Currently, all of the HH pathway therapeutics in clinical development act by inhibiting SMO (Table I) and thus would be predicted to be ineffective against tumors harboring molecular lesions that lie downstream (including loss of SUFU or gain-of-function mutations in SMO that abrogate the inhibitor binding site). However, several groups are attempting to develop agents to target GLI that would have wider application82
. In addition, arsenic tri-oxide (ATO) has recently been proposed to inhibit GLI proteins directly by two distinct mechanisms83, 84
. ATO is proposed to block accumulation of GLI2 in cilia, ultimately resulting in reduced protein levels83
and ATO is proposed to bind directly to GLI1, inhibiting its transcriptional function, even in the absence of cilia84
. As ATO is an approved therapeutic, it may provide an alternative treatment for tumors that develop resistance to SMO inhibitors and could potentially be used in combination therapy with SMO inhibitors.
Cyclopamine, and other naturally occurring inhibitors of SMO, are not suitable as therapeutic agents because of poor solubility, low potency, rapid clearance, non-specific toxicity and chemical instability85
. This encouraged a search for novel inhibitors with preferential characteristics for drug development. HH-mediated GLI transcription activity provided a highly appropriate biomarker for identification of small molecule inhibitors as GLI1 and GLI2 contribute directly to oncogenic activity55, 86–88
. Cell-based screening approaches, using GLI-dependent transcriptional reporters, proved remarkably adept at identifying small molecule inhibitors of SMO89, 90
. Thus, it appears that SMO is a highly druggable target and a range of compounds, many with distinct structures from cyclopamine, have been uncovered that are capable of binding to the same site to block signaling (reviewed in91
). Medicinal chemistry approaches optimized the properties of several lead compounds resulting in a 100-fold increase in potency compared to cyclopamine as measured in gene expression assays8, 92, 93
Pre-clinical proof-of-concept studies of SMO inhibitors, to establish efficacy in cancer models, have been both challenging and controversial. The advent of GEM models of cancer offered hope that new approaches would be more predictive than the venerable xenograft models. Indeed, a study of acute myeloid leukemia (AML) allograft transplant models, expressing different translocation fusion genes, shows close concordance with responses seen in the respective pediatric populations94
. Nevertheless, the issue remains hotly debated with strong proponents arguing in favor of xenograft models, others supporting GEM models and some adamant that mouse models of any kind cannot predict clinical outcome.
Most studies of HH pathway activity in cancer employed either human tumor cell lines or xenograft transplantation models treated with cyclopamine to demonstrate dependency on HH pathway activity. Reduced rates of cell proliferation or tumor growth were interpreted to mean that SMO activity was critical for growth of the respective tumors74, 75, 95
. However, different groups have reported contradictory results, even when using the same cell lines or mouse strains carrying identical transplantable tumors8, 96–100
. Taken together, a number of variables in the experimental approaches and application of the model systems used can account for these discrepancies.
There are no standard approaches to determining whether a particular cell line or tumor depends on HH pathway activity. Therefore, different marker genes have been used to assess pathway activity, and a range of quantitative and semi-quantitative approaches employed to measure gene expression levels, making it problematic to compare results from different studies. Cyclopamine is a toxic compound85
and it can cause growth inhibition, independently of HH pathway activity, when used at high concentrations depending on the cell type8, 101
. This means that it is critical to use cyclopamine at a concentration that inhibits the HH pathway but does not result in non-specific growth inhibition. This is very hard to do because there is no agreed up standard method to measure HH pathway activity. As a consequence, while some studies report concentrations of cyclopamine of 3 μM and above result in non-specific growth inhibition8, 101
, others claim that cyclopamine can function as a specific HH pathway inhibitor at levels up to 30 μM100
. Although several studies employed complementary genetic approaches, such as short interfering RNAs to inhibit GLI1 or GLI2, as independent confirmation of the effects of cyclopamine, this approach does not demonstrate dependence on SMO. For example, GLI1 and GLI2 have been shown to function independently of HH pathway activity and SMO in B-cell chronic lymphocytic leukemia81
. Thus, the fact that GLI1 and GLI2 are required for tumor cell viability does not necessarily mean that SMO is active in these cells and therefore does not confirm the effects of cyclopamine. In vivo, it is difficult to reach systemic levels of cyclopamine that completely inhibit HH pathway activity because of associated toxicities85, 101
. Thus, it is likely that the role of HH pathway activity in human cancer has been overestimated as a consequence of the prevalent use of cyclopamine at toxic concentrations.
