In relapsed or refractory diffuse large B-cell lymphoma, CD19 expression by immunohistochemistry alone is not a predictor of response to loncastuximab tesirine.

CD19-targeting treatments have shown promise in relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). Loncastuximab tesirine (loncastuximab tesirine-lpyl [Lonca]) is a CD19-targeting antibody-drug conjugate indicated for R/R DLBCL after at least two systemic treatments. CD19 expression was evaluated in patients receiving Lonca in the LOTIS-2 clinical trial with available tissue samples obtained after last systemic therapy/before Lonca treatment. Lonca cytotoxicity was evaluated in a panel of six lymphoma cell lines with various CD19 expression levels. Quantitative systems pharmacology (QSP) modelling was used to predict Lonca responses. Lonca responses were seen in patients across all CD19 expression levels, including patients with low/no detectable CD19 expression and H-scores at baseline. Similarly, Lonca induced cytotoxicity in cell lines with different levels of CD19 expression, including one with very low expression. QSP modelling predicted that CD19 expression by immunohistochemistry alone does not predict Lonca response, whereas inclusion of CD19 surface density improved response prediction. Virtual patients responded to Lonca with estimated CD19 as low as 1000 molecules/cell of CD19, normally below the immunohistochemistry detection level. We found Lonca is an effective treatment for R/R DLBCL regardless of CD19 expression by immunohistochemistry. These results provide the basis for future studies addressing CD19-targeted agent sequencing.

Omega-3 polyunsaturated fatty acids reverse the impact of western diets on regulatory T cell responses through averting ceramide-mediated pathways.

Western diet (WD), high in sugar and fat, promotes obesity and associated chronic low-grade pro-inflammatory environment, leading to impaired immune function, reprogramming of innate and adaptive immune cells, and development of chronic degenerative diseases, including cardiovascular disease. Increased concentrations of circulating and tissue ceramides contribute to inflammation and cellular dysfunction common in immune metabolic and cardiometabolic disease. Therefore, ceramide-lowering interventions have been considered as strategies to improve adipose tissue health. Here, we report the ability of omega-3 polyunsaturated fatty acids (n-3PUFA) to attenuate inflammatory phenotypes promoted by WD, through ceramide-dependent pathways. Using an animal model, we show that enrichment of WD diet with n-3PUFA, reduced the expression of ceramide synthase 2 (CerS2), and lowered the concentration of long-chain ceramides (C23-C26) in plasma and adipose tissues. N-3PUFA also increased prevalence of the anti-inflammatory CD4+Foxp3+ and CD4+Foxp3+CD25+ Treg subtypes in lymphoid organs. The CerS inhibitor FTY720 mirrored the effect of n-3PUFA. Treatment of animal and human T cells with ceramide C24 in vitro, reduced CD4+Foxp3+ Treg polarisation and IL-10 production, and increased IL-17, while it decreased Erk and Akt phosphorylation downstream of T cell antigen receptors (TCR). These findings suggest that molecular mechanisms mediating the adverse effect of ceramides on regulatory T lymphocytes, progress through reduced TCR signalling. Our findings suggest that nutritional enrichment of WD with fish oil n-3PUFA can partially mitigate its detrimental effects, potentially improving the low-grade inflammation associated with immune metabolic disease. Compared to pharmacological interventions, n-3PUFA offer a simpler approach that can be accommodated as lifestyle choice.

The emerging relationship between metabolism and DNA repair.

The DNA damage response (DDR) consists of multiple specialized pathways that recognize different insults sustained by DNA and repairs them where possible to avoid the accumulation of mutations. While loss of activity of genes in the DDR has been extensively associated with cancer predisposition and progression, in recent years it has become evident that there is a relationship between the DDR and cellular metabolism. The activity of the metabolic pathways can influence the DDR by regulating the availability of substrates required for the repair process and the function of its players. Additionally, proteins of the DDR can regulate the metabolic flux through the major pathways such as glycolysis, tricarboxylic acid cycle (TCA) and pentose phosphate pathway (PPP) and the production of reactive oxygen species (ROS). This newly discovered connection bears great importance in the biology of cancer and represents a new therapeutic opportunity. Here we describe the nature of the relationship between DDR and metabolism and its potential application in the treatment of cancer. Keywords: DNA repair, metabolism, mitochondria.

