The Ah Receptor from Toxicity to Therapeutics: Report from the 5th AHR Meeting at Penn State University, USA, June 2022.

The aryl hydrocarbon receptor (AHR) is a sensor of low-molecular-weight molecule signals that originate from environmental exposures, the microbiome, and host metabolism. Building upon initial studies examining anthropogenic chemical exposures, the list of AHR ligands of microbial, diet, and host metabolism origin continues to grow and has provided important clues as to the function of this enigmatic receptor. The AHR has now been shown to be directly involved in numerous biochemical pathways that influence host homeostasis, chronic disease development, and responses to toxic insults. As this field of study has continued to grow, it has become apparent that the AHR is an important novel target for cancer, metabolic diseases, skin conditions, and autoimmune disease. This meeting attempted to cover the scope of basic and applied research being performed to address possible applications of our basic knowledge of this receptor on therapeutic outcomes.

Bypassing evolutionary dead ends and switching the rate-limiting step of a human immunotherapeutic enzyme.

The Trp metabolite kynurenine (KYN) accumulates in numerous solid tumours and mediates potent immunosuppression. Bacterial kynureninases (KYNases), which preferentially degrade kynurenine, can relieve immunosuppression in multiple cancer models, but immunogenicity concerns preclude their clinical use, while the human enzyme (HsKYNase) has very low activity for kynurenine and shows no therapeutic effect. Using fitness selections, we evolved a HsKYNase variant with 27-fold higher activity, beyond which exploration of >30 evolutionary trajectories involving the interrogation of >109 variants led to no further improvements. Introduction of two amino acid substitutions conserved in bacterial KYNases reduced enzyme fitness but potentiated rapid evolution of variants with ~500-fold improved activity and reversed substrate specificity, resulting in an enzyme capable of mediating strong anti-tumour effects in mice. Pre-steady-state kinetics revealed a switch in rate-determining step attributable to changes in both enzyme structure and conformational dynamics. Apart from its clinical significance, our work highlights how rationally designed substitutions can potentiate trajectories that overcome barriers in protein evolution.

Discovery and Characterization of a Novel Aryl Hydrocarbon Receptor Inhibitor, IK-175, and Its Inhibitory Activity on Tumor Immune Suppression.

Aryl hydrocarbon receptor (AHR) is a transcription factor that regulates the activity of multiple innate and adaptive immune cells subsequent to binding to numerous endogenous and exogenous ligands. For example, AHR is activated by the metabolite kynurenine, which is secreted into the tumor microenvironment by cancer cells leading to broad immunosuppression. Therefore, AHR inhibition provides a novel and ideal approach to stimulate immune-mediated recognition and subsequent eradication of tumor cells. We report here the discovery and characterization of IK-175, a novel, potent and selective AHR antagonist with favorable ADME and pharmacokinetic profiles in preclinical species. IK-175 inhibits AHR activity in experimental systems derived from multiple species including mouse, rat, monkey, and humans. In human primary immune cells, IK-175 decreased AHR target gene expression and anti-inflammatory cytokine release and increased proinflammatory cytokine release. Moreover, IK-175 led to a decrease in suppressive IL17A-, IL-22+ expressing T cells in a Th17 differentiation assay. IK-175 dose dependently blocks ligand-stimulated AHR activation of Cyp1a1 transcription in mouse liver and spleen, demonstrating on-target in vivo activity. IK-175 increases proinflammatory phenotype of the tumor microenvironment in mouse syngeneic tumors and in adjacent tumor-draining lymph nodes. As a monotherapy and combined with an anti-PD-1 antibody, IK-175 demonstrates antitumor activity in syngeneic mouse models of colorectal cancer and melanoma. IK-175 also demonstrates antitumor activity combined with liposomal doxorubicin in syngeneic mouse tumors. These studies provide rationale for targeting AHR in patients with cancer. IK-175 is being evaluated in a phase I clinical trial in patients with advanced solid tumors.

Blockade of the AHR restricts a Treg-macrophage suppressive axis induced by L-Kynurenine.

