Jynho Kim

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.

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.

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 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.

The role of ciliary trafficking in Hedgehog receptor signaling

Pathway activation by Hedgehog can occur without ciliary exit of the receptor Patched. No need to leave In mammals, regulation of the Hedgehog pathway, which is involved in development and tissue homeostasis, occurs in a cellular sensory process called the primary cilium. Binding of Hedgehog to the receptor Patched not only alleviates repression of the transmembrane protein Smoothened but also stimulates Patched to exit from the primary cilium. Kim et al. determined that the C-terminal domain of Patched contained the ciliary localization sequence and was necessary and sufficient to mediate localization of proteins to the primary cilium. Unexpectedly, Patched mutants that were unable to exit the cilia not only repressed Smoothened activity but also enabled Hedgehog to alleviate this repression and stimulate Smoothened activity despite Patched not exiting the cilia in the presence of Hedgehog. Thus, Patched must reach the cilium to inhibit Smoothened in the absence of Hedgehog, but exit from the primary cilium is not required for cells to respond to Hedgehog. 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.

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.

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.

Abstract 5644: Itraconazole and arsenic trioxide inhibit hedgehog pathway activation and tumor growth associated with acquired resistance to vismodegib.

Recognition of the multiple roles of Hedgehog signaling in cancer has prompted intensive efforts to develop targeted pathway inhibitors, most recently culminating in the FDA-approval of vismodegib (Genentech/Roche) for the treatment of locally advanced or metastatic basal cell carcinoma. Vismodegib and other hedgehog pathway antagonists currently being evaluated in the clinical setting are small molecules that act by binding to a common site within Smoothened (SMO), a critical pathway component. Acquired mutations in SMO that map to these overlapping binding pockets compromise the potency and clinical efficacy of vismodegib and other clinically relevant agents in this space through an apparent class effect. As a consequence there are currently no visible therapeutic options for maintaining profound on-target efficacies in patients that present with recurrent disease harboring these point mutations. We report here that itraconazole and arsenic trioxide, two FDA-approved agents recently reported to inhibit Hedgehog signaling through mechanisms distinct from that of current SMO antagonists, maintain potent inhibition of hedgehog signaling in the face of acquired SMO mutations, both as single agents and when used in combination. Using a preclinical mouse model of medulloblastoma that mimics clinical mechanisms of vismodegib-associated acquired resistance, we demonstrate that itraconazole and arsenic trioxide inhibit pathway activation, proliferation, and tumor growth of medulloblastoma harboring SMO-D477G mutation, both in vitro and in vivo. These drugs act as effectively in syngeneic models of medulloblastoma demonstrating constitutive signaling through wild-type SMO. Combination of itraconazole and arsenic trioxide further suppress tumor growth and improves survival in an orthotopic model of vismodegib-resistant medulloblastoma compared to either single agent alone. Furthermore, itraconazole and arsenic trioxide retain activity in all reported drug-resistant SMO mutants, including those reported in relation to NVP-LDE225 (Novartis). Taken together, these results support clinical evaluation of itraconazole and arsenic trioxide for the treatment of Hh-dependent tumors, most notably those with acquired resistance to cyclopamine-mimics. These readily available FDA-approved agents have well defined pharmacokinetic and toxicity profiles. Our data support the rapid entry of itraconazole and arsenic trioxide into clinical testing to address patient populations for which vismodegib may not be available or is no longer efficacious. Citation Format: Blake T. Aftab, James Kim, Jean Y. Tang, Emily M. King, Daniel Kim, Jynho Kim, Ervin H. Epstein, Philip A. Beachy, Charles M. Rudin. Itraconazole and arsenic trioxide inhibit hedgehog pathway activation and tumor growth associated with acquired resistance to vismodegib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5644. doi:10.1158/1538-7445.AM2013-5644