Qualitative RT-PCR analysis of Hh pathway gene expression in precursor B-ALL cell lines. HFB, human fetal brain, used as positive control for all experiments. For each cell line, positive and negative control reactions were run with B-actin primers with (to demonstrate equal amounts of cDNA template across each cell line) and without (to rule out contamination with genomic DNA) reverse transcriptase. B-ACTIN- denotes PCR results from mock cDNA synthesis reactions lacking RT. Precursor B-ALL cell lines: HB 1119 (t11;19), Nalm6 (t2;6), REH (t12;21), RS4;11 (MLL gene rearrangement); SEM-K2 (MLL gene rearrangement), TOM-1 (Ph⁺). Electrophoresis was performed with each gene separately across the panel of cell lines. The separate gels are shown together for the purpose of clarity across cell lines in the figure.

Precursor B-ALL cell lines REH and RS4;11 were transiently transfected with a Gli-responsive luciferase reporter or pGL3-basic, a plasmid lacking GLI binding site, as a negative control. Relative luciferase levels were measured following 48 hours of treatment with vehicle control, purified, dual lipid-modified Shh ligand (Shh-NP), SHh antibody 5E1 (10 µg/ml), cyclopamine (5 µM) or IPI-926 (1 µM). Hh pathway stimulation with SHh (p<0.005) and inhibition with 5E1 (p<0.0001), cyclopamine (p<0.0005) and IPI-926 (p<0.0002) was statistically significant in both the REH and RS4;11 cell lines.

(A)ALDH⁺ and ALDH^neg cells were isolated from the REH cell line by FACS using the Aldefluor reagent and DEAB control. The gated area represents the ALDH⁺ cells, measuring 1.18% of total REH population. (B) ALDH⁺ and ALDH^neg cells from REH and RS4;11 were plated in methylcellulose to evaluate clonogenic growth. Colony formation was measured at 10–14 days. There was a 5-fold difference in colony number following initial plating for REH (p = 0.05) and RS4;11 (p<0.01). (C) A representative plate from initial plating was washed with media and cells resuspended prior to replating in methylcellulose for an additional 10-14 days. Secondary plating of ALDH⁺ and ALDH^neg cells demonstrated an 8-fold difference in colony-formation between the ALDH⁺ and ALDH^neg cell fractions for REH (p<0.002) and RS4;11 (p<0.001). (D)Unfractionated REH and RS4;11 cells were treated with cyclopamine (5 µM), IPI-926 (1 µM) or vehicle control for 10 days and the percentage of ALDH⁺ and ALDH^neg cells was measured using the Aldefluor staining kit and FACS. The reduction in ALDH^neg cells following treatment with cyclopamine and IPI-926 was statistically significant for REH (p<0.01) and RS4;11 (p<0.03).

Clonogenic recovery of REH and RS4;11 cells following treatment with Hh pathway agonist and inhibitors. B-ALL cells were treated with Hh pathway modulators for 72 hours, washed free of drug and plated in quadruplicate in methylcellulose. At 10–14 days, colonies were counted (represented as initial plating). A representative plate was then washed and cells resuspended and replated. After an additional 10–14 days, colonies were counted (represented as secondary replating). Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups are expressed as % control ± SEM. (A)Colony-formation of REH and RS4;11 cells following treatment with the Hh pathway ligand SHhNP. Increased colony-formation was statistically significant in both cell lines at initial and secondary replating despite no further addition of drug (p<0.001 for all groups). (B) Colony-formation of REH and RS4;11 cells following treatment with Hh pathway inhibitors 5E1 (10 µg/ml), cyclopamine (5 µM), IPI-926 (1 µM). Inhibition of clonogenic growth at secondary replating for both REH and RS4;11 cells was statistically significant (p<0.02) with Hh pathway inhibition with 5E1, cyclopamine and IPI-926.

Representative flow cytometry results of mouse bone marrow demonstrating engraftment of human leukemia. Mice were injected with REH cells and then treated with either vehicle control or IPI-926 for 21 days. Mouse bone marrow was harvested after development of symptoms and analyzed by flow cytometry for the presence of human leukemia cells. All mice in both treatment groups had engraftment of human leukemia cells that were mouse CD45 negative, human CD45 positive and human CD19 positive.

NOD-Scid mice were injected by tail vein with REH cells that had been pre-treated with either IPI-926 or vehicle in vitro prior to injection. Mice were followed for progression of symptoms and overall survival, and recipient mice of pre-treated cells had statistically significantly longer overall survival (70 days versus 50 days, p<0.001).