Tryptophan catabolism by the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase 2 (IDO/TDO) promotes immunosuppression across different cancer types. The tryptophan metabolite L-Kynurenine (Kyn) interacts with the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) to drive the generation of Tregs and tolerogenic myeloid cells and PD-1 up-regulation in CD8+ T cells. Here, we show that the AHR pathway is selectively active in IDO/TDO-overexpressing tumors and is associated with resistance to immune checkpoint inhibitors. We demonstrate that IDO-Kyn-AHR-mediated immunosuppression depends on an interplay between Tregs and tumor-associated macrophages, which can be reversed by AHR inhibition. Selective AHR blockade delays progression in IDO/TDO-overexpressing tumors, and its efficacy is improved in combination with PD-1 blockade. Our findings suggest that blocking the AHR pathway in IDO/TDO expressing tumors would overcome the limitation of single IDO or TDO targeting agents and constitutes a personalized approach to immunotherapy, particularly in combination with immune checkpoint inhibitors.

Inhibition of de novo lipogenesis targets androgen receptor signaling in castration-resistant prostate cancer.

A hallmark of prostate cancer progression is dysregulation of lipid metabolism via overexpression of fatty acid synthase (FASN), a key enzyme in de novo fatty acid synthesis. Metastatic castration-resistant prostate cancer (mCRPC) develops resistance to inhibitors of androgen receptor (AR) signaling through a variety of mechanisms, including the emergence of the constitutively active AR variant V7 (AR-V7). Here, we developed an FASN inhibitor (IPI-9119) and demonstrated that selective FASN inhibition antagonizes CRPC growth through metabolic reprogramming and results in reduced protein expression and transcriptional activity of both full-length AR (AR-FL) and AR-V7. Activation of the reticulum endoplasmic stress response resulting in reduced protein synthesis was involved in IPI-9119-mediated inhibition of the AR pathway. In vivo, IPI-9119 reduced growth of AR-V7-driven CRPC xenografts and human mCRPC-derived organoids and enhanced the efficacy of enzalutamide in CRPC cells. In human mCRPC, both FASN and AR-FL were detected in 87% of metastases. AR-V7 was found in 39% of bone metastases and consistently coexpressed with FASN. In patients treated with enzalutamide and/or abiraterone FASN/AR-V7 double-positive metastases were found in 77% of cases. These findings provide a compelling rationale for the use of FASN inhibitors in mCRPCs, including those overexpressing AR-V7.

The phosphoinositide-3 kinase (PI3K)-δ,γ inhibitor, duvelisib shows preclinical synergy with multiple targeted therapies in hematologic malignancies.

Duvelisib is an orally active dual inhibitor of PI3K-δ and PI3K-γ in clinical development in hematologic malignancies (HM). To identify novel pairings for duvelisib in HM, it was evaluated alone and in combination with 35 compounds comprising a diverse panel of standard-of-care agents and emerging drugs in development for HM. These compounds were tested in 20 cell lines including diffuse large B-cell, follicular, T-cell, and mantle cell lymphomas, and multiple myeloma. Single agent activity was seen in fourteen cell lines, with a median GI50 of 0.59 µM. A scalar measure of the strength of synergistic drug interactions revealed a synergy hit rate of 19.3% across the matrix of drug combinations and cell lines. Synergy with duvelisib was prominent in lymphoma lines with approved and emerging drugs used to treat HM, including dexamethasone, ibrutinib, and the BCL-2 inhibitor venetoclax. Western blotting revealed that certain duvelisib-treated cell lines showed inhibition of phosphorylated (p) AKT at serine 473 only out to 12 hours, with mTORC2 dependent re-phosphorylation of pAKT evident at 24 hours. Combination with dexamethasone or ibrutinib, however, prevented this reactivation leading to durable inhibition of pAKT. The combination treatments also inhibited downstream signaling effectors pPRAS40 and pS6. The combination of duvelisib with dexamethasone also significantly reduced p-4EBP1, which controls cap dependent translation initiation, leading to decreased levels of c-MYC 6 hours after treatment. In support of the in vitro studies, in vivo xenograft studies revealed that duvelisib in combination with the mTOR inhibitor everolimus led to greater tumor growth inhibition compared to single agent administration. These data provide a rationale for exploring multiple combinations in the clinic and suggest that suppression of mTOR-driven survival signaling may be one important mechanism for combination synergy.

Reversal of indoleamine 2,3-dioxygenase-mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme.

