Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance

Abstract

Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common malignant brain tumor in children. GDC-0449 is an Hh pathway inhibitor (HPI) currently under clinical investigation as an anticancer agent. Treatment of a MB patient with GDC-0449 initially regressed tumors, but this individual ultimately relapsed with a D473H resistance mutation in Smoothened (SMO), the molecular target of GDC-0449. To explore the role of the mutated aspartic acid residue in SMO function, we substituted D473 with every amino acid and found that all functional mutants were resistant to GDC-0449, with positively charged residues conferring potential oncogenic properties. Alanine scan mutagenesis of SMO further identified E518 as a novel prospective mutation site for GDC-0449 resistance. To overcome this form of acquired resistance, we screened a panel of chemically diverse HPIs and identified several antagonists with potent in vitro activity against these GDC-0449-resistant SMO mutants. The bis-amide compound 5 was of particular interest, as it was able to inhibit tumor growth mediated by drug resistant SMO in a murine allograft model of MB. However, focal amplifications of the Hh pathway transcription factor Gli2 and the Hh target gene cyclin D1 (Ccnd1) were observed in two additional resistant models, indicating that resistance may also occur downstream of SMO. Importantly, these HPI resistant MB allografts retained their sensitivity to PI3K inhibition, presenting additional opportunities for the treatment of such tumors. Cancer Res; 71(2); 435-44. Ó2010 AACR.

Arhgap36-dependent activation of Gli transcription factors

“…ARHGAP36-induced GLI activation may be a mechanism by which group 2 medulloblastomas acquire resistance to Smo antagonists, in addition to SMO mutations and chromosomal GLI2 amplicons (29,36,44). Furthermore, the up-regulation of ARHGAP36 in group 3 and 4 tumors alludes to a more complex relationship between GLI activity and medulloblastoma biology than has been discerned through the bulk transcriptional profiling of advanced-stage tumors.…”

Inactivation of the Transcription Factor GLI1 Accelerates Pancreatic Cancer Progression

“…This is important because several clinical trials inhibiting pathways known to induce GLI1 activity (e.g. Hedgehog) are underway (12,48,49,(57)(58)(59). The success of these trials has been mixed, possibly due to the complexity of the signaling processes involved in pancreatic cancer progression.…”

GPCRs and cancer

“…However, the response of this patient to GDC-0449 treatment was only transient due to a mutation of Smo [71] . Recently, a number of Hh pathway antagonists targeting Smo mutants [72] as well as inhibitors able to block both wild-type and Smo mutants have been identified [73] . In addition to Smo antagonists, other inhibitors were used to block Hh signaling like the small molecule inhibitor of GLI1 and GLI2 transcription factors, GANT61, which induced colon carcinoma cell death in a higher extent respect to the conventional Smo inhibitor cyclopamin [74] .…”