Endothelial cell and T-cell crosstalk: Targeting metabolism as a therapeutic approach in chronic inflammation.

The role of metabolic reprogramming in the coordination of the immune response has gained increasing consideration in recent years. Indeed, it has become clear that changes in the metabolic status of immune cells can alter their functional properties. During inflammation, T cells need to generate sufficient energy and biomolecules to support growth, proliferation, and effector functions. Therefore, T cells need to rearrange their metabolism to meet these demands. A similar metabolic reprogramming has been described in endothelial cells, which have the ability to interact with and modulate the function of immune cells. In this overview, we will discuss recent insights in the complex crosstalk between endothelial cells and T cells as well as their metabolic reprogramming following activation. We highlight key components of this metabolic switch that can lead to the development of new therapeutics against chronic inflammatory disorders. LINKED ARTICLES: This article is part of a themed issue on Cellular metabolism and diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.10/issuetoc.

Omega-3 polyunsaturated fatty acids impinge on CD4+ T cell motility and adipose tissue distribution via direct and lipid mediator-dependent effects.

AIMS: Adaptive immunity contributes to the pathogenesis of cardiovascular metabolic disorders (CVMD). The omega-3 polyunsaturated fatty acids (n-3PUFA) are beneficial for cardiovascular health, with potential to improve the dysregulated adaptive immune responses associated with metabolic imbalance. We aimed to explore the mechanisms through which n-3PUFA may alter T cell motility and tissue distribution to promote a less inflammatory environment and improve lymphocyte function in CVMD. METHODS AND RESULTS: Using mass spectrometry lipidomics, cellular, biochemical, and in vivo and ex vivo analyses, we investigated how eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the main n-3PUFA, modify the trafficking patterns of activated CD4+ T cells. In mice subjected to allogeneic immunization, a 3-week n-3PUFA-enriched diet reduced the number of effector memory CD4+ T cells found in adipose tissue, and changed the profiles of eicosanoids, octadecanoids, docosanoids, endocannabinoids, 2-monoacylglycerols, N-acyl ethanolamines, and ceramides, in plasma, lymphoid organs, and fat tissues. These bioactive lipids exhibited differing chemotactic properties when tested in chemotaxis assays with activated CD4+ T cells in vitro. Furthermore, CD4+ T cells treated with EPA and DHA showed a significant reduction in chemokinesis, as assessed by trans-endothelial migration assays, and, when implanted in recipient mice, demonstrated less efficient migration to the inflamed peritoneum. Finally, EPA and DHA treatments reduced the number of polarized CD4+ T cells in vitro, altered the phospholipid composition of membrane microdomains and decreased the activity of small Rho GTPases, Rhoα, and Rac1 instrumental in cytoskeletal dynamics. CONCLUSIONS: Our findings suggest that EPA and DHA affect the motility of CD4+ T cells and modify their ability to reach target tissues by interfering with the cytoskeletal rearrangements required for cell migration. This can explain, at least in part, the anti-inflammatory effects of n-3PUFA supporting their potential use in interventions aiming to address adipocyte low-grade inflammation associated with cardiovascular metabolic disease.

Lactate Buildup at the Site of Chronic Inflammation Promotes Disease by Inducing CD4+ T Cell Metabolic Rewiring.

Accumulation of lactate in the tissue microenvironment is a feature of both inflammatory disease and cancer. Here, we assess the response of immune cells to lactate in the context of chronic inflammation. We report that lactate accumulation in the inflamed tissue contributes to the upregulation of the lactate transporter SLC5A12 by human CD4+ T cells. SLC5A12-mediated lactate uptake into CD4+ T cells induces a reshaping of their effector phenotype, resulting in increased IL17 production via nuclear PKM2/STAT3 and enhanced fatty acid synthesis. It also leads to CD4+ T cell retention in the inflamed tissue as a consequence of reduced glycolysis and enhanced fatty acid synthesis. Furthermore, antibody-mediated blockade of SLC5A12 ameliorates the disease severity in a murine model of arthritis. Finally, we propose that lactate/SLC5A12-induced metabolic reprogramming is a distinctive feature of lymphoid synovitis in rheumatoid arthritis patients and a potential therapeutic target in chronic inflammatory disorders.

