Canonical and noncanonical Hedgehog pathway in the pathogenesis of multiple myeloma

The Hedgehog (Hh) pathway regulates multiple processes involved in development and differentiation of tissues and organs during embryonic life.¹  Recently, it has become evident that Hh signaling retains some activity even during adult life: in mature tissues, it regulates tissue homeostasis and repair and, in those tissues undergoing constant renewal, such as skin, colon, liver, and blood, it is also implicated in maintaining a stem/progenitor cell compartment,^2,3  explaining how the Hh pathway deregulation may cause developmental defects during the embryonic life.^1,4  Its abnormal activity can also lead to tumorigenesis during adult life either by stem cell pool expansion^2,3  or mutations affecting the normal growth-regulatory mechanisms.^5-7  An aberrant expression of developmental genes from Wnt and Hh pathways has been reported during malignant transformation of multiple myeloma (MM) cells.⁸  Several findings support a role of Hh signaling in regulating a stem cell niche also in MM⁹  and in modulating clinical response to conventional and novel therapeutic agents.¹⁰  Indeed, Hh ligands produced by murine bone marrow stromal cells (BMSCs) support growth and survival of human primary CD19⁺ lymphoma and CD138⁺ MM cells, demonstrating a role of the Hh pathway both in lymphoma and in terminally differentiated MM cells.¹¹  Finally, we recently showed ciliary protein overexpression as a possible cause of constitutive and noncanonical Hh pathway activation, suggesting a cilia-dependent mode of Hh signaling in MM.¹² 

Aberrant Hh signaling has been described in almost all tumors and is associated with 3 possible mechanisms: genetic alterations, autocrine and/or paracrine Hh activity, and alternative and synergistic pathways leading to Hh gene activation.^13,14  Sonic (Shh), Desert (Dhh), and Indian (Ihh) hedgehog are the ligands for the pathway. The signaling is triggered by binding of endogenously or exogenously produced ligand to Patched1 (Ptch1) on target cells. This leads to inhibition of Ptch1 via cellular internalization and Smoothened (Smo) localization on the cell surface, both by a cilium-mediated mechanism.¹⁵  Smo activation leads to nuclear translocation of Glioma (Gli) transcription factors followed by expression of Gli target genes, including Ptch1 and Gli1, in a negative and positive feedback loop, respectively. The biologic effect is cell proliferation, with deregulation contributing to tumorigenesis. Abnormal Hh pathway activation occurs not only through ligand-dependent or receptor-induced signaling, also known as canonical Hh signaling, but also by mechanisms of activation downstream to Smo known as noncanonical or ligand-independent Hh signaling. Genetic alterations or ciliary protein overexpression leading to functional redundancy of Gli transcription factors, crosstalk between Hh signaling and unrelated pathways, are all causes of noncanonical Hh signaling activation.

In the present study, we first show Hh gene overexpression in CD138⁺ plasma cells (PCs) from persons with monoclonal gammopathy of undetermined significance (MGUS) compared with CD138⁺ PCs from healthy persons, MM and plasma cell leukemia (PCL) patients, suggesting that aberrant Hh activation is important in disease initiation. We further demonstrate that both canonical and Smo-dependent as well as noncanonical and Smo-independent mechanisms can contribute to Hh signaling activation in MM. Finally, we provide evidence that NVP-LDE225, a novel synthetic Smo antagonist currently in clinical development,^16,17  decreases MM cell viability in vitro by inducing specific down-regulation of Gli1 and Ptch1, hallmarks of cell response to Hh pathway. Moreover, the combination of NVP-LDE225 with bortezomib in vitro has a modest additive effect compared with either drug alone, and the in vivo study shows an increased antitumor activity by the combination compared with bortezomib alone.

The gene expression data were generated using our Affymetrix HG-U133A GeneChip Arrays (Affymetrix) from 4 healthy donors, 11 MGUS, 133 MM, and 9 PCL patients and from 23 human MM cell lines (HMCLs), obtained as previously described¹⁸  and deposited in National Center for Biotechnology Information's Gene Expression Omnibus (http://www.ncbi.nlm.mih.gov/geo) as accession no. GSE13591 and GSE6205, respectively. All samples were normalized and analyzed using the bioconductor function for Robust Multi-array Analysis¹⁹  in which perfect match intensities were background adjusted and normalized by means of quantile-quantile normalization. Gene expression levels were expressed as mean ± SEM. The statistical significance of differences among the groups was assessed using Kruskal-Wallis test. P values < .05 were considered significant.

