CML arises owing to a translocation between the BCR serine/threonine kinase gene and the ABL1 tyrosine kinase³. Imatinib mesylate, which binds to the ABL1 kinase domain and inhibits phosphorylation of substrates, has been used to treat CML, but it is not curative because the cancer stem cells that propagate the leukaemia are resistant to therapy and are not eradicated^5–7. Furthermore, imatinib resistance due to mutations in the drug-binding site can occur, especially in advanced stage disease, leading to disease relapse and progression⁴. The design of effective new therapies thus critically depends on the identification of signals that are required for CML propagation and in particular signals required for CML cancer stem cell maintenance.

Given the parallels in signalling between development and cancer^8,9, we examined whether Hh signalling, an important regulator of development and oncogenesis in many tissues^1,10, might also have a role in CML. To this end we conditionally deleted Smo—a protein that is essential for Hh signal transduction². Smo is negatively regulated by the Hh receptor Patched (Ptch) in the absence of Hh protein signals. This inhibition is relieved when Ptch is bound by the Hh proteins Shh, Ihh or Dhh. Subsequently, Smo causes activation of Hh pathway targets via the Gli family of transcriptional effectors².

Smo was deleted in the haematopoietic system by crossing mice carrying a loxP-flanked Smo allele¹¹ to mice in which Cre is driven by Vav regulatory elements¹². Smo was efficiently deleted in haematopoietic cells of Vav-Cre;Smo^loxP/loxP mice as compared to control Smo^loxP/loxP mice lacking Cre (Fig. 1a, b). Although the frequency of haematopoietic stem cells (HSCs) and differentiated cells was unchanged in the Smo-deficient (Smo^−/−) mice (Supplementary Fig. 1), these animals had a clear defect in long-term HSC function in primary and secondary transplants (Fig. 1c and Supplementary Fig. 1). Decreased reconstitution was not due to decreased homing or to a preferential loss of any specific lineage (Supplementary Figs 1 and 2), and could be recapitulated with whole bone marrow transplants (Supplementary Fig. 3). Cumulatively, these data indicate that Smo is required for HSC renewal in vivo.

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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.

We next investigated the role of Hh signalling in CML initiation and propagation. CML can be modelled by transplanting BCR–ABL1-transduced haematopoietic progenitors into irradiated mice¹³. Whereas control cells transduced with BCR–ABL1 caused CML in 94% (16 out of 17) of recipients within 3 months, similarly transduced Smo^−/− cells caused CML in only 47% (8 out of 17) of recipients (Fig. 1d), and these tumours showed an increased latency. The reduced CML incidence was not due to altered homing or engraftment (Supplementary Fig. 4). These data suggest that Hh activity is required for the initiation and propagation of CML. The propagation of several cancers has been shown to depend on cancer stem cells, a critical subset of cancer cells that are capable of transferring the disease to a new host⁸. Because deletion of Smo led to impaired CML growth, we postulated that the loss of Smo might affect CML stem cells. As shown in Fig. 1e, f, the frequency of c-Kit⁺ Lin⁻ Sca-1⁺ (KLS) cells (which have previously been shown to be responsible for propagating CML⁶) was significantly reduced in the absence of Smo.

The fact that loss of Smo led to decreased numbers of CML stem cells led us to investigate whether activation of the Hh pathway might lead to an increased frequency of CML stem cells and acceleration of disease. To test this, we used a transgenic mouse expressing an activated form of Smo (SmoM2) fused to yellow fluorescent protein (YFP)¹⁴; expression of this gene from the Rosa26 locus promoter is blocked by a loxP-flanked stop sequence, and is thus inducible by Cre. When SmoM2–YFP mice were crossed with Vav-Cre mice, progeny expressed YFP in >70% of c-Kit⁺ Lin^−/lo Sca-1⁺ Flk2⁻ (KLSF) cells (Fig. 2a). Although the infection of both control and SmoM2 KLSF cells with BCR–ABL1 resulted in CML, the frequency of CML stem cells in animals receiving SmoM2 cells was fourfold greater than in mice receiving control cells (Fig. 2b, c). As expected, SmoM2 was expressed in transgenic but not in control CML stem cells (Fig. 2d). Most importantly, the increased CML stem cell frequency led to a significant acceleration of CML progression (Fig. 2e). SmoM2 expression also accelerated CML growth when activated post-embryonically (Supplementary Fig. 5). Thus genetic loss- and gain-of-function experiments indicate that Hh pathway activity controls the frequency and maintenance of CML stem cells, and consequently the incidence and latency of CML formation.

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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).

Our previous studies suggested that in normal haematopoietic stem cells, maintenance of the undifferentiated state depends on low levels of the cell fate determinant Numb¹⁵. We therefore tested whether Numb may act similarly in CML stem cells, and whether the effects of loss of Smo might be due to altered levels of Numb. We found that a greater frequency of Smo^−/− CML KLS cells had high levels of Numb as compared to control CML KLS cells (Fig. 3a–c). Additionally, ectopically expressed Numb inhibited in vitro propagation of BCR–ABL1-infected haematopoietic cells (Fig. 3d) and of CML stem cells from established leukaemias (Fig. 3e). These data suggest that increased Numb expression may contribute to the loss of CML stem cells in the absence of Smo.