KCTD15 inhibits the Hedgehog pathway in Medulloblastoma cells by increasing protein levels of the oncosuppressor KCASH2.

Medulloblastoma (MB) is the most common malignant childhood brain tumor. About 30% of all MBs belong to the I molecular subgroup, characterized by constitutive activation of the Sonic Hedgehog (Hh) pathway. The Hh pathway is involved in several fundamental processes during embryogenesis and in adult life and its deregulation may lead to cerebellar tumorigenesis. Indeed, Hh activity must be maintained via a complex network of activating and repressor signals. One of these repressor signals is KCASH2, belonging to the KCASH family of protein, which acts as negative regulators of the Hedgehog signaling pathway during cerebellar development and differentiation. KCASH2 leads HDAC1 to degradation, allowing hyperacetylation and inhibition of transcriptional activity of Gli1, the main effector of the Hh pathway. In turn, the KCASH2 loss leads to persistent Hh activity and eventually tumorigenesis. In order to better characterize the physiologic role and modulation mechanisms of KCASH2, we have searched through a proteomic approach for new KCASH2 interactors, identifying Potassium Channel Tetramerization Domain Containing 15 (KCTD15). KCTD15 is able to directly interact with KCASH2, through its BTB/POZ domain. This interaction leads to increase KCASH2 stability which implies a reduction of the Hh pathway activity and a reduction of Hh-dependent MB cells proliferation. Here we report the identification of KCTD15 as a novel player in the complex network of regulatory proteins, which modulate Hh pathway, this could be a promising new target for therapeutic approach against MB.

MLH1 deficiency leads to deregulated mitochondrial metabolism.

The DNA mismatch repair (MMR) pathway is responsible for the repair of base-base mismatches and insertion/deletion loops that arise during DNA replication. MMR deficiency is currently estimated to be present in 15-17% of colorectal cancer cases and 30% of endometrial cancers. MLH1 is one of the key proteins involved in the MMR pathway. Inhibition of a number of mitochondrial genes, including POLG and PINK1 can induce synthetic lethality in MLH1-deficient cells. Here we demonstrate for the first time that loss of MLH1 is associated with a deregulated mitochondrial metabolism, with reduced basal oxygen consumption rate and reduced spare respiratory capacity. Furthermore, MLH1-deficient cells display a significant reduction in activity of the respiratory chain Complex I. As a functional consequence of this perturbed mitochondrial metabolism, MLH1-deficient cells have a reduced anti-oxidant response and show increased sensitivity to reactive oxidative species (ROS)-inducing drugs. Taken together, our results provide evidence for an intrinsic mitochondrial dysfunction in MLH1-deficient cells and a requirement for MLH1 in the regulation of mitochondrial function.

Fatty acids - from energy substrates to key regulators of cell survival, proliferation and effector function.

Recent advances in immunology and cancer research show that fatty acids, their metabolism and their sensing have a crucial role in the biology of many different cell types. Indeed, they are able to affect cellular behaviour with great implications for pathophysiology. Both the catabolic and anabolic pathways of fatty acids present us with a number of enzymes, receptors and agonists/antagonists that are potential therapeutic targets, some of which have already been successfully pursued. Fatty acids can affect the differentiation of immune cells, particularly T cells, as well as their activation and function, with important consequences for the balance between anti- and pro-inflammatory signals in immune diseases, such as rheumatoid arthritis, psoriasis, diabetes, obesity and cardiovascular conditions. In the context of cancer biology, fatty acids mainly provide substrates for energy production, which is of crucial importance to meet the energy demands of these highly proliferating cells. Fatty acids can also be involved in a broader transcriptional programme as they trigger signals necessary for tumorigenesis and can confer to cancer cells the ability to migrate and generate distant metastasis. For these reasons, the study of fatty acids represents a new research direction that can generate detailed insight and provide novel tools for the understanding of immune and cancer cell biology, and, more importantly, support the development of novel, efficient and fine-tuned clinical interventions. Here, we review the recent literature focusing on the involvement of fatty acids in the biology of immune cells, with emphasis on T cells, and cancer cells, from sensing and binding, to metabolism and downstream effects in cell signalling.

