Scott Weir

Wnt Inhibitor Screen Reveals Iron Dependence of β-Catenin Signaling in Cancers

Excessive signaling from the Wnt pathway is associated with numerous human cancers. Using a high throughput screen designed to detect inhibitors of Wnt/β-catenin signaling, we identified a series of acyl hydrazones that act downstream of the β-catenin destruction complex to inhibit both Wnt-induced and cancer-associated constitutive Wnt signaling via destabilization of β-catenin. We found that these acyl hydrazones bind iron in vitro and in intact cells and that chelating activity is required to abrogate Wnt signaling and block the growth of colorectal cancer cell lines with constitutive Wnt signaling. In addition, we found that multiple iron chelators, desferrioxamine, deferasirox, and ciclopirox olamine similarly blocked Wnt signaling and cell growth. Moreover, in patients with AML administered ciclopirox olamine, we observed decreased expression of the Wnt target gene AXIN2 in leukemic cells. The novel class of acyl hydrazones would thus be prime candidates for further development as chemotherapeutic agents. Taken together, our results reveal a critical requirement for iron in Wnt signaling and they show that iron chelation serves as an effective mechanism to inhibit Wnt signaling in humans.

DNAJA1 controls the fate of misfolded mutant p53 through the mevalonate pathway

Stabilization of mutant p53 (mutp53) in tumours greatly contributes to malignant progression. However, little is known about the underlying mechanisms and therapeutic approaches to destabilize mutp53. Here, through high-throughput screening we identify statins, cholesterol-lowering drugs, as degradation inducers for conformational or misfolded p53 mutants with minimal effects on wild-type p53 (wtp53) and DNA contact mutants. Statins preferentially suppress mutp53-expressing cancer cell growth. Specific reduction of mevalonate-5-phosphate by statins or mevalonate kinase knockdown induces CHIP ubiquitin ligase-mediated nuclear export, ubiquitylation, and degradation of mutp53 by impairing interaction of mutp53 with DNAJA1, a Hsp40 family member. Knockdown of DNAJA1 also induces CHIP-mediated mutp53 degradation, while its overexpression antagonizes statin-induced mutp53 degradation. Our study reveals that DNAJA1 controls the fate of misfolded mutp53, provides insights into potential strategies to deplete mutp53 through the mevalonate pathway–DNAJA1 axis, and highlights the significance of p53 status in impacting statins’ efficacy on cancer therapy.

Drug Repurposing for Gastrointestinal Stromal Tumor

Despite significant treatment advances over the past decade, metastatic gastrointestinal stromal tumor (GIST) remains largely incurable. Rare diseases, such as GIST, individually affect small groups of patients but collectively are estimated to affect 25 to 30 million people in the United States alone. Given the costs associated with the discovery, development, and registration of new drugs, orphan diseases such as GIST are often not pursued by mainstream pharmaceutical companies. As a result, “drug repurposing” or “repositioning,” has emerged as an alternative to the traditional drug development process. In this study, we screened 796 U.S. Food and Drug Administration (FDA)-approved drugs and found that two of these compounds, auranofin (Ridaura) and fludarabine phosphate, effectively and selectively inhibited the proliferation of GISTs, including imatinib-resistant cells. One of the most notable drug hits, auranofin, an oral, gold-containing agent approved by the FDA in 1985 for the treatment of rheumatoid arthritis, was found to inhibit thioredoxin reductase activity and induce reactive oxygen species (ROS) production, leading to dramatic inhibition of GIST cell growth and viability. Importantly, the anticancer activity associated with auranofin was independent of imatinib-resistant status, but was closely related to the endogenous and inducible levels of ROS. Coupled with the fact that auranofin has an established safety profile in patients, these findings suggest for the first time that auranofin may have clinical benefit for patients with GIST, particularly in those suffering from imatinib-resistant and recurrent forms of this disease. Mol Cancer Ther; 12(7); 1299–309. ©2013 AACR.

A Novel, Unusually Efficacious Duocarmycin Carbamate Prodrug That Releases No Residual Byproduct

A unique heterocyclic carbamate prodrug of seco-CBI-indole(2) that releases no residual byproduct is reported as a new member of a class of hydrolyzable prodrugs of the duocarmycin and CC-1065 family of natural products. The prodrug was designed to be activated by hydrolysis of a carbamate releasing the free drug without the cleavage release of a traceable extraneous group. Unlike prior carbamate prodrugs examined that are rapidly cleaved in vivo, the cyclic carbamate was found to be exceptionally stable to hydrolysis under both chemical and biological conditions providing a slow, sustained release of the exceptionally potent free drug. An in vivo evaluation of the prodrug found that its efficacy exceeded that of the parent drug, that its therapeutic window of efficacy versus toxicity is much larger than the parent drug, and that its slow free drug release permitted the safe and efficacious use of doses 150-fold higher than the parent compound.

Antigen-Specific Suppression of Experimental Autoimmune Encephalomyelitis by a Novel Bifunctional Peptide Inhibitor: Structure Optimization and Pharmacokinetics

The objective of this study was to optimize the in vivo activity of proteolipid protein (PLP)-bifunctional peptide inhibitor (BPI) molecule to suppress experimental autoimmune encephalomyelitis (EAE) in SJL/J mice and evaluate pharmacokinetic profiles of PLP-BPI. PLP-BPI is constructed via conjugation of myelin PLP139-151 with CD11a237-246-derived peptide (LABL) via a spacer. The hypothesis is that PLP-BPI binds simultaneously to major histocompatibility complex-II and intercellular adhesion molecule-1 on the antigen-presenting cell (APC) and inhibits the formation of the immunological synapse during T-cell and APC interactions. In this study, the structure of BPI was modified by varying the spacer and was evaluated in the EAE model. Intravenous injections of BPI derivatives inhibited the onset, severity, and incidence of EAE more effectively and induced a lower incidence of anaphylaxis than that produced by unmodified PLP-BPI. As anticipated, production of interleukin-17, a proinflammatory cytokine commonly found in elevated levels among multiple sclerosis (MS) patients, was significantly lower in Ac-PLP-BPI-PEG6- or Ac-PLP-BPI-NH2-2-treated mice than in phosphate-buffered saline-treated mice. These results suggest that BPI-type molecules can be modified to achieve more efficient and better tolerated BPI-based derivatives for the treatment of MS.

