Effects of Combined Treatment With Arsenic Trioxide and Itraconazole in Patients With Refractory Metastatic Basal Cell Carcinoma.

IMPORTANCE: Tumor resistance is an emerging problem for Smoothened (SMO) inhibitor-treated metastatic basal cell carcinoma (BCC). Arsenic trioxide and itraconazole antagonize the hedgehog (HH) pathway at sites distinct from those treated by SMO inhibitors. OBJECTIVE: To determine whether administration of intravenous arsenic trioxide and oral itraconazole in patients with metastatic BCC is associated with a reduction in GLI1 messenger RNA expression in tumor and/or normal skin biopsy samples. DESIGN, SETTING, AND PARTICIPANTS: Five men with metastatic BCC who experienced relapse after SMO inhibitor treatment underwent intravenous arsenic trioxide treatment for 5 days, every 28 days, and oral itraconazole treatment on days 6 to 28. Data were collected from April 10 to November 14, 2013. Follow-up was completed on October 3, 2015, and data were analyzed from June 5 to October 6, 2015. MAIN OUTCOMES AND MEASURES: The primary outcome was the change in messenger RNA levels of the GLI family zinc finger 1 (GLI1) gene (HH-pathway target gene) in biopsy specimens of normal skin or BCC before and after treatment. Secondary objectives were evaluation of tumor response and tolerability. RESULTS: Of the 5 patients (mean [SD] age, 52 [9] years; age range, 43-62 years), 3 completed 3 cycles of treatment and 2 discontinued treatment early owing to disease progression or adverse events. Adverse effects included grade 2 transaminitis and grade 4 leukopenia with a grade 3 infection. Overall, arsenic trioxide and itraconazole reduced GLI1 messenger RNA levels by 75% from baseline (P < .001). The best overall response after 3 treatment cycles was stable disease in 3 patients. CONCLUSIONS AND RELEVANCE: Targeting the HH pathway with sequential arsenic trioxide and itraconazole treatment is a feasible treatment for metastatic BCC. Although some patients experienced stable disease for 3 months, none had tumor shrinkage, which may be owing to transient GLI1 suppression with sequential dosing. Continuous dosing may be required to fully inhibit the HH pathway and achieve clinical response.

The role of ciliary trafficking in Hedgehog receptor signaling.

Defects in the biogenesis of or transport through primary cilia affect Hedgehog protein signaling, and many Hedgehog pathway components traffic through or accumulate in cilia. The Hedgehog receptor Patched negatively regulates the activity and ciliary accumulation of Smoothened, a seven-transmembrane protein that is essential for transducing the Hedgehog signal. We found that this negative regulation of Smoothened required the ciliary localization of Patched, as specified either by its own cytoplasmic tail or by provision of heterologous ciliary localization signals. Surprisingly, given that Hedgehog binding promotes the exit of Patched from the cilium, we observed that an altered form of Patched that is retained in the cilium nevertheless responded to Hedgehog, resulting in Smoothened activation. Our results indicate that whereas ciliary localization of Patched is essential for suppression of Smoothened activation, the primary event enabling Smoothened activation is binding of Hedgehog to Patched, and Patched ciliary removal is secondary.

Simultaneous measurement of smoothened entry into and exit from the primary cilium.

Ciliary accumulation of signaling proteins must result from a rate of ciliary entry that exceeds ciliary exit, but approaches for distinguishing ciliary entry vs. exit are lacking. Using a photoconvertible fluorescent protein tag, we establish an assay that allows a separate but simultaneous examination of ciliary entry and exit of the Hedgehog signaling protein Smoothened in individual cells. We show that KAAD-cyclopamine selectively blocks entry, whereas ciliobrevin interferes initially with exit and eventually with both entry and exit of ciliary Smoothened. Our study provides an approach to understanding regulation of ciliary entry vs. exit of Hedgehog signaling components as well as other ciliary proteins.

Itraconazole and arsenic trioxide inhibit Hedgehog pathway activation and tumor growth associated with acquired resistance to smoothened antagonists.

Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors. Leading inhibitors in clinical development act by binding to a common site within Smoothened, a critical pathway component. Acquired Smoothened mutations, including SMO(D477G), confer resistance to these inhibitors. Here, we report that itraconazole and arsenic trioxide, two agents in clinical use that inhibit Hedgehog signaling by mechanisms distinct from that of current Smoothened antagonists, retain inhibitory activity in vitro in the context of all reported resistance-conferring Smoothened mutants and GLI2 overexpression. Itraconazole and arsenic trioxide, alone or in combination, inhibit the growth of medulloblastoma and basal cell carcinoma in vivo, and prolong survival of mice with intracranial drug-resistant SMO(D477G) medulloblastoma.

Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector.