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

The Selective Phosphoinoside-3-Kinase p110δ Inhibitor IPI-3063 Potently Suppresses B Cell Survival, Proliferation, and Differentiation.

The class I phosphoinoside-3-kinases (PI3Ks) are important enzymes that relay signals from cell surface receptors to downstream mediators driving cellular functions. Elevated PI3K signaling is found in B cell malignancies and lymphocytes of patients with autoimmune disease. The p110δ catalytic isoform of PI3K is a rational target since it is critical for B lymphocyte development, survival, activation, and differentiation. In addition, activating mutations in PIK3CD encoding p110δ cause a human immunodeficiency known as activated PI3K delta syndrome. Currently, idelalisib is the only selective p110δ inhibitor that has been FDA approved to treat certain B cell malignancies. p110δ inhibitors can suppress autoantibody production in mouse models, but limited clinical trials in human autoimmunity have been performed with PI3K inhibitors to date. Thus, there is a need for additional tools to understand the effect of pharmacological inhibition of PI3K isoforms in lymphocytes. In this study, we tested the effects of a potent and selective p110δ inhibitor, IPI-3063, in assays of B cell function. We found that IPI-3063 potently reduced mouse B cell proliferation, survival, and plasmablast differentiation while increasing antibody class switching to IgG1, almost to the same degree as a pan-PI3K inhibitor. Similarly, IPI-3063 potently inhibited human B cell proliferation in vitro. The p110γ isoform has partially overlapping roles with p110δ in B cell development, but little is known about its role in B cell function. We found that the p110γ inhibitor AS-252424 had no significant impact on B cell responses. A novel dual p110δ/γ inhibitor, IPI-443, had comparable effects to p110δ inhibition alone. These findings show that p110δ is the dominant isoform mediating B cell responses and establish that IPI-3063 is a highly potent molecule useful for studying p110δ function in immune cells.

Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells.

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

PI3Kγ is a molecular switch that controls immune suppression.

Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer. In response to pathogens or injury, inflammatory macrophages express cytokines that stimulate cytotoxic T cells, whereas macrophages in neoplastic and parasitic diseases express anti-inflammatory cytokines that induce immune suppression and may promote resistance to T cell checkpoint inhibitors. Here we show that macrophage PI 3-kinase γ controls a critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ signalling through Akt and mTor inhibits NFκB activation while stimulating C/EBPß activation, thereby inducing a transcriptional program that promotes immune suppression during inflammation and tumour growth. By contrast, selective inactivation of macrophage PI3Kγ stimulates and prolongs NFκB activation and inhibits C/EBPß activation, thus promoting an immunostimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity. PI3Kγ synergizes with checkpoint inhibitor therapy to promote tumour regression and increased survival in mouse models of cancer. In addition, PI3Kγ-directed, anti-inflammatory gene expression can predict survival probability in cancer patients. Our work thus demonstrates that therapeutic targeting of intracellular signalling pathways that regulate the switch between macrophage polarization states can control immune suppression in cancer and other disorders.

Integration of Hedgehog and mutant FLT3 signaling in myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild-type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing signal transducer and activator of transcription 5 (STAT5) signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathway inhibition limited leukemic growth in vitro and in vivo, and this approach may serve as a therapeutic strategy for FLT3-ITD AML.

HSP90 inhibition enhances antimitotic drug-induced mitotic arrest and cell death in preclinical models of non-small cell lung cancer.

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.

Impact of the Smoothened inhibitor, IPI-926, on smoothened ciliary localization and Hedgehog pathway activity.

