Combination of the Hedgehog Pathway Inhibitor LDE225 and Nilotinib Eliminates Chronic Myeloid Leukemia Stem and Progenitor Cells.

Abstract 1428

Poster Board I-451

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder arising in a hematopoietic stem cell (HSC). CML is associated with expression of the Philadelphia chromosome (Ph) and its fusion gene product, BCR-ABL, a constitutively active tyrosine kinase. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib, all show impressive rates of complete cytogenetic response in chronic phase (CP) CML. However, the majority of responding CP CML patients have detectable BCR-ABL transcripts which might arise from a population of quiescent CML stem cells not effectively targeted by TKIs. Recent studies have indicated that the Hedgehog (Hh) pathway, a developmental pathway with roles in primitive and adult hematopoiesis, is activated in CML stem cells via upregulation of Smoothened (SMO), a seven-transmembrane domain receptor protein (Dierks et al, Cancer Cell 2008;14:238). We found that Gli1, a downstream target of Hh signalling, is significantly upregulated (6-fold) at the mRNA level in CD34+ enriched CP CML cells compared to normal CD34+ hematopoietic cells by Taqman quantitative RT-PCR. Thus, inhibition of SMO may be an effective therapeutic strategy to reduce the quiescent CML stem cell pool. LDE225 (Novartis Pharma) is a small molecule SMO antagonist which has recently entered Phase 1 clinical evaluation in patients with solid tumors. We assessed the efficacy of LDE225, alone and in combination with nilotinib, in primary CD34+ CML cells in vitro using short- and long-term culture techniques, including colony forming cell (CFC) assays with replates, long-term culture-initiating cell (LTC-IC) assays, CFSE-based flow cytometry to track cell division, Annexin/Viaprobe to measure apoptosis and Ki-67/7AAD cell cycle analysis. In short-term culture, incremental concentrations of LDE225 (0.5 - 1000nM) had no effect on total viable cell counts up to 12 days and did not inhibit cell proliferation as assessed by BrDU incorporation and CFSE staining. LDE225 did not increase apoptosis or alter the cell cycle profile of CD34+ CML cells compared to untreated controls. The lack of response in short-term culture experiments is explained by the stem cell selective nature of LDE225 which affects self-renewal and not proliferation or apoptosis. For long-term culture experiments, CD34+ CML cells were cultured for either 3 or 7 days in serum-free media supplemented with physiological growth factors (IL-3, IL-6, G-CSF, FLT-3 and SCF) with and without LDE225; cells were then washed and put into CFC assays with no added LDE225. Increasing concentrations of LDE225 did not significantly reduce colony read-out in the CFC assays. However, when the CFCs were serially replated there was a highly significant reduction in secondary colony formation and replating efficiency with increasing concentrations of LDE225 up to a plateau at 50nM during the initial 72 hour culture. In the first re-plate, colony formation was reduced by 38% with 5nM (P=0.07) and 62% with 50nM LDE225 (P=0.011; n=3) compared to a “no drug” control. Further reductions in colony formation were seen in second and subsequent replates. In further CFC replating experiments, after 3 and 7 days initial exposure to the combination of LDE225 10nM + nilotinib 5μM, replating efficiency was reduced by 50% (P<0.03) and 74%,(P<0.005) respectively (n=3). Single agent nilotinib resulted in a non-significant increase in colony formation and replating efficiency. In LTC-IC assays, compared to the “no drug” control, CD34+ CML cells showed increased colony formation in the nilotinib arm, indicating that, by inhibiting proliferation, nilotinib exerts a protective effect on CML stem cells (as shown previously for imatinib and dasatinib; Copland et al, Blood 2008;111:2843). The arm containing LDE225 + nilotinib showed a reduction in colony formation compared to both the nilotinib arm (85% reduction) and the “no drug” control (50% reduction). These results confirm the stem cell selectivity of LDE225. In conclusion, LDE225 targets CML stem cells and, in combination with nilotinib, represents a novel therapeutic strategy for targeting both the primitive quiescent CML stem cell population and the bulk CML progenitor population. In vivo studies in murine models of CML are ongoing to help further evaluate the clinical potential of LDE225 in combination with nilotinib.