Flow cytometry-based evaluation of Hh-protein expression was performed using a panel of MM cell lines and MM patient-derived BMSCs. U266, NCIH929, RPMI8226, and MM1R were obtained from ATCC. KMS12BM and KMS12PE were from German Collection of Microorganisms and Cell Cultures (DSMZ). MM1S was a gift from Steven Rosen (Northwestern University). OPM1 and OPM2 were provided by Dr P. Leif Bersagel (Mayo Clinic-Tucson). S6B45 and KMS11 were kindly provided by Dr T. Kishimoto (Osaka University) and the Kawasaki Medical School, respectively. All MM cells were cultured at 37°C in RPMI 1640 medium containing 10% heat inactivated FBS (Sigma-Aldrich), 2mM/L l-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen). CD138⁻ mononuclear cells obtained by microbead selection of primary MM samples were used to establish long-term BMSCs. To analyze the expression of Ptch1 and Smo receptors on MM cells, a single surface staining was performed by incubating MM cells in PBS/1% FBS in the presence of anti-Ptch1 or anti-Smo antibody (Ab) for 1 hour at 4°C. After washing, the cells were stained with specific secondary FITC-conjugated Ab for 45 minutes at 4°C and after final washes analyzed by a FACSCanto II flow cytometer (BD Biosciences). Cells stained with secondary Ab alone were used as negative controls to define background fluorescence and to gate the positive population. To confirm the specificity of Abs, we used 2 different primary Abs for each receptor: purified goat anti-Smo and rabbit anti-Ptch1 from Santa Cruz Biotechnology; and rabbit anti-Smo and anti-Ptch1 from Abcam. As secondary Abs, we used anti–rabbit FITC-conjugated (BD Biosciences) and antigoat AlexaFluor-488–conjugated (Invitrogen). To analyze Shh expression in the CD138⁺ population, we performed a double staining using anti-CD138 allophycocyanin (APC)–conjugated rat monoclonal Ab (R&D Systems) for surface staining followed after fixation with Cytofix/Cytoperm solution (BD Biosciences) for 30 minutes at 4°C, by intracellular staining with anti-Shh-PE–conjugated mouse monoclonal Ab (R&D System) in Perm/Wash buffer (BD Biosciences) for 1 hour at 4°C. After final washing, the cells were analyzed by flow cytometry. Isotype-matched control for both Abs (R&D Systems) was used as negative control. For the MM patient-derived BMSCs, single intracellular staining with anti-Shh PE-conjugated Ab was performed.

For Western blot analysis, MM cell lines and BMSCs from MM patients were harvested and lysed as previously described.¹²  For protein detection, we used anti-Smo and anti-Ptch1 Abs from Abcam and anti-Gli1Ab from Santa Cruz Biotechnology. Human heart lysate (Abcam) was used as positive control to detect Smo protein and lysate from HeLa and MCF-7 cell lines for detecting Ptch1 and Gli1, respectively. To study Ptch1 and Gli1 down-regulation, we treated cells in 1% FBS medium with 5μM NVP-LDE225 (Novartis) for 12, 24, and 36 hours. In addition, we assessed, by Western blotting analysis, time- and dose-dependent Hh-induced modifications of genes involved in cell cycle/apoptosis regulation and signal transduction, focusing on Hh-target genes in other cancers, such as Bcl-2, c-myc, cyclin-D1, caspase-3, ERK/pERK, and JNK/pJNK. The Abs used for immunoblotting included: rabbit anti-phosphop42/p44 MAPK (ERK1/2) and anti-p42/p44 MAPK (ERK1/2), anticaspase-3 from Cell Signaling Technology; rabbit anticyclin-D1, mouse anti-JNK, antiphospho JNK, anti–c-myc, anti-bcl2, and anti–α-tubulin as well as the secondary HRP-conjugated Abs were from Santa Cruz Biotechnology.