A requisite step for canonical Hedgehog (Hh) pathway activation by Sonic Hedgehog (Shh) ligand is accumulation of Smoothened (Smo) to the primary cilium (PC). Activation of the Hh pathway has been implicated in a broad range of cancers, and several Smo antagonists are being assessed clinically, one of which is approved for the treatment of advanced basal cell carcinoma. Recent reports demonstrate that various Smo antagonists differentially impact Smo localization to the PC while still exerting inhibitory activity. In contrast to other synthetic small molecule Smo antagonists, the natural product cyclopamine binds to and promotes ciliary accumulation of Smo and "primes" cells for Hh pathway hyper-responsiveness after compound withdrawal. We compared the properties of IPI-926, a semi-synthetic cyclopamine analog, to cyclopamine with regard to potency, ciliary Smo accumulation, and Hh pathway activity after compound withdrawal. Like cyclopamine, IPI-926 promoted accumulation of Smo to the PC. However, in contrast to cyclopamine, IPI-926 treatment did not prime cells for hyper-responsiveness to Shh stimulation after compound withdrawal, but instead demonstrated continuous inhibition of signaling. By comparing the levels of drug-induced ciliary Smo accumulation with the degree of Hh pathway activity after compound withdrawal, we propose that a critical threshold of ciliary Smo is necessary for "priming" activity to occur. This "priming" appears achievable with cyclopamine, but not IPI-926, and is cell-line dependent. Additionally, IPI-926 activity was evaluated in a murine tumor xenograft model and a pharmacokinetic/pharmacodynamic relationship was examined to assess for in vivo evidence of Hh pathway hyper-responsiveness. Plasma concentrations of IPI-926 correlated with the degree and duration of Hh pathway suppression, and pathway activity did not exceed baseline levels out to 96 hours post dose. The overall findings suggest that IPI-926 possesses unique biophysical and pharmacological properties that result in Hh pathway inhibition in a manner that differentiates it from cyclopamine.

Hedgehog pathway inhibition in chondrosarcoma using the smoothened inhibitor IPI-926 directly inhibits sarcoma cell growth.

Hedgehog (Hh) pathway inhibition in cancer has been evaluated in both the ligand-independent and ligand-dependent settings, where Hh signaling occurs either directly within the cancer cells or within the nonmalignant cells of the tumor microenvironment. Chondrosarcoma is a malignant tumor of cartilage in which there is ligand-dependent activation of Hh signaling. IPI-926 is a potent, orally delivered small molecule that inhibits Hh pathway signaling by binding to Smoothened (SMO). Here, the impact of Hh pathway inhibition on primary chondrosarcoma xenografts was assessed. Mice bearing primary human chondrosarcoma xenografts were treated with IPI-926. The expression levels of known Hh pathway genes, in both the tumor and stroma, and endpoint tumor volumes were measured. Gene expression profiling of tumors from IPI-926-treated mice was conducted to identify potential novel Hh target genes. Hh target genes were studied to determine their contribution to the chondrosarcoma neoplastic phenotype. IPI-926 administration results in downmodulation of the Hh pathway in primary chondrosarcoma xenografts, as demonstrated by evaluation of the Hh target genes GLI1 and PTCH1, as well as inhibition of tumor growth. Chondrosarcomas exhibited autocrine and paracrine Hh signaling, and both were affected by IPI-926. Decreased tumor growth is accompanied by histopathologic changes, including calcification and loss of tumor cells. Gene profiling studies identified genes differentially expressed in chondrosarcomas following IPI-926 treatment, one of which, ADAMTSL1, regulates chondrosarcoma cell proliferation. These studies provide further insight into the role of the Hh pathway in chondrosarcoma and provide a scientific rationale for targeting the Hh pathway in chondrosarcoma.

Involvement and targeted intervention of dysregulated Hedgehog signaling in osteosarcoma.

BACKGROUND: During development, the Hedgehog pathway plays important roles regulating the proliferation and differentiation of chondrocytes, providing a template for growing bone. In this study, the authors investigated the components of dysregulated Hedgehog signaling as potential therapeutic targets for osteosarcoma. METHODS: Small-molecule agonists and antagonists that modulate the Hedgehog pathway at different levels were used to investigate the mechanisms of dysregulation and the efficacy of Hedgehog blockade in osteosarcoma cell lines. The inhibitory effect of a small-molecule Smoothened (SMO) antagonist, IPI-926 (saridegib), also was examined in patient-derived xenograft models. RESULTS: An inverse correlation was identified in osteosarcoma cell lines between endogenous glioma-associated oncogene 2 (GLI2) levels and Hedgehog pathway induction levels. Cells with high levels of GLI2 were sensitive to GLI inhibition, but not SMO inhibition, suggesting that GLI2 overexpression may be a mechanism of ligand-independent activation. In contrast, cells that expressed high levels of the Hedgehog ligand gene Indian hedgehog (IHH) and the target genes patched 1 (PTCH1) and GLI1 were sensitive to modulation of both SMO and GLI, suggesting ligand-dependent activation. In 2 xenograft models, active autocrine and paracrine, ligand-dependent Hedgehog signaling was identified. IPI-926 inhibited the Hedgehog signaling interactions between the tumor and the stroma and demonstrated antitumor efficacy in 1 of 2 ligand-dependent models. CONCLUSIONS: The current results indicate that both ligand-dependent and ligand-independent Hedgehog dysregulation may be involved in osteosarcoma. It is the first report to demonstrate Hedgehog signaling crosstalk between the tumor and the stroma in osteosarcoma. The inhibitory effect of IPI-926 warrants additional research and raises the possibility of using Hedgehog pathway inhibitors as targeted therapeutics to improve treatment for osteosarcoma.

