Inhibitors of Raf Kinase Activity Block Growth of Thyroid Cancer Cells with RET/PTC or BRAF Mutations In vitro and In vivo

Abstract

Purpose: Papillary thyroid carcinomas are associated with nonoverlapping activating mutations of RET, NTRK, RAS and BRAF, which altogether are present in f70% of cases.We postulated that compounds that inhibit a distal effector in the mitogen-activated protein kinase (MAPK) pathway would inhibit growth and tumorigenicity of human thyroid cancer cell lines with mutations of RET or BRAF. Experimental Design and Results: We first examined the effects of AAL-881 and LBT-613, two inhibitors of RAF kinase activity, on RAF-MAPK/extracellular signal^regulated kinase (ERK) kinase (MEK)-ERK activation in thyroid PCCL3 cells after conditional induction of expression of H-RAS G12V or BRAF V600E. Both compounds blocked RAS and RAF-dependent MEK and ERK phosphorylation. They also potently blocked MEK phosphorylation in human thyroid cancer cell lines with either RET/PTC1 (TPC1) or BRAF V600E (NPA, ARO, and FRO) mutations. Inhibition of ERK phosphorylation was transient inTPC1and ARO cells, with recovery of ERK phosphorylation associated with concomitant down-regulation of the MAPK phosphatases MKP-3 and DUSP5. Both compounds inhibited growth of all cell lines, with LBT-613 being f10-fold more potent than AAL-881. TPC1 cells were more sensitive to growth inhibition (IC 50 0.1-0.25 and f0.05 Amol/L for AAL-881 and LBT-613, respectively) than BRAF (+) lines (IC 50 2.5-5 and 0.1-0.5 Amol/L, respectively). Growth inhibition was associated with G 1 arrest, and induction of cell death. Growth of ARO and NPA tumor xenografts was inhibited by LBT-613 or AAL-881. MEK and ERK phosphorylation was inhibited by both compounds in ARO but not in NPA cell xenografts. Conclusions: Compounds that inhibit kinase activity are effective growth inhibitors for poorly differentiated thyroid cancer cell lines with either RET or RAF mutations, and hold promise for treatment of most forms of papillary thyroid carcinoma.

Targeting BRAFV600E in thyroid carcinoma: therapeutic implications

“…Also, Ouyang et al showed that the Raf kinase inhibitors AAL881 and LBT-613 suppress the growth of BRAF V600E thyroid carcinoma cells (18). We extended these studies to compare the effect of protein depletion and enzymatic inhibition between BRAF wt and BRAF V600E thyroid carcinoma cell lines.…”

“…To obtain further insight into how B-Raf function can be therapeutically targeted, we expanded on our B-Raf inhibition studies using the small molecule B-Raf kinase inhibitor AAL881 (18,19). AAL881 had more potent antitumor activity against BRAF V600E than BRAF wt cell lines, and in further confirmation of its selective effect on mutant B-Raf, AAL881 suppressed MEK and ERK phosphorylation more efficiently in BRAF V600E cell lines, suggesting higher affinity of this inhibitor for the threedimensional structure of the mutant kinase domain.…”

“…This selectivity is supported by early data on the inhibition of enzymatic activity of various Raf forms by AAL881 (IC 50 for enzymatic activity of BRAF V600E and BRAF wt was 0.22 and 0.94 Amol/L, respectively; ref. 18). This represents a paradigm for an alternative therapeutic approach, in which selectively targeting the mutant kinase form lowers the risk of side effects related to inhibition of the wild-type kinase in normal cells, at the expense of narrowing the therapeutic spectrum of such an inhibitor only to cancers harboring the respective mutation.…”

“…The B-Raf inhibitor AAL881 (18,19) was kindly provided by Dr. David Batt (Novartis Institutes for BioMedical Research, Inc., Cambridge, MA). The anti-Fas agonistic monoclonal antibody CH-11 was obtained from Panvera (Madison, WI).…”

An update on molecular biology of thyroid cancers

“…The efficiency of AAL-81 and LBT-613, other inhibitors of RAF kinases, have been tested in thyroid cells in preclinical studies [230]. Both agents were determined to inhibit MAPK No study results posted yet [264] Motesanib Phase II completed Inhibits VEGF 14% [262] signaling and growth of human thyroid tumor cell lines and rat thyroid cells carrying the V600E BRAF and RET/PTC1 mutations.…”