Aberrant Hedgehog (Hh) pathway activation has been implicated in cancers of diverse tissues and organs, and the tumor growth-inhibiting effects of pathway antagonists in animal models have stimulated efforts to develop pathway antagonists for human therapeutic purposes. These efforts have focused largely on cyclopamine derivatives or other compounds that mimic cyclopamine action in binding to and antagonizing Smoothened, a membrane transductory component. We report here that arsenicals, in contrast, antagonize the Hh pathway by targeting Gli transcriptional effectors; in the short term, arsenic blocks Hh-induced ciliary accumulation of Gli2, the primary activator of Hh-dependent transcription, and with prolonged incubation arsenic reduces steady-state levels of Gli2. Arsenicals active in Hh pathway antagonism include arsenic trioxide (ATO), a curative agent in clinical use for acute promyelocytic leukemia (APL); in our studies, ATO inhibited growth of Hh pathway-driven medulloblastoma allografts derived from Ptch+/-p53-/- mice within a range of serum levels comparable to those achieved in treatment of human APL. Arsenic thus could be tested rapidly as a therapeutic agent in malignant diseases associated with Hh pathway activation and could be particularly useful in such diseases that are inherently resistant or have acquired resistance to cyclopamine mimics.

Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth.

In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus.

Stimulation by the extracellular Hedgehog (Hh) protein signal has been shown to alter ciliary localization of the mammalian Hh receptor components Smoothened (Smo) and Patched (Ptc), and mutations that disrupt the structure and function of the cilium also disrupt Hh-induced changes in gene expression. But how ciliary events affect gene expression in the nucleus is not known, and to address this question we have characterized the cellular trafficking of Gli2, the principal mediator of Hh-dependent transcriptional activation. From a combination of pharmacological and genetic manipulations we find in resting cells that both Gli2 and Smo appear to shuttle in and out of the cilium, with Gli2 but not Smo requiring intact cytoplasmic microtubules for ciliary entry and both requiring the ciliary retrograde motor, cytoplasmic dynein 2, for ciliary exit. We also find that changes in ciliary and nuclear trafficking of Gli2 are triggered by the Hh-dependent accumulation of activated Smo in the cilium, resulting in a shift from primarily cytoplasmic localization to accumulation at the distal tip of the cilium and within the nucleus. Gli2 thus functions as a dynamic monitor of Smo activity in the cilium and thereby links Hh pathway activation in the cilium to transcriptional activation in the nucleus.

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

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.

Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma.

The cancer stem cell hypothesis suggests that malignant growth depends on a subset of tumor cells with stem cell-like properties of self-renewal. Because hedgehog (Hh) signaling regulates progenitor cell fate in normal development and homeostasis, aberrant pathway activation might be involved in the maintenance of such a population in cancer. Indeed, mutational activation of the Hh pathway is associated with medulloblastoma and basal cell carcinoma; pathway activity is also critical for growth of other tumors lacking such mutations, although the mechanism of pathway activation is poorly understood. Here we study the role and mechanism of Hh pathway activation in multiple myeloma (MM), a malignancy with a well defined stem cell compartment. In this model, rare malignant progenitors capable of clonal expansion resemble B cells, whereas the much larger tumor cell population manifests a differentiated plasma cell phenotype that pathologically defines the disease. We show that the subset of MM cells that manifests Hh pathway activity is markedly concentrated within the tumor stem cell compartment. The Hh ligand promotes expansion of MM stem cells without differentiation, whereas the Hh pathway blockade, while having little or no effect on malignant plasma cell growth, markedly inhibits clonal expansion accompanied by terminal differentiation of purified MM stem cells. These data reveal that Hh pathway activation is heterogeneous across the spectrum of MM tumor stem cells and their more differentiated progeny. The potential existence of similar relationships in other adult cancers may have important biologic and clinical implications for the study of aberrant Hh signaling.

The Fes tyrosine kinase: a signal transducer that regulates myeloid-specific gene expression through transcriptional activation.

The c-fps/fes protooncogene encodes a 92-kDa protein tyrosine kinase that is involved in myeloid cell development and immune responses of granulocytes and macrophages. To help define its biological role and mechanism of action, we have developed a gain of function allele of Fes that has potent biological activity in myeloid cells. Introduction of constitutively active Fes into myeloid progenitors induced the appearance of fully differentiated macrophages or granulocytes depending on the lineage commitment of the transduced cells. We found that Fes-induced macrophage differentiation correlated with activation of the ets family transcription factor PU.1, which is essential for macrophage development. On the other hand, granulocyte differentiation by Fes was mediated through activation of CCAAT/enhancer-binding protein alpha (C/EBP-alpha) and STAT3, two transcription factors that are critical for granulocytic differentiation. We postulate that Fes transduces inductive signals for terminal macrophage and granulocyte differentiation, and that this biological activity is mediated through the activation of lineage-specific transcription factors.

Human herpesvirus 8-encoded vGPCR activates nuclear factor of activated T cells and collaborates with human immunodeficiency virus type 1 Tat.

