining subsets of pediatric tumors by genotyping and expression profiling. 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 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. For pediatric astrocytomas, low-grade tumors are associated with the activation of BRAF through a tandem duplication that results in the KIAA1549-BRAF fusion 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), and in 60% of xanthoastrocytomas. 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. Mutations in PIK3CA are reported to be rare in these tumors. Findings for BRAF mutation, similar to other tumors with activated BRAF (e.g. melanoma), suggest that activated BRAF may provide a potential drug target.

BRAF is a component of the mitogen-activated protein kinase (MAPK) signaling pathway that induces multiple proliferative or differentiation signals within tumor cells. In many adult carcinomas, MAPK activation occurs through activating mutations in RAS or RAF. The frequency of BRAFV600E mutations ranges from ≥90% in Hairy Cell leukemia and 60-80% in melanoma to around 10% in colon cancer, with other tumors in between. 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.

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: Initial testing (stage 1) of AZD6244 (ARRY-142886) by the Pediatric Preclinical Testing Program.: 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.

Development

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 reverted 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 the 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 sequelae from radiation treatment can be severe. As many childhood gliomas are associated with the 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 leads to 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.

Preventing the Emergence of Drug Resistance

Regulation of TORC1 by MAPK Signaling Determines Sensitivity and Acquired Resistance to Trametinib in Pediatric BRAFV600E Brain Tumor Models. Fuyang Li, Kathryn M. Bondra, Samson Ghilu, Adam Studebaker Qianqian Liu Joel E. Michalek, Mari Kogiso, Xiao-Nan Li, John A. Kalapurakal, C. David James, Sandeep Burma, Raushan T. Kurmasheva, and Peter J. Houghton. Clin Cancer Res. 2022 Sep 1;28(17):3836-3849. doi: 10.1158/1078-0432.CCR-22-1052.PMID: 35797217

PURPOSE: We investigated why three patient-derived xenografts (PDX) childhood BRAFV600E-mutant brain tumor models are highly sensitive to trametinib. Mechanisms of acquired resistance selected in situ and approaches to prevent resistance were also examined, which may translate to both LGG molecular subtypes.

EXPERIMENTAL DESIGN: Sensitivity to trametinib (MEKi) alone or in combination with rapamycin (TORC1 inhibitor) was evaluated in pediatric PDX models. The effect of combined treatment of trametinib with rapamycin on the development of trametinib resistance in vivo was examined. PDX tissue and tumor cells from trametinib-resistant xenografts were characterized.

RESULTS: In pediatric models, TORC1 is activated through ERK-mediated inactivation of the tuberous sclerosis complex TSC: consequently, inhibition of MEK also suppressed TORC1 signaling. Trametinib-induced tumor regression correlated with dual inhibition of MAPK/TORC1 signaling and decoupling TORC1 regulation from BRAF/MAPK control conferred trametinib resistance. In mice, acquired resistance to trametinib developed within 3 cycles of therapy in all three PDX models. Resistance to trametinib developed in situ is tumor cell-intrinsic, and the mechanism was tumor-line specific. Rapamycin retarded or blocked the development of resistance.

CONCLUSIONS: In these three pediatric BRAF-mutant brain tumors, TORC1 signaling is controlled by the MAPK cascade. Trametinib suppressed both MAPK/TORC1 pathways leading to tumor regression. While low-dose intermittent rapamycin to enhance inhibition of TORC1 only modestly enhanced the antitumor activity of trametinib, it prevented or retarded the development of trametinib resistance, suggesting future therapeutic approaches using rapamycin analogs in combination with MEK  inhibitors that may be therapeutically beneficial in both KIAA1549::BRAF and BRAFV600E driven gliomas.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A. Responses of BT-40 patient-derived xenograft. Mice bearing subcutaneous BT-40 tumors were randomized to receive no treatment, trametinib (1 mg/kg daily x 42 days), rapamycin (5 mg/kg daily x 5 for 6 consecutive weeks), or the combination. Each curve shows the growth of an individual tumor;

B. Activity of the trametinib-rapamycin combination on intracranial BT-40/Luc tumors. 105 BT-40/luc cells expressing luciferase were implanted intracranially. Mice were randomized into control or treatment groups when the bli value reached >1×105 photons/sec/cm3 and received combination treatment as in (A). Mice were imaged once weekly to ascertain tumor growth.

Individual tumor growth curves based on Bli emission. Control (black), Treated (Red) Bli values plotted against time for control and treatment groups;

C. Luciferase images of cranial tumor growth in control and treatment groups.

D. Schema for developing drug resistance in mice.

E. Upper panels: Responses of BT-40 xenografts to 3 cycles of trametinib (Tram) treatment (1 mg/kg/day for 42 consecutive days). The arrows indicate the tumor that was transplanted into recipient mice for the subsequent cycle of treatment. The top right panel shows mean tumor volume (± SD) for cycles 1 to 4 of treatment; Center panels: Responses of BT-40 xenografts for two cycles of trametinib + rapamycin (Rap) (trametinib 1 mg/kg daily x 42, rapamycin 5 mg/kg daily x 5 for 6 consecutive weeks). After 2 cycles of treatment, single agent trametinib was administered for 3 further cycles; Lower panels: Responses of BT-40 xenografts for six cycles of trametinib + rapamycin (trametinib 1 mg/kg daily x 42, rapamycin 5 mg/kg daily x 5 for 6 consecutive weeks). Each curve represents the growth of a single tumor. The arrows indicate the tumor that was transplanted into recipient mice for the subsequent cycle of treatment.

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 leads to 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.