MM cell lines (MM1S,U266) previously growth overnight in 1% FBS RPMI medium were cultured for 24 hours in the presence or absence of 10μM forskolin (Sigma-Aldrich) or 5μM NVP-LDE225. After cytospin, cells were fixed in acetone and methanol (1:1) for 15 minutes at room temperature; after 3 washes in PBS for 10 minutes each, they were blocked in PBS with 5% FBS for 1 hour at room temperature. After washing, the cells were cultured overnight at 4°C in a humidified chamber with the primary anti-Gli1Ab 1:50 in blocking solution. After 3 washes on the following day, cells were incubated for 1 hour at 4°C in a dark chamber with the secondary antibody (anti–rabbit FITC-conjugated, 1:800). After final washes, 4′-6′-diamidine-2phenylindole was used to stain nuclei followed by analysis using an epifluorescence microscope (Nikon Eclipse E800, Nikon) and a Photometrics Coolsnap CF color camera (Nikon).

MM cell lines, BMSCs, and CD138⁺ PCs from MM patients, peripheral blood mononuclear cells (PBMCs) were treated for 24, 48, and 72 hours in RPMI medium supplemented with 1% FBS in the presence and absence of NVP-LDE225 in a range of doses from 1 to 20μM. The inhibitory effect of NVP-LDE225 on cell growth was assayed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) dye absorbance. In addition, we used annexin V–FITC assay for apoptosis qualification and flow cytometric analysis of DNA content of nuclei labeled with propidium iodide (PI) for evaluation of cell cycle changes, as described in the manufacturer's protocol (BD Biosciences PharMingen). MTT experiments have been performed in triplicate and repeated at least 3 times: data are presented as mean ± SD. Flow cytometry data are representative of 3 independent experiments. We also tested by MTT the effect on MM cell growth of 5μM NVP-LDE225 in combination with 5nM and 10nM bortezomib. Finally, to assess whether MM cell sensitivity to NVP-LDE225 is attenuated by coculture with cytokines and BMSCs, we used MTT assay to evaluate MM cell survival after adding BM supernatant and CFSE staining to quantify proliferation of MM cells cultured alone and cocultured with BMSCs, as previously described.²⁰ 

CB-17 SCID mice were purchased from Charles River Laboratories and maintained and monitored in our Animal Research Facility. All animal studies were conducted according to protocols approved by the National Directorate of Veterinary Services. Animals were killed when their tumors reached 2 cm in diameter or when paralysis or major compromise in their life occurred. CB-17 SCID mice were subcutaneously inoculated in the interscapular area with 2 × 10⁶ OPM1 cells in 100 μL RPMI medium. When tumors were measurable ∼ 3 weeks after the injection, animals were treated with vehicle alone (n = 6), NVP-LDE225 40 mg/kg (n = 7), NVP-LDE225 80 mg/kg (n = 6), bortezomib 1 mg/kg (n = 6), and bortezomib1 mg/kg + NVP-LDE225 80 mg/kg (n = 5). Vehicle and NVP-LDE225 alone were administered daily orally. Bortezomib was administered intraperitoneally on days 1, 4, 8, and 11. In the combination therapy group, NVP-LDE225 was added on day 8 and administered daily orally. All the drugs were given until the day of sacrifice or death. Tumor sizes were measured every 2 days in 2 dimensions using an electronic caliper, and tumor volume was calculated using the following formula: V = 0.5a × b², where a and b was the long and short diameter of the tumor, respectively. The statistical significance of differences among the groups was assessed using the Student t test. P values < .05 were considered significant.

We first evaluated the Hh-gene expression in PCs isolated from BM aspirate of 4 healthy donors, as well as 11 MGUS, 133 MM, and 9 PCL patients and 23 MM cell lines using oligonucleotide microarray analysis. Compared with normal healthy donors (Figure 1), PCs from MGUS patients express higher levels of Ihh/Shh, Smo, and Ptch1 receptors, as well as Gli1 and Gli2 transcription factors, but lower levels of the repressor genes Ptch2 and Gli3, with statistically significant difference (P < .05) for Smo, Ptch1, and Gli2; suggesting that Hh pathway activation may play a role in malignant transformation of PCs and disease initiation. Up-regulation of Ptch1, Gli1, and Gli2 and down-regulation of Ptch2 and Gli3 with statistically significant difference for the last 2 genes (P < .05) have also been observed in PCs from MM patients supporting a Gli-dependent Hh activation in MM pathogenesis. Conversely, the relative down-regulation of Hh-gene expression observed in PCL, a more advanced and BM-independent disease, and in MM cell lines with statistically significant difference (P < .05) for the majority of the analyzed genes, suggests a role for stroma-derived Hh-signals in triggering paracrine Hh activity in MM.

