Blockade of the Hedgehog Signaling Pathway by the Novel Agent NVP-LDE225 Induces Differentiation, Prevents De-Differentiation and Inhibits Proliferation of Multiple Myeloma Stem Cells in Vitro

Abstract 3950

Chemotherapy-resistant cancer stem cells are a major cause of relapse. It is considered that the small population of CD38++ cells which also exhibit “side population” (SP) characteristics after Hoescht 33342 staining contain chemotherapy resistant multiple myeloma (MM) stem cells. The hedgehog (Hh) signalling pathway is a key regulator of stem cell proliferation and differentiation. A range of Hh inhibitors are currently under development as therapeutics. Our aim was to determine the effect that NVP-LDE225, a novel Hh pathway inhibitor has on MM stem cells.

NVP-LDE225 was kindly supplied by Novartis. Side population (SP) cells, considered to contain the MM stem cells, were detected after Hoescht 33342 staining using a BD ARIA II flow cytometer, Proliferation was monitored with CFSE tracking and viability with propidium iodide. MM cell lines RPMI8226, KMS-11, OPM2, U266 and primary MM cells were included. Expression of Hh pathway proteins PTCH1, Smo and Gli1 was determined by flow cytometry. Bone marrow samples were obtained from patients with myeloma after informed consent.

CD38++ SP cells were identified in all 4 MM cell lines tested and 90% (26/29) of MM BM samples with the percentage of SP cells ranging from 0 to 9.5% of CD38++ cells. There was no correlation between %SP and %CD38++ and no significant difference in %SP during therapy. PTCH1 expression was higher on SP cells of KMS-11, OPM2 and U266 than non-SP cells (80.5% vs 51.7%) suggesting that the canonical Hh pathway was upregulated in CD38++ SP cells. PTCH1 expression varied from 0.1 – 87% on patient's CD38++ cells. In vitro dose response curves demonstrated increased killing of plasma cell lines above 1μM NVP-LDE225. The IC₅₀ was 4.7uM for KMS-11 SP cells and 3.4uM for KMS-11 non-SP cells. During culture of flow-sorted CD38++ SP+ and non-SP cells (RPMI 8226 and KMS11), fluctuations in %SP suggest that SP cells differentiated to non-SP cells but also that the SP phenotype is unstable. This is supportive of the concept that the stem cell compartment is in a state of flux. The presence of NVP-LED225 caused a 90% inhibition of non-SP returning to SP and induced an additional 79% differentiation of SP to non-SP KMS11 cells on day 2. Proliferation studies using CFSE tracking of sorted SP cells demonstrated a 34% reduction in proliferation after exposure to NVP-LDE225. Studies with 9 primary human bone marrow samples from patients with MM confirmed the data from cell lines demonstrating that differentiation was significantly induced (t=2.78; p<0.0275) and de-differentiation was blocked.

The studies confirm there is flux, to and from the SP and presumably MM stem cell compartment, that the canonical Hh pathway is upregulated in MM SP cells, and that the Hh pathway inhibitor NVP-LDE225 can induce SP differentiation, block de-differentiation, inhibit SP proliferation and at high concentrations is cytotoxic to all cells including MM stem cells. NVP-LDE25 could synergise with other maintenance therapy to reduce drug resistance by inducing the differentiation of chemo-resistant MM stem cells.