Lactate transporters as therapeutic targets in cancer and inflammatory diseases.

INTRODUCTION: Inflammation is associated with the accumulation of lactate at sites of tumor-growth and inflammation. Lactate initiates tissue-responses contributing to disease. We discuss the potential of targeting lactate transporters in the treatment of cancer and inflammatory conditions. Areas covered: Lactate is the end product of glycolysis, often considered a waste metabolite but also a fuel for oxidative cells. It is however an active signaling molecule with immunomodulatory and angiogenic properties. They are the consequence of lactate binding to membrane receptor(s) or being transported through specific carrier-mediated-transporters across the cellular membrane. Carriers are distinct in proton-linked-monocarboxylate-transporters (MCTs) and Na+-coupled- electrogenic-transporters, expressed by several tissues including immune-system, endothelium and epithelium. Several tumors and inflammatory sites show accumulation of lactate and altered expression of its transporters, thus suggesting a role of this metabolite in cancer and inflammation. We review the most recent evidence on lactate biology, focusing on transporter expression and function in health and disease. Expert opinion: Lactate-initiated signaling is gaining attention for its implications in cancer and inflammation. This review discusses the therapeutic potential of targeting lactate transporters and drugs that are already in clinical use for cancer and discusses the opportunity to develop new therapeutics for inflammation and cancer.

ß-arrestin1-mediated acetylation of Gli1 regulates Hedgehog/Gli signaling and modulates self-renewal of SHH medulloblastoma cancer stem cells.

BACKGROUND: Aberrant Sonic Hedgehog/Gli (Hh/Gli) signaling pathway is a critical regulator of Sonic hedgehog medulloblastoma (SHH-MB). Cancer stem cells (CSCs), thought to be largely responsible for tumor initiation, maintenance, dissemination and relapse, have been identified in SHH-MB. Since we previously demonstrated that Hh/Gli signaling controls CSCs features in SHH-MB and that in these tumors miR-326 is down regulated, here we investigated whether there is a functional link between Hh/Gli signaling and miR-326. METHODS: We evaluated ß-arrestin1 (Arrb1) and its intragenic miR-326 levels in CSCs derived from SHH-MB. Subsequently, we modulated the expression of Arrb1 and miR-326 in CSCs in order to gain insight into their biological role. We also analyzed the mechanism by which Arrb1 and miR-326 control Hh/Gli signaling and self-renewal, using luciferase and protein immunoprecipitation assays. RESULTS: Low levels of Arrb1 and miR-326 represent a feature of CSCs derived from SHH-MB. We observed that re-expression of Arrb1 and miR-326 inhibits Hh/Gli signaling pathway at multiple levels, which cause impaired proliferation and self-renewal, accompanied by down regulation of Nanog levels. In detail, miR-326 negatively regulates two components of the Hh/Gli pathway the receptor Smoothened (Smo) and the transcription factor Gli2, whereas Arrb1 suppresses the transcriptional activity of Gli1, by potentiating its p300-mediated acetylation. CONCLUSIONS: Our results identify a new molecular mechanism involving miR-326 and Arrb1 as regulators of SHH-MB CSCs. Specifically, low levels of Arrb1 and miR-326 trigger and maintain Hh/Gli signaling and self-renewal.

BCAT1 controls metabolic reprogramming in activated human macrophages and is associated with inflammatory diseases.

Branched-chain aminotransferases (BCAT) are enzymes that initiate the catabolism of branched-chain amino acids (BCAA), such as leucine, thereby providing macromolecule precursors; however, the function of BCATs in macrophages is unknown. Here we show that BCAT1 is the predominant BCAT isoform in human primary macrophages. We identify ERG240 as a leucine analogue that blocks BCAT1 activity. Selective inhibition of BCAT1 activity results in decreased oxygen consumption and glycolysis. This decrease is associated with reduced IRG1 levels and itaconate synthesis, suggesting involvement of BCAA catabolism through the IRG1/itaconate axis within the tricarboxylic acid cycle in activated macrophages. ERG240 suppresses production of IRG1 and itaconate in mice and contributes to a less proinflammatory transcriptome signature. Oral administration of ERG240 reduces the severity of collagen-induced arthritis in mice and crescentic glomerulonephritis in rats, in part by decreasing macrophage infiltration. These results establish a regulatory role for BCAT1 in macrophage function with therapeutic implications for inflammatory conditions.

Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4+ T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals.

Low-grade systemic inflammation associated to obesity leads to cardiovascular complications, caused partly by infiltration of adipose and vascular tissue by effector T cells. The signals leading to T cell differentiation and tissue infiltration during obesity are poorly understood. We tested whether saturated fatty acid-induced metabolic stress affects differentiation and trafficking patterns of CD4+ T cells. Memory CD4+ T cells primed in high-fat diet-fed donors preferentially migrated to non-lymphoid, inflammatory sites, independent of the metabolic status of the hosts. This was due to biased CD4+ T cell differentiation into CD44hi-CCR7lo-CD62Llo-CXCR3+-LFA1+ effector memory-like T cells upon priming in high-fat diet-fed animals. Similar phenotype was observed in obese subjects in a cohort of free-living people. This developmental bias was independent of any crosstalk between CD4+ T cells and dendritic cells and was mediated via direct exposure of CD4+ T cells to palmitate, leading to increased activation of a PI3K p110δ-Akt-dependent pathway upon priming.

The centrosomal deubiquitylase USP21 regulates Gli1 transcriptional activity and stability.

USP21 is a centrosome-associated deubiquitylase (DUB) that has been implicated in the formation of primary cilia - crucial organelles for the regulation of the Hedgehog (Hh) signaling pathway in vertebrates. Here, we identify KCTD6 - a cullin-3 E3-ligase substrate adapter that has been previously linked to Hh signaling - as well as Gli1, the key transcription factor responsible for Hh signal amplification, as new interacting partners of USP21. We identify a cryptic structured protein interaction domain in KCTD6, which is predicted to have a similar fold to Smr domains. Importantly, we show that both depletion and overexpression of catalytically active USP21 suppress Gli1-dependent transcription. Gli proteins are negatively regulated through protein kinase A (PKA)-dependent phosphorylation. We provide evidence that USP21 recruits and stabilises Gli1 at the centrosome where it promotes its phosphorylation by PKA. By revealing an intriguing functional pairing between a spatially restricted deubiquitylase and a kinase, our study highlights the centrosome as an important hub for signal coordination.

Intermediates of Metabolism: From Bystanders to Signalling Molecules.

The integration of biochemistry into immune cell biology has contributed immensely to our understanding of immune cell function and the associated pathologies. So far, most studies have focused on the regulation of metabolic pathways during an immune response and their contribution to its success. More recently, novel signalling functions of metabolic intermediates are being discovered that might play important roles in the regulation of immunity. Here we describe the three long-known small metabolites lactate, acetyl-CoA, and succinate in the context of immunometabolic signalling. Functions of these ubiquitous molecules are largely dependent on their intra- and extracellular concentrations as well as their subcompartmental localisation. Importantly, the signalling functions of these metabolic intermediates extend beyond self-regulatory roles and include cell-to-cell communication and sensing of microenvironmental conditions to elicit stress responses and cellular adaptation.

In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors.

Aberrant Hedgehog (Hh)/glioma-associated oncogene (GLI) signaling has been implicated in cancer progression. Here, we analyzed GLI1, Sonic Hedgehog (Shh) and NF-κB expression in 51 breast cancer (ductal carcinoma) tissues using immunohistochemistry. We found a positive correlation between nuclear GLI1 expression and tumor grade in ductal carcinoma cases. Cytoplasmic Shh staining significantly correlated with a lower tumor grade. Next, the in vitro effects of two Hh signaling pathway inhibitors on breast cancer cell lines were evaluated using the Smoothened (SMO) antagonist GDC-0449 and the direct GLI1 inhibitor GANT-61. GDC-0449 and GANT-61 exhibited the following effects: a) inhibited breast cancer cell survival; b) induced apoptosis; c) inhibited Hh pathway activity by decreasing the mRNA expression levels of GLI1 and Ptch and inhibiting the nuclear translocation of GLI1; d) increased/decreased EGFR and ErbB2 protein expression, reduced p21-Ras and ERK1/ERK2 MAPK activities and inhibited AKT activation; and e) decreased the nuclear translocation of NF-κB. However, GANT-61 exerted these effects more effectively than GDC-0449. The in vivo antitumor activities of GDC-0449 and GANT-61 were analyzed in BALB/c mice that were subcutaneously inoculated with mouse breast cancer (TUBO) cells. GDC-0449 and GANT-61 suppressed tumor growth of TUBO cells in BALB/c mice to different extents. These findings suggest that targeting the Hh pathway using antagonists that act downstream of SMO is a more efficient strategy than using antagonists that act upstream of SMO for interrupting Hh signaling in breast cancer.