Although gene profiling showed a relatively lower Hh-gene expression in MM cell lines compared with normal PCs, we screened 11 MM cell lines for Hh protein. Because CD138 antigen is expressed, especially on terminally differentiated PCs, we used anti-CD138 APC-conjugated Ab for surface staining and anti-Shh PE-conjugated Ab for intracellular staining, followed by flow cytometric analysis for the percentage of double-positive cells. As seen in Figure 2A, Shh ligand was detected in both CD138⁺ as well as CD138⁻ MM cell population. In addition, using anti-Ptch1 and anti-Smo mAb, we observed cell surface expression of Ptch1 and Smo receptors on MM cells showing similar staining patterns for these 2 proteins using 2 different Abs (Figure 2B-D). We have also confirmed basal levels of Smo expression by Western blot analysis (Figure 2C bottom panel). Because Ptch1 is the Shh binder and Smo is the Hh signal transducer of this receptor complex, their coexpression is indicative of cell responsiveness to Hh ligands.

In the absence of ligand, the Hh pathway is turned off and Gli transcription factors remain cytosolic. In the presence of ligand and after its binding to Ptch1, the pathway is turned on and Gli1 is activated and migrates to the nucleus, thereby inducing activation of a plethora of genes involved in signal transduction, apoptosis, and cell cycle regulation. In the MM1S cell line, we observed both cytosolic and nuclear Gli1 using immunofluorescence; the nuclear presence of Gli1 suggests constitutive Hh pathway activation (Figure 2E top panel). This is completely abrogated by 24-hour treatment with 10μM forskolin, which inactivates Gli1 via PKA (Figure 2E bottom panel), indicating that Smo-independent mechanisms might contribute to Hh activation in MM.

To further investigate additional mechanisms of Hh signaling in MM, we used NVP-LDE225, a novel synthetic Smo inhibitor currently in clinical development.^16,17  At micro molar doses, NVP-LDE225 significantly reduced MM cell viability in vitro. In the majority of MM cell lines screened, IC₅₀ was 3-5μM after 48 hours (Figure 3A); after treatment with 5μM NVP-LDE225 up to 36 hours, specific down-regulation of Gli1 and Ptch1 proteins was observed (Figure 3B), suggesting the existence of canonical and Smo-dependent Hh activation in MM and also a specific Hh inhibition by NVP-LDE225. The down-regulation of Ptch1 in MM cells was observed at 12 hours, whereas down-regulation of Gli1 was observed at 36 hours. Indeed, Ptch1 down-regulation also occurred in the MM1R cell line, which does not express Gli1, and in U266 cells where Gli1 down-regulation was not observed. This indicates that Gli1 is not the only transcription factor involved in Hh-signal transduction in MM and suggests Gli2 involvement, in agreement with previous data showing a critical role of Gli2 in infiltrating PCs and in human plasmacytoma tumors.²¹  Despite Smo expression in the remaining cell lines screened, the observed inhibition of survival (IC₅₀ higher than 5μM) was not associated with Gli1 and/or Ptch1 down-regulation, suggesting that alternative, noncanonical, and Smo-independent mechanisms might contribute to aberrant expression of Hh components (Figure 3C-D). We did not find any correlation between Shh expression level and sensitivity to Smo inhibitor because cell lines highly positive for Shh (eg, RPMI8226 and S6B45; Figure 2A), were relatively less sensitive to NVP-LDE225 (Figure 3D), suggesting that some MM cell lines do not have autocrine Hh activity. Similarly, despite high expression levels of both receptors detected in all MM cell lines, not all MM cell lines were sensitive to Smo inhibitor, suggesting that protein expression levels do not reflect functional activity of the receptor in all cases. Indeed, in U266 cells with Smo-dependent activation, 5μM NVP-LDE225 treatment abrogated the nuclear localization of Gli1 at 24 hours (Figure 3E). PCs from MM patients also showed sensitivity to Smo inhibitor (Figure 3F left panel), whereas viability of PBMCs from 5 different healthy persons was not affected by treatment with 20μM NVP-LDE225 for 72 hours (Figure 3F right panel). Finally, we found that 24-hour treatment with 10μM NVP-LDE225 induces early as well as late apoptosis (Figure 4A), with accumulation of cells in G₂/M phase, decreased S phase, and increased sub-G₁ cell fractions (Figure 4B-C). Using NVP-LDE225 at dose and exposure time (5μM for 24 hours) that did not cause apoptosis and cell cycle modification (Figure 4A-C), we observed decreased c-myc, cyclin-D1, and Bcl-2 protein expression, ERK and JNK phosphorylation, and increased caspase-3 cleavage as earlier consequences of NVP-LDE225 treatment. These results suggest a potential involvement of these proteins in Hh-signal transduction in MM, as already described in other tumors.