Human herpesvirus 8 (HHV-8), the etiologic agent of Kaposi's sarcoma (KS), encodes a chemokine receptor homologue, the viral G protein-coupled receptor (vGPCR), that has been implicated in KS pathogenesis. Expression of vGPCR constitutively activates several signaling pathways, including NF-kappa B, and induces the expression of proinflammatory and angiogenic factors, consistent with the inflammatory hyperproliferative nature of KS lesions. Here we show that vGPCR also constitutively activates the nuclear factor of activated T cells (NF-AT), another transcription factor important in regulation of the expression of inflammatory cytokines and related factors. NF-AT activation by vGPCR depended upon signaling through the phosphatidylinositol 3-kinase-Akt-glycogen synthetase kinase 3 (PI3-K/Akt/GSK-3) pathway and resulted in increased expression of NF-AT-dependent cell surface molecules (CD25, CD29, Fas ligand), proinflammatory cytokines (interleukin-2 [IL-2], IL-4), and proangiogenic factors (granulocyte-macrophage colony-stimulating factor GMCSF and TNF alpha). vGPCR expression also increased endothelial cell-T-cell adhesion. Although infection with HHV-8 is necessary to cause KS, coinfection with human immunodeficiency virus type 1 (HIV-1), in the absence of antiretroviral suppressive therapy, increases the risk of KS by many orders of magnitude. NF-AT and NF-kappa B activation by vGPCR was greatly increased by the HIV-1 Tat protein, although Tat alone had little effect on NF-AT. The enhancement of NF-AT by Tat appears to be mediated through collaborative stimulation of the PI3-K/Akt/GSK-3 pathway by vGPCR and Tat. Our data further support the idea that vGPCR contributes to the pathogenesis of KS by a paracrine mechanism and, in addition, provide the first evidence of collaboration between an HIV-1 protein and an HHV-8 protein.

Fes tyrosine kinase promotes survival and terminal granulocyte differentiation of factor-dependent myeloid progenitors (32D) and activates lineage-specific transcription factors.

The c-fps/fes proto-oncogene encodes a 92-kDa protein-tyrosine kinase that is involved in myeloid cell development and function. We have recently shown that expression of an activated allele of Fes (Fes(act)) in monocyte precursors resulted in their differentiation into functional macrophages through the activation of lineage-specific transcription factors. We now report that this kinase also plays a role in the survival and terminal differentiation of granulocyte progenitors. The expression of Fes(act) in factor-dependent 32D cells prevented their apoptotic death after interleukin-3 removal, but Fes(act)-expressing cells remained factor-dependent for proliferation. Removal of interleukin-3 from the Fes(act)-expressing cells was followed by granulocytic differentiation in the absence of granulocyte colony-stimulating factor within 4-8 days. The differentiated cells had distinctive granulocyte morphology and there was up-regulation of CD11b, Gr-1, and late differentiation markers such as lactoferrin, suggesting that this kinase induced terminal granulocytic differentiation. Concomitantly, Fes(act) down-regulated the macrophage marker F4/80, suggesting that the biological activity of Fes was coordinated in a lineage-specific manner. Further analysis showed that Fes(act) caused activation of CCAAT/enhancer-binding protein-alpha and STAT3, two transcription factors that are involved in granulocyte differentiation. Our results provide evidence that Fes may be a key component of the granulocyte differentiation machinery, and suggest a potential mechanism by which this kinase may regulate granulocyte-specific gene expression.

Activated Fes protein tyrosine kinase induces terminal macrophage differentiation of myeloid progenitors (U937 cells) and activation of the transcription factor PU.1.

The c-fps/fes proto-oncogene encodes a 92-kDa protein tyrosine kinase that is preferentially expressed in myeloid and endothelial cells. Fes is believed to play a role in vascular development and myelopoiesis and in the inflammatory responses of granulocytes and macrophages. To help define the biological role of this kinase and identify its downstream targets, we have developed a gain-of-function allele of Fes that has potent biological activity in myeloid cell progenitors. Introduction of constitutively active Fes into bipotential U937 cells induced the appearance of fully differentiated macrophages within 6 to 12 days. The Fes-expressing differentiated cells became adherent, had distinctive macrophage morphology, and exhibited increased expression of myelomonocytic differentiation markers, including CD11b, CD11c, CD18, CD14, and the macrophage colony-stimulating factor receptor. These cells acquired phagocytic properties and exhibited NADPH oxidase and nonspecific esterase activities, confirming that they were functionally active macrophages. Concomitantly, there was downregulation of the granulocytic marker granulocyte colony-stimulating factor receptor, indicating that the biological activity of Fes was coordinated in a lineage-specific manner. A constitutively active Src did not induce macrophage morphology or upregulation of myelomonocytic markers in U937 cells, suggesting that the biological activity we observed was not a general consequence of expression of an activated nonreceptor tyrosine kinase. Analysis of possible downstream targets of Fes revealed that this kinase activated the ets family transcription factor PU.1, which is essential for macrophage development. Our results strongly implicate Fes as a key regulator of terminal macrophage differentiation and identify PU.1 as a transcription factor that may mediate some of its biological activities in myeloid cells.