The energy sensor AMPK regulates Hedgehog signaling in human cells through a unique Gli1 metabolic checkpoint.

Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs) and its aberrant activation is a leading cause of Medulloblastoma, the most frequent pediatric brain tumor. We show here that the energy sensor AMPK inhibits Hh signaling by phosphorylating a single residue of human Gli1 that is not conserved in other species. Studies with selective agonists and genetic deletion have revealed that AMPK activation inhibits canonical Hh signaling in human, but not in mouse cells. Indeed we show that AMPK phosphorylates Gli1 at the unique residue Ser408, which is conserved only in primates but not in other species. Once phosphorylated, Gli1 is targeted for proteasomal degradation. Notably, we show that selective AMPK activation inhibits Gli1-driven proliferation and that this effect is linked to Ser408 phosphorylation, which represents a key metabolic checkpoint for Hh signaling. Collectively, this data unveil a novel mechanism of inhibition of Gli1 function, which is exclusive for human cells and may be exploited for the treatment of Medulloblastoma or other Gli1 driven tumors.

Gli1/DNA interaction is a druggable target for Hedgehog-dependent tumors.

Hedgehog signaling is essential for tissue development and stemness, and its deregulation has been observed in many tumors. Aberrant activation of Hedgehog signaling is the result of genetic mutations of pathway components or other Smo-dependent or independent mechanisms, all triggering the downstream effector Gli1. For this reason, understanding the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, representing a critical goal in tumor biology. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify Glabrescione B as the first small molecule binding to Gli1 zinc finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, Glabrescione B inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. The identification of the structural requirements of Gli1/DNA interaction highlights their relevance for pharmacologic interference of Gli signaling.

Hedgehog signaling pathway and its targets for treatment in basal cell carcinoma.

Basal cell carcinoma (BCC) of the skin is the most common type of cancer and accounts for up to 40% of all cancers in the US, with a growing incidence rate over recent decades in all developed countries. Surgery is curative for most patients, although it leaves unaesthetic scars, but those that develop locally advanced or metastatic BCC require different therapeutic approaches. Furthermore, patients with BCC present a high risk of developing additional tumors. The increasing economic burden and the morbidity of BCC render primary interest in the development of targeted treatments for this disease. Among the molecular signals involved in the development of BCC, the critical role of the morphogenetic Hedgehog (Hh) pathway has become evident. This pathway is found altered and activated in almost all BCCs, both sporadic and inherited. Given the centrality of the Hh pathway in the pathophysiology of BCC, the primary efforts to identify molecular targets for the topical or systemic treatment of this cancer have focused on the Hh components. Several Hh inhibitors have been so far identified - from the first identified natural cyclopamine to the recently Food and Drug Administration-approved synthetic vismodegib - most of which target the Hh receptor Smoothened (either its function or its translocation to the primary cilium). Other molecules await further characterization (bisamide compounds), while drugs currently approved for other diseases such as itraconazole (an antimicotic agent) and vitamin D3 have been tested on BCC with encouraging results. The outcomes of the numerous ongoing clinical trials are expected to expand the field in the very near future. Further research is needed to obtain drugs targeting downstream components of the Hh pathway (eg, Gli) or to exploit combinatorial therapies (eg, with phosphatidylinositol 3-kinase inhibitors or retinoids) in order to overcome potential drug resistance.