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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.

Our genetic data showing that CML stem cells depend on Hh pathway activity raised the possibility that these cells might be targeted by pharmacological blockade of this pathway. We therefore tested the effect of cyclopamine, which inhibits Hh signalling by stabilizing Smo in its inactive form¹⁶. Exposure of CML stem cells to cyclopamine at a dose determined to minimize off-target effects and toxicity (Supplementary Fig. 6) led to a twofold inhibition of colony growth (Fig. 4a). This could be recapitulated using the Hh-blocking antibody 5E1 (Supplementary Fig. 7), suggesting that Hh pathway activation in CML may be ligand dependent^17,18. We also delivered cyclopamine to animals transplanted with BCR–ABL1-infected HSCs; whereas all of the control animals succumbed to CML within 4 weeks, 60% of the cyclopamine-treated mice were still alive after 7 weeks (Fig. 4b and Supplementary Fig. 8). Furthermore cyclopamine-treated mice had up to a 14-fold reduction in the CML stem cell population (Fig. 4c, d); consistent with this depletion, leukaemias from cyclopamine-treated mice were unable to propagate disease effectively when transplanted (Supplementary Fig. 9). These data suggest that cyclopamine can target the CML stem cell compartment critical for propagation of CML. Notably cyclopamine was most effective when initiated at early stages after CML establishment (Supplementary Fig. 10).

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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.

One rationale for identifying new pathways involved in CML is that targeting these pathways may be used to prevent or overcome imatinib resistance¹⁹. To test whether cyclopamine could impair the growth of imatinib-resistant CML, we infected cells with virus encoding either wild-type BCR–ABL1 or the T315I mutant^4,6,20 that is resistant to imatinib and other tyrosine kinase inhibitors used at present. Cells infected with mutant BCR–ABL1 were unresponsive to imatinib (Fig. 4e), but their growth was reduced 2.5-fold by cyclopamine (Fig. 4e). Progression of drug-resistant CML in vivo was also significantly impaired in the presence of cyclopamine (Fig. 4f) as well as in the genetic absence of Smo (Supplementary Fig. 11). These data indicate that Hh pathway activity is required for maintenance of CML stem cells, and raise the possibility that small molecule inhibitors of the pathway could be useful for targeting both normal and imatinib-resistant CML.

Finally we tested whether targeting this pathway could inhibit the growth of human CML. Freshly isolated primary human blast crisis CML samples expressed SMO (Fig. 4g). To determine whether SMO may functionally contribute to human CML growth, we tested the effect of cyclopamine on both a human CML cell line and patient samples in a methylcellulose assay. Cyclopamine decreased colony formation in an imatinib-resistant human CML cell line (Fig. 4h) and in three independent primary patient samples (Fig. 4i and Supplementary Fig. 12), but not in untransformed cord blood cells (Supplementary Fig. 13). Consistent with this, inhibition of SMO by the delivery of a new SMO antagonist also impaired the propagation of imatinib-resistant human CML cells in xenografts (A.A., M.M., T.V.A. and C.H.J., manuscript in preparation). Cumulatively, these data suggest that Hh signalling is important for human CML growth and that it may continue to have a role even as the disease progresses into blast crisis phase.

The studies discussed earlier demonstrate a dependence of normal and neoplastic stem cells on Hh signalling. Previously, mammalian Hh proteins have been reported to preserve and increase the short-term repopulating capacity of human HSCs²¹. Our genetic loss-of-function studies indicate that intact Hh signalling is needed for HSC renewal. This requirement may be shared with the nervous system, in which progenitor development is dependent on Hh pathway activation^22,23. Just as Hh signalling seems to be required for stem cells, its aberrant activation has been associated with the development of several solid cancers^17,18,24,25. In the haematopoietic system, Hh signalling has been implicated in the growth of B cell lymphoma²⁶ and multiple myeloma²⁷. Our observations provide critical genetic evidence for a role of Hh signalling in myeloid leukaemia, and together with previous work, suggest that this pathway may have a broader role in haematological malignancies. Our data are also unique in demonstrating that loss of endogenous cancer stem cells impairs in vivo cancer progression, supporting the notion that the leukaemia stem cells identified by transplantation are the relevant drivers of endogenous cancer growth^28–30. The real impetus for identification of new pathways that drive cancer is the opportunity to inhibit these pathways instead of, or in addition to, conventional therapies. This is especially true for CML, in which imatinib is incapable of eradicating the disease, not only necessitating its continual use but also increasing the likelihood of resistance and relapse. Our findings that the pharmacological inhibition of Hh signalling can impair growth of CML driven by wild-type and imatinib-resistant BCR–ABL1, as well as human CML, raise the possibility that Hh antagonists may be useful as a therapy for normal and drug resistant CML.