Targeting the Hedgehog Signaling Pathway in Therapy-Resistant BCR-ABL1 Positive Leukemia with Ponatinib

Abstract 63

The hedgehog signaling pathway is a key regulator of cell growth and differentiation during development. While the hedgehog pathway is inactive in most normal adult tissues, hedgehog pathway reactivation has been implicated in the pathogenesis of several neoplasms. Recent studies demonstrated that hedgehog pathway is involved in the development of B cell acute lymphoblastic leukemia (B-ALL), as well as self-renewal and survival of B-ALL. Vismodegib is a selective hedgehog pathway inhibitor that blocks hedgehog signaling by binding to Smo and inhibiting activation of downstream hedgehog target genes. In the present study, we investigated the combined effects of vismodegib and ponatinib, a pan-ABL1 kinase inhibitor, in mutant forms of BCR-ABL1-expressing BaF3 cells and T315I-expressing human leukemia cell line, SK-9 (Exp Hematol. 2010; 38:765). We observed that the treatments with sonic hedgehog (Shh) enhanced the proliferation of SK-9 cells, correlated with the up regulation of Cyclin D2 and Bcl-2. The treatment with Shh significantly reduced the induction of apoptosis in ponatinib-treated SK-9 cells, however, co-tratment with vismodegib and ponatinib resulted in significantly more induction of apoptosis in Shh-treated SK-9 cells. Combined treatment with vismodegib and ponatinib in SK-9 cells also associated with the reduction of Cyclin D2 and Bcl-2, and more PARP cleavage, resulting from increased activation of caspase-3 and -9 during apoptosis. We next conducted the experiments to further evaluate the mechanism of cooperation between vismodegib and ponatinib in SK-9 cells. SK-9 cells were transfected with control siRNA or Smo siRNA or Gli1 siRNA. At 48 h after transfection, Shh co-cultured SK-9 cells were treated with indicated concentration of ponatinib for 48 h, and viable cells were counted. In the presence of Smo siRNA or Gli1 siRNA, SK-9 cells increased antiproliferative activity with ponatinib. These results demonstrated that hedgehog signaling activation impairs the efficacy of pan-ABL1 kinase inhibitor. To assess the in vivo efficacy of ponatinib and vismodegib, athymic nude mice were injected s.c. with BaF3 cells expressing wild-type (WT)-BCR-ABL1 and BCR-ABL1 mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). 5 days after injection (average tumor volume, 100 mm³), the mice were randomized into four groups (5 mice per group), with each group receiving either vehicle, ponatinib (30 mg/kg; q.d.), vismodegib (10 mg/kg; q.d.), ponatinib (30 mg/kg; q.d.) + vismodegib (10 mg/kg; q.d.). The ponatinib and vismodegib combination more effectively inhibited tumor growth in mice compared to either vehicle- or ponatinib- or vismodegib-treated mice. Histopathologic analysis of tumor tissue from ponatinib + vismodegib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL stain. To investigate combined effects of vismodegib and ponatinib on T315I-expressing human leukemia cell line, NOD/SCID mice were injected intravenously with SK-9 cells. Treatment with vismodegib and ponatinib demonstrated a marked segregation of apoptotic cells in both the central bone-marrow cavity, the endosteal surface, spleen and liver. These results suggest that the combination with a Smo inhibitor and ABL1 tyrosine kinase inhibitors (TKIs) may help to eliminate the therapy-resistant T315I BCR-ABL1 positive ALL cells. In summary, our preclinical results indicate that vismodegib has potential as an important option for controlling minimal residual cells in BCR-ABL1-positive ALL. The combined results of cell-based, and in vivo studies suggest that vismodegib exhibits sufficient activity against mutants form of BCR-ABL1 to warrant consideration for combined use with pan-ABL1 TKIs.