In many cases, alternate routes of administration were used to deliver cyclopamine. For example, in some instances cyclopamine was delivered by subcutaneous inoculation95, 96
. This often caused ulceration at the injection site as the vehicle contained alcohol. This route is not directly comparable to systemic delivery and it is not clear if it resulted in the same amount of drug delivery to the tumor cells. When the route of administration was changed to oral delivery, it was not possible to achieve the same degree of tumor inhibition102
. In most cases the level of cyclopamine achieved in tumor tissues was not determined, however, subcutaneous inoculation may have resulted in greater bioavailability, increased levels of exposure and higher specific, as well as non-specific, toxic effects.
Of much greater concern are studies in which cyclopamine was injected directly into the tumor mass103
. In some studies the administration route was described as both proximal and directly into the tumor mass104, 105
. In these cases, the actual concentration of cyclopamine to which tumor cells are exposed is extremely high and would likely result in non-specific toxicity. Finally, although the standard xenograft approach recommends treating tumors only after they are fully established as transplants (approximately 200 – 400 mm3
) some studies used much smaller tumors, even as little as 10 mm3
, before they are fully established105
. Thus, a series of methodological differences in preclinical experimental design, combined with the high degree of non-specific toxicity associated with cyclopamine, may account for some of the conflicting findings reported in the literature. These reports may have significantly overstated the potential of SMO inhibitors for treating human cancer.
Compounding these problems is the fact that pharmaceutical companies generally decline to supply compounds currently in development for such studies, because of fears that negative results would compromise the approval process. Therefore, most investigators relied on the use of cyclopamine, despite reservations about its properties, to test the contribution of SMO to tumor growth in vitro and in vivo. The notable exception was a set of compounds identified by Curis Inc., and subsequently developed in collaboration with Genentech Inc., that were used to show efficacy of SMO inhibitors in BCC explant cultures29, 106
and in mouse models of medulloblastoma52, 101
. One particular compound, a benzimidazole termed HhAntag, provided the most compelling preclinical data on the efficacy of SMO inhibitors. Oral delivery of HhAntag eradicated large medulloblastomas arising spontaneously in the cerebellum of Ptch1+/−
. However, tumor cell lines derived from these medulloblastomas, as well as allograft tumors made using these cell lines, were completely resistant to the inhibitory effects of HhAntag because the HH pathway was dramatically downregulated as soon as these cells were propagated in culture. By contrast, allografts derived directly from the medulloblastomas that were never grown in culture exhibited dramatic sensitivity, with large tumor masses (200 mm3
) regressing after only 4 days of treatment52
. These model studies predicted that BCC and the HH pathway subtype of medulloblastoma would exhibit dramatic responses to SMO inhibitors in the clinic and led to the inclusion of patients with medulloblastoma in the initial clinical trials.
The results of the first phase I trial of the SMO inhibitor GDC-0449 reported that 19 out of 33 patients with BCC, and one patient with medulloblastoma, exhibited either a partial or complete response to this novel therapy6
. An unprecedented 50% response rate was observed in patients with metastatic BCC3, 6
. A dramatic, albeit transient, responses was also reported in an adult with metastatic medulloblastoma4
, and encouraging results in a Phase I pediatric medulloblastoma clinical trial were reported at the 2010 American Society of Clinical Oncology meeting107
, indicating that the preclinical data on BCC and medulloblastoma were indicative of a response. In the one case of metastatic adult medulloblastoma, the patient subsequently relapsed because of mutation of the drug-binding site in SMO2
. This unfortunate circumstance provided strong affirmation of SMO as a drug target and echoed early experiences with Imatinib (Gleevec), the prototypic molecular targeted therapy108
. Resistance to inhibitors also arises readily in animal models as a consequence of mutations in SMO, amplification of GLI2 or amplification of CCND1
(encoding cyclin D1)2, 109, 110
. It is likely that drug resistance will be an important aspect of clinical treatment with HH pathway inhibitors and compounds have already been identified that can overcome resistance resulting from SMO mutations110
. However, this approach will not be successful in resistant tumors that acquire mutations downstream of SMO. Some hope has been offered by the recent finding that blocking phosphatidylinositol-3 kinase (PI3K) activity inhibits the growth of certain resistant tumors109
In contrast to the dramatic clinical results reported in early BCC and medulloblastoma patients, no major responses to SMO inhibitors have yet been reported in other cancers6