Intracellular staining of MM patient-derived BMSCs with anti-Shh Ab showed significantly higher Shh expression in BMSCs (Figure 5A). Moreover, NVP-LDE225 treatment at 20μM for up to 48 hours had minimal effect on BMSC survival (Figure 5B). Western blot analysis demonstrated that BMSCs are Smo-defective and have variable Gli1 expression (Figure 5C) and Ptch1 up-regulation, which did not change after treatment with 5μM NVP-LDE225 (Figure 5C-D). In the absence of Smo, which is required to activate Hh-target genes, Shh-producing BMSCs do not have autocrine Hh activity but provide Shh ligand in a paracrine fashion. Importantly, MM-BMSC interaction or MM cell growth in the presence of BMSC supernatant did not induce resistance to NVP-LDE225 treatment (Figure 5E-F).

Recent data demonstrated that bortezomib exerts antitumor activity in medulloblastoma, by Ptch1 protein accumulation leading to down-regulation of Hh signaling.²²  Therefore, we evaluated the in vitro and in vivo antitumor efficacy of NVP-LDE225 alone and in combination with bortezomib. In vitro combination of 5nM or 10nM bortezomib with 5μM NVP-LDE225 for 24 hours showed only modest effect on MM cell viability compared with either drug alone (Figure 6A). In a mouse subcutaneous xenograft model of MM, daily oral dosing of NVP-LDE225 at 40 mg/kg showed only modest antitumor efficacy, with 21% tumor growth inhibition by 12 days compared with the control mice (P = .08); whereas bortezomib as a single agent treatment produced 57% tumor reduction (P = .009). Importantly, the combination of NVP-LDE225 with bortezomib showed additive cytotoxicity with 78% tumor growth inhibition compared with control mice (P = .0001) and with bortezomib as a single agent (P = .029), suggesting that combination therapy with the Smo inhibitor can improve bortezomib response.

The processes regulating self-renewal of both normal and cancer cells are poorly understood, but several pathways and genes, including Notch, Wingless (Wnt), and Hh, usually required for generation and maintenance of normal stem cells during embryonic development, have emerged as potential candidates.^2,3  The Hh pathway is particularly attractive because several small molecules, acting as agonists and antagonists of Hh signaling, are currently under development²³  and some of these have already showed clinical efficacy.^17,24  These developmental pathways are generally silenced in most adult tissues, except during tissue homeostasis and repair, but are reactivated in many diseases^1,4  and human cancers¹³  and therefore represent potential novel therapeutic targets. Here we provide evidence of the role of Hh signaling in MM pathogenesis and the therapeutic potential of targeting Hh pathway in MM.

To date, contrasting results, arising mostly from mice studies, are available about the role of Hh signaling in hematologic system and hematologic malignancies.^25-34  Several data demonstrate a specific involvement of Hh signaling in hematologic malignancies both in supporting cancer stem cells and in promoting growth and survival of mature malignant cells. Specifically Hh gene overexpression during progenitor cell expansion is associated with malignant transformation in CD133⁺/CD34⁺ cells.³⁵  The Hh pathway inhibition also induces apoptosis and reduces drug resistance in human acute myeloid and lymphoid leukemia.^36,37  Indeed, in chronic myeloid leukemia, Hh signaling plays a role in maintaining bcr-abl⁺ leukemic stem cells^38,39  through Smo up-regulation, as well as in the blastic transformation of CD34⁺ chronic myeloid leukemia cells by Ptch1 overexpression.⁴⁰  Indeed, pharmacologic Smo inhibition reduces leukemic stem cells in vivo, enhances time to relapse, and impairs the growth of imatinib-resistant cells.⁴¹  Shh/Gli1 signaling is also active in some types of non-Hodgkin lymphoma,^42,43  where perturbation of Hh pathway by Shh or cyclopamine influences cell proliferation/apoptosis and Gli1 down-regulation increases susceptibility to doxorubicin. Finally Smo-dependent as well Smo-independent Hh activation support B-CLL cell survival, correlates with adverse indicators of clinical outcome, and Gli1 inhibition increases susceptibility to fludarabine-induced cytotoxicity.^44,45  Altogether, these data indicate that molecular targeting of Hh pathway is a potential therapeutic approach to improve treatment of hematologic malignancies.