The pre-clinical absorption, distribution, metabolism and excretion properties of IPI-926, an orally bioavailable antagonist of the hedgehog signal transduction pathway.

1. IPI-926 is a novel semisynthetic cyclopamine derivative that is a potent and selective Smoothened inhibitor that blocks the hedgehog signal transduction pathway. 2. The in vivo clearance of IPI-926 is low in mouse and dog and moderate in monkey. The volume of distribution is high across species. Oral bioavailability ranges from moderate in monkey to high in mouse and dog. Predicted human clearance using simple allometry is low (24 L h(-1)), predicted volume of distribution is high (469 L) and predicted half-life is long (20 h). 3. IPI-926 is highly bound to plasma proteins and has minimal interaction with human α-1-acid glycoprotein. 4. In vitro metabolic stability ranges from stable to moderately stable. Twelve oxidative metabolites were detected in mouse, rat, dog, monkey and human liver microsome incubations and none were unique to human. 5. IPI-926 is not a potent reversible inhibitor of CYP1A2, 2C8, 2C9 or 3A4 (testosterone). IPI-926 is a moderate inhibitor of CYP2C19, 2D6 and 3A4 (midazolam) with KI values of 19, 16 and 4.5 µM, respectively. IPI-926 is both a substrate and inhibitor (IC50 = 1.9 µM) of P-glycoprotein. 6. In summary, IPI-926 has desirable pre-clinical absorption, distribution, metabolism and excretion properties.

Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.

Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

Hedgehog pathway inhibitor saridegib (IPI-926) increases lifespan in a mouse medulloblastoma model.

The Sonic Hedgehog (Shh) pathway drives a subset of medulloblastomas, a malignant neuroectodermal brain cancer, and other cancers. Small-molecule Shh pathway inhibitors have induced tumor regression in mice and patients with medulloblastoma; however, drug resistance rapidly emerges, in some cases via de novo mutation of the drug target. Here we assess the response and resistance mechanisms to the natural product derivative saridegib in an aggressive Shh-driven mouse medulloblastoma model. In this model, saridegib treatment induced tumor reduction and significantly prolonged survival. Furthermore, the effect of saridegib on tumor-initiating capacity was demonstrated by reduced tumor incidence, slower growth, and spontaneous tumor regression that occurred in allografts generated from previously treated autochthonous medulloblastomas compared with those from untreated donors. Saridegib, a known P-glycoprotein (Pgp) substrate, induced Pgp activity in treated tumors, which likely contributed to emergence of drug resistance. Unlike other Smoothened (Smo) inhibitors, the drug resistance was neither mutation-dependent nor Gli2 amplification-dependent, and saridegib was found to be active in cells with the D473H point mutation that rendered them resistant to another Smo inhibitor, GDC-0449. The fivefold increase in lifespan in mice treated with saridegib as a single agent compares favorably with both targeted and cytotoxic therapies. The absence of genetic mutations that confer resistance distinguishes saridegib from other Smo inhibitors.

New developments in the discovery of small molecule Hedgehog pathway antagonists.

The Hedgehog (Hh) signaling pathway is crucial for normal embryonic development. Aberrant Hh signaling is implicated in numerous pathologic conditions including proliferative diseases such as cancer. During the past decade, academic and industrial research efforts have resulted in the discovery of a variety of Hh pathway antagonists. This review focuses on the most recent advances in this field with particular emphasis on the medicinal chemistry approaches used to discover these Hh antagonists. While most of the small molecule modulators of the Hh pathway were discovered through screening and subsequent medicinal chemistry, a number of them originated from rational design or natural products.