. Although Phase I trials are designed to test drug safety, not efficacy, the positive effects on BCC and medulloblastoma were very obvious. In addition, two trials of GDC-0449 have been closed to patient accrual (NCT00739661 and NTC00636610, listed on http://clinicaltrials.gov
). In the case of advanced ovarian cancer, GDC-0449 was being used in a maintenance setting as a single agent in a phase II clinical trial, but it did not sufficiently extend the median time to recurrence. In the case of colon carcinoma, a phase II combination therapy clinical trial of GDC-0449 plus bevacizumab (Avastin) failed because treatment did not extend the time from randomization to disease progression or death. A major caveat in the interpretation of the results of the initial clinical trials is that patients were not selected based on the presence of HH pathway activity in tumor tissue (Box 2
). Previous experience with protein kinase inhibitors showed that therapeutic effects are often not detected without molecular stratification of patients. It is important to learn from these experiences by including tests that determine the suitability of patient populations for treatment with HH pathway inhibitors, as well as assays for monitoring the effect of treatment on the target at all stages of the drug development process111
Box 2. Personalizing HH-based therapies
One of the major challenges in the use of SMO inhibitors for treating cancer is how to identify the tumors that are capable of responding. Although genetic testing can reveal the presence of PTCH1
mutations, this would only identify a subset of susceptible cases. Biomarkers for HH pathway activity can be employed, but this also identifies tumors with activating mutations in SMO, as well as those with lesions downstream of SMO, that would be resistant to treatment. Currently, RNA expression signatures are used as the primary biomarker, as there are no antibodies specific for HH pathway target genes that work in reliably immunohistochemistry assays30
. However, additional biomarkers were recently proposed that could discriminate between medulloblastoma subsets133, 134
. In the case of advanced BCC, molecular diagnostics is not a major issue as most tumors exhibit an activated HH pathway. However, it is a concern for medulloblastoma, in which only 30% of tumors have an activated HH pathway and only half of these have PTCH1
mutations. In addition, different biomarkers will be needed to identify tumors in which HH signaling functions through stromal cells as the presence of HH ligands in these tumors is also not sufficient to predict responses8
. The lack of appropriate biomarkers makes it challenging to develop robust criteria for stratification of patients with tumors other than BCC or medulloblastoma for treatment with SMO inhibitors. Therefore, it is important to revisit preclinical studies of SMO inhibitors in both genetic and xenograft models of these other tumors, to understand the mechanism of action and develop diagnostic markers.
Currently, seven compounds that bind and inhibit SMO are listed for use in a range of advanced cancers in more than 40 different clinical trials (). The most widely used compound is the first-in-class Vismodegib (GDC-0449), developed by Curis and Genentech6, 92, 112
In mammals, HH pathway activity depends on the primary cilia113
. HH binding to PTCH1 allows SMO to translocate to the ciliary membrane where it relieves repression of GLI1 and GLI2 by SUFU. Although cyclopamine binding to SMO promotes translocation to the primary cilia through the intraflagellar transport pathway, it does not activate GLI1 or GLI2 114, 115
. In contrast, the antagonists SANT-1 and SANT-2, which bind to the same site on SMO, do not promote cilial translocation of SMO114, 115
. These findings imply that SMO has multiple conformations that could be exploited for drug development. Future research on the mechanisms responsible for SMO function and the downstream events leading to GLI activation will be particularly important if gain-of-function mutations in SMO occur frequently in drug-resistant tumors. At present, it is not clear which of the many SMO inhibitors currently in drug development would be least affected by acquired mutations in SMO.
SMO inhibitors have exhibited remarkably few and only relatively modest side effects in adult patients ()6
. However, when given to young mice, for as little as 2–4 days, HhAntag caused dramatic and permanent defects in bone growth116
. Short-term treatment of 10-day old mice resulted in malformation of the epiphysis and growth plate. The columnar organization of chondrocytes in the growth plate was disrupted, and the cartilage structure appeared dysplastic. These observations are consistent with the critical role the hedgehog pathway is known to have in bone development117
. Deletion of Indian Hedgehog (Ihh
) causes embryonic lethality in mice118
, conditional ablation results in a phenotype similar to that seen in mice treated with HhAntag119
, and hypomorphic mutations of IHH
in humans cause acrocapitofemoral dysplasia 120
. The several roles that HH signaling has in development, including the postnatal formation of the cerebellum121, 122
, raise many concerns about potential side effects that may be seen in the youngest patients ().
Most Frequent Toxicities Associated with SMO Inhibitorsb