In the present study, we provide data supporting a role of Hh pathway also in CD138⁺ PCs, and we show canonical as well as noncanonical mechanisms leading to its activation in MM. We previously demonstrated overexpression of ciliary proteins, such as OFD1, as one of the possible causes of constitutive, noncanonical, or Smo-independent Hh activation, and highlighted the important role of primary cilium in regulating cellular responses to Hh signaling in MM.¹²  Here we investigated whether the canonical Hh pathway was also active in MM. We first demonstrated that CD138⁺ PCs from MGUS patients have a significant up-regulation of Hh-activating genes, such as Smo, Ptch1, and Gli2 compared with CD138⁺ PCs from healthy persons. This supports a role of Hh pathway in malignant transformation of PCs and in the pathogenesis of the precursor condition MGUS. In contrast, we observed a significant down-regulation of Hh-repressor genes, such as Ptch2 and Gli3 in MM PCs compared with their normal cellular counterpart. In addition, MM tumor cells overexpress Gli1/Gli2 and Ptch1, which is itself a Gli1 target gene, suggesting a Gli-dependent Hh activation. These results are in agreement with our previous findings showing Gli1/Ptch1 down-regulation after OFD1 specific knockdown¹²  and also with recent evidence demonstrating that Gli1 overexpression increases MM cells resistance to the antiproliferative effects of the Smo inhibitor cyclopamine.⁹  These data suggest that Hh activity may be induced in MM cells in a Smo-independent manner. Finally, we found a relatively reduced Hh-gene expression in MM cell lines and PCL, a more advanced and BM-independent disease, suggesting a critical role for stroma-derived Hh signals consistent with a paracrine model of Hh pathway in MM. Despite the Hh-gene expression, Hh-protein analysis in MM cell lines revealed that Shh ligand is expressed at significant levels in both CD138⁺ as well as CD138⁻ cells. The CD138⁺ MM cell population probably also includes the side population, which is a CD138^low+ subpopulation with stem cell properties.⁴⁶  Matsui et al^47,48  have previously reported predominant effect and activity of Hh signaling in CD138⁻ MM stem cells, although we observed that CD138⁺ cells are also susceptible to inhibition by NVP-LDE225. Various molecular explanations can be considered for this observation, including a lower level or differential expression of some Hh genes in CD138⁺ MM cells versus CD138^low+ side population. Moreover, a wide spectrum of genetic alterations have been described in MM and are differently associated with MM disease initiation and progression; disease stage and previous treatment may also impact the biology of the cancer and hence Hh signaling in MM cells.⁴⁸  Therefore, it is possible that MM represents a number of biologically distinct disease, each containing different initiating cells.⁴⁸  These factors may all contribute to the reported differences.

We did not find correlation between Shh expression and Hh responsiveness, suggesting the absence of link among expression level and functional activity; therefore, autocrine and/or paracrine mechanisms can both contribute to Hh activation. Importantly, MM cell lines strongly coexpress Ptch1 and Smo receptors, suggesting their potential Hh responsiveness. We have confirmed Smo-dependent Hh signaling in MM using an Smo inhibitor NVP-LDE225,^16,17  which decreased MM cell viability in a range of 3-5μM in the majority of MM cell lines tested. This was associated with specific down-regulation of Gli1 and/or Ptch1, hallmarks of cell response to the Hh pathway. In the remaining MM cell lines, despite Smo expression, Gli1 and/or Ptch1 down-regulation was not observed after treatment, suggesting lack of correlation between Smo expression level and functional protein activity. Importantly, in those MM cell lines not responding to Smo inhibitor, Gli1 nuclear localization indicates that the Hh pathway is constitutively activated, suggesting that alternative, noncanonical and Gli-dependent mechanisms may contribute to Hh signaling activation in MM. Therefore, the combination of Gli-modulating agents with Smo inhibitors may provide an alternative and more effective strategy for Hh inhibition.

