Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia

Abstract

Although the role of Hedgehog (Hh) signalling in embryonic pattern formation is well established, its functions in adult tissue renewal and maintenance remain unclear, and the relationship of these functions to cancer development has not been determined. Here we show that the loss of Smoothened (Smo), an essential component of the Hh pathway, impairs haematopoietic stem cell renewal and decreases induction of chronic myelogenous leukaemia (CML) by the BCR-ABL1 oncoprotein. Loss of Smo causes depletion of CML stem cells--the cells that propagate the leukaemia--whereas constitutively active Smo augments CML stem cell number and accelerates disease. As a possible mechanism for Smo action, we show that the cell fate determinant Numb, which depletes CML stem cells, is increased in the absence of Smo activity. Furthermore, pharmacological inhibition of Hh signalling impairs not only the propagation of CML driven by wild-type BCR-ABL1, but also the growth of imatinib-resistant mouse and human CML. These data indicate that Hh pathway activity is required for maintenance of normal and neoplastic stem cells of the haematopoietic system and raise the possibility that the drug resistance and disease recurrence associated with imatinib treatment of CML might be avoided by targeting this essential stem cell maintenance pathway.

Figure 1. Conditional deletion of Smo impairs the development of BCR–ABL1-induced CML and depletes CML stem cells

a, Real-time PCR (rtPCR) using primers specific for the loxP sites was carried out on Smo^−/− or Smo^+/+ bone marrow genomic DNA (n = 3, *P = 0.03). RFU, relative fluorescent units. b, rtPCR for Smo using complementary DNA from Smo^−/− or Smo^+/+ KLSF cells (n = 7, *P < 0.00001). c, KLSF cells from Smo^−/− or Smo^+/+ mice were transplanted together with competing bone marrow cells into congenic recipients and donor-derived chimaerism was monitored. The graph shows the average donor-derived chimaerism after long-term reconstitution (n = 2 with 20 mice, P = 0.0002). d, Survival curve of mice transplanted with BCR–ABL1-infected Smo^−/− or Smo^+/+ KLSF cells (n = 3 with 34 mice, P = 0.0002). e, Representative example of CML stem cells (GFP⁺ Lin⁻c-Kit⁺ Sca⁺) in the bone marrow of mice transplanted with BCR–ABL1-infected Smo^−/− or Smo^+/+ KLS cells. f, Graph of the average percentage of CML stem cells in mice receiving BCR–ABL1-infected Smo^+/+ (8 mice) or Smo^−/− KLS cells (9 mice). Frequency shows c-Kit⁺Sca⁺ cells as as a percentage of the GFP⁺Lin⁻ population. *P = 0.006. Error bars in all bar graphs are s.e.m.

Figure 2. The presence of constitutively active Smo increases the frequency of CML stem cells and accelerates disease

a, Analysis of YFP fluorescence, which reflects SmoM2 expression in control (con) and SmoM2 KLSF cells (n = 4). b, Analysis of CML stem cells (GFP⁺ KLS) in mice transplanted with BCR–ABL1-infected control (left) and constitutively activated Smo (SmoM2, right) KLSF cells. c, The average percentage of CML stem cells in mice receiving BCR–ABL1-transduced control (n = 4) and SmoM2 KLSF cells (n = 12). Error bars are s.e.m., *P = 0.048. d, rtPCR analysis of SmoM2 expression in CML stem cells. e, Survival curve of mice receiving BCR–ABL1 transduced control (solid line) or SmoM2 KLSF cells (dashed line; n = 2, 16 mice, P = 0.0082).

Figure 3. Loss of Smo increases frequency of cells with high levels of Numb and contributes to decreased CML growth

a, CML stem cells from Smo^+/+ and Smo^−/− leukaemias were stained with Numb (red, top panel) and 4,6-diamidino-2-phenylindole (DAPI; blue, bottom panel). Scale bar, 10 μM. b, The average fluorescence intensity (AFI) was determined by dividing the overall mean fluorescence intensity by the area of the cell (P = 0.002). c, The frequency of cells with high expression levels of Numb was calculated by designating cells above a mean fluorescence intensity value of 1,000 as high expressors (n = 3, using either CML KLS or CML c-Kit⁺ cells). d, KLSF cells were infected with MSCV-BCR-ABL1-IRES-GFP and either vector MSCV-IRES-YFP or MSCV-Numb-IRES-YFP. GFP and YFP double-positive cells were plated in methylcellulose, colonies were counted and cells were serially replated (n = 2, *P < 0.006). e, CML KLS cells were infected with viruses expressing control vector or Numb IRES-YFP. Infected cells were sorted and plated in methylcellulose (n = 2, P = 0.03). Error bars in b–e are s.e.m.

Figure 4. Pharmacological inhibition of Smo impairs CML development and propagation

a, CML stem cells were plated in methylcellulose in the presence of 1 μM tomatidine (control) or cyclopamine (n = 2, *P = 0.005). b, Survival curve of mice transplanted with BCR–ABL1-infected KLS cells in the presence of vehicle or cyclopamine (n = 3 with 32 mice, P = 0.02). c, Representative example of CML stem-cell frequency in vehicle- or cyclopamine-treated CML. d, Average CML stem cell frequency in vehicle-or cyclopamine-treated mice (n = 4, *P = 0.03). e, KLSF cells were infected with wild-type BCR–ABL1 or the imatinib-resistant T315I mutant BCR–ABL1, and plated in methylcellulose with imatinib (2 μM) or cyclopamine (1 μM) (n = 2, *P < 0.004). f, Survival curve of mice receiving T3151 BCR–ABL1-infected KLSF cells and either vehicle or cyclopamine, 7 days after transplantation (n = 2 with 16 mice, P = 0.021). g, SMO PCR on primary uncultured human CML samples. h, Imatinib-resistant K562 blast crisis CML cells were plated in methycellulose in the presence of 1 μM tomatidine or cyclopamine (n = 2, *P < 0.01). i, CD34⁺ cells were sorted from primary uncultured human blast crisis CML samples and plated in methylcellulose in the presence of 1 μM tomatidine or cyclopamine (n = 3, *P < 0.05). Error bars in all bar graphs are s.e.m.