NVP-LDE225 similarly inhibited Gli1 nuclear translocation in MM cells with Smo-dependent Hh activity. Cytotoxicity of NVP-LDE225 was also observed in primary MM cells, whereas no toxicity has been observed in PBMCs from healthy persons, suggesting a specific antitumor activity and a favorable therapeutic index. We observed that MM patient-derived BMSCs produce Shh ligand and overexpress Ptch1 but do not express Smo and rarely express Gli1. Recent reports show a role for Ptch1 in the BM compartment in inducing B-lymphocyte differentiation in mouse model,³²  leading to the hypothesis that Ptch1 might mediate MM-BM microenvironment interactions and PC differentiation also in humans. Defective Smo expression, Ptch1 up-regulation, and absence of Gli1 may all contribute to Hh inhibitor resistance observed in MM patient-derived BMSCs. Importantly, Shh production by BMSCs suggests a paracrine role for stroma-derived Hh signals and also that Shh ligand is a novel cytokine supporting growth and survival of human PCs. Therefore, Shh-blocking agents may be useful to interrupt MM-BM interactions. MM-BMSC interaction does not induce resistance to NVP-LDE225. Finally, in vitro as well in vivo studies showed antitumor activity of NVP-LDE225 in combination with bortezomib, demonstrating that NVP-LDE225 can potentiate the efficacy of well-established anti-MM agents.

In conclusion, our findings suggest that both canonical (Smo-dependent) and noncanonical (Smo-independent) mechanisms are crucial regulators of Hh activation in MM cells. Canonical and noncanonical pathways probably work in parallel, with a possible crosstalk between them.⁴⁹  Directed analyses of the noncanonical Hh signaling as well as a better understanding of all the mechanisms contributing to the noncanonical Hh activation, such as genetic mutations, ciliary protein overexpression, crosstalk between Hh signaling and unrelated pathways, and MM-BMSCs interactions, are therefore needed (Figure 7). Taken together, our findings provide the framework for novel therapeutic strategies targeting the Hh pathway to improve patient outcome in MM.

The authors thank Novartis Institute for Bio-Medical Research (Cambridge, MA) for providing the NVP-LDE225 compound.

This work was supported by the Specialized Program of Research Excellence in Myeloma–National Institutes of Health (Career Development Award grant P50CA-100707, S.B.), Department of Veterans Affairs (Merit Review Award I01-BX001584), National Institutes of Health (grants RO1-124929 and PO1-155258, N.C.M.; grants P50-100007 and PO1-78378, N.C.M. and K.C.A.; and grant RO1-50947 to K.C.A. and C.S.M.), the American Cancer Society Clinical Research Professor (K.C.A.), and the Italian Association for Cancer Research (grant 9980, P.T.).

National Institutes of Health

Contribution: S.B. conceived the project, designed and performed research, analyzed and interpreted the data, and wrote the manuscript; N.C.M. conceived and designed the project, analyzed and interpreted the data, supervised the research, and critically reviewed the manuscript; K.C.A. supervised the research and critically reviewed the manuscript; J.J. helped with the flow cytometry; T.C. helped with the animal study; A.M.R., N.A., A.K.A., and U.F.contributed to the research; C.S.M., M.R., P. Tagliaferri, and P. Tassone analyzed the data; K.T., S.M., F.M., and A.N. provided primary tumor specimens for the microarray experiment and performed the statistical analysis; and all authors reviewed and approved the final manuscript.

Conflict-of-interest disclosure: K.C.A. is on the advisory board of Celgene, Millennium, Onyx, and Bristol-Myers Squibb and is Scientific Founder of Acetylon and Oncopep. N.C.M. is on the advisory board of Celgene, Millennium, Onyx, and Merck and is Scientific Founder of Oncopep. C.S.M. received consultant honoraria from Millennium, Celgene, Novartis, Bristol-Myers Squibb, Merck & Co, Centocor, and Arno Therapeutics; he received research funding from Amgen, AVEO Pharma, OSI, EMD Serono, Sunesis, Genzyme, and Johnson & Johnson; and he has an uncompensated role as scientific advisor for Axios Biosciences. The remaining authors declare no competing financial interests.

Correspondence: Nikhil C. Munshi, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney St, M1B28, Boston, MA 02115; e-mail: nikhil_munshi@dfci.harvard.edu.