Oral ciclopirox olamine displays biological activity in a phase I study in patients with advanced hematologic malignancies

The antimycotic ciclopirox olamine is an intracellular iron chelator that has anticancer activity in vitro and in vivo. We developed an oral formulation of ciclopirox olamine and conducted the first‐in‐human phase I study of this drug in patients with relapsed or refractory hematologic malignancies (Trial registration ID: NCT00990587). Patients were treated with 5–80 mg/m2 oral ciclopirox olamine once daily for five days in 21‐day treatment cycles. Pharmacokinetic and pharmacodynamic companion studies were performed in a subset of patients. Following definition of the half‐life of ciclopirox olamine, an additional cohort was enrolled and treated with 80 mg/m2 ciclopirox olamine four times daily. Adverse events and clinical response were monitored throughout the trial. Twenty‐three patients received study treatment. Ciclopirox was rapidly absorbed and cleared with a short half‐life. Plasma concentrations of an inactive ciclopirox glucuronide metabolite were greater than those of ciclopirox. Repression of survivin expression was observed in peripheral blood cells isolated from patients treated once daily with ciclopirox olamine at doses greater than 10 mg/m2, demonstrating biological activity of the drug. Dose‐limiting gastrointestinal toxicities were observed in patients receiving 80 mg/m2 four times daily, and no dose limiting toxicity was observed at 40 mg/m2 once daily. Hematologic improvement was observed in two patients. Once‐daily dosing of oral ciclopirox olamine was well tolerated in patients with relapsed or refractory hematologic malignancies, and further optimization of dosing regimens is warranted in this patient population. Am. J. Hematol. 89:363–368, 2014.

The repositioning of the anti‐fungal agent ciclopirox olamine as a novel therapeutic agent for the treatment of haematologic malignancy

What is known and Objective:  6‐Cyclohexyl‐1‐hydroxy‐4‐methyl‐2(1H)‐pyridone (ciclopirox) and specifically its olamine salt 6‐cyclohexyl‐1‐hydroxy‐4‐methyl‐2(1H)‐pyridone 2‐aminoethanol salt (ciclopirox olamine) are anti‐fungal agents currently used for the treatment of mild to moderate cutaneous fungal infection. Our objective is to comment on the opportunity to rapidly reposition ciclopirox and its olamine for the treatment of haematologic malignancy by leveraging its prior published toxicology and pharmacology data.

Drug Repurposing Identifies a Synergistic Combination Therapy with Imatinib Mesylate for Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is a rare and therefore often neglected disease. Introduction of the kinase inhibitor imatinib mesylate radically improved the clinical response of patients with GIST; however, its effects are often short-lived, with GISTs demonstrating a median time-to-progression of approximately two years. Although many investigational drugs, approved first for other cancers, have been subsequently evaluated for the management of GIST, few have greatly affected the overall survival of patients with advanced disease. We employed a novel, focused, drug-repurposing effort for GIST, including imatinib mesylate–resistant GIST, evaluating a large library of FDA-approved drugs regardless of current indication. As a result of the drug-repurposing screen, we identified eight FDA-approved drugs, including fludarabine phosphate (F-AMP), that showed synergy with and/or overcame resistance to imatinib mesylate. F-AMP induces DNA damage, Annexin V, and caspase-3/7 activities as the cytotoxic effects on GIST cells, including imatinib mesylate–resistant GIST cells. F-AMP and imatinib mesylate combination treatment showed greater inhibition of GIST cell proliferation when compared with imatinib mesylate and F-AMP alone. Successful in vivo experiments confirmed the combination of imatinib mesylate with F-AMP enhanced the antitumor effects compared with imatinib mesylate alone. Our results identified F-AMP as a promising, repurposed drug therapy for the treatment of GISTs, with potential to be administered in combination with imatinib mesylate or for treatment of imatinib mesylate–refractory tumors. Mol Cancer Ther; 13(10); 2276–87. ©2014 AACR.

Gender-specific reduction of hepatic Mrp2 expression by high-fat diet protects female mice from ANIT toxicity

Emerging evidence suggests that feeding a high-fat diet (HFD) to rodents affects the expression of genes involved in drug transport. However, gender-specific effects of HFD on drug transport are not known. The multidrug resistance-associated protein 2 (Mrp2, Abcc2) is a transporter highly expressed in the hepatocyte canalicular membrane and is important for biliary excretion of glutathione-conjugated chemicals. The current study showed that hepatic Mrp2 expression was reduced by HFD feeding only in female, but not male, C57BL/6J mice. In order to determine whether down-regulation of Mrp2 in female mice altered chemical disposition and toxicity, the biliary excretion and hepatotoxicity of the Mrp2 substrate, α-naphthylisothiocyanate (ANIT), were assessed in male and female mice fed control diet or HFD for 4weeks. ANIT-induced biliary injury is a commonly used model of experimental cholestasis and has been shown to be dependent upon Mrp2-mediated efflux of an ANIT glutathione conjugate that selectively injures biliary epithelial cells. Interestingly, HFD feeding significantly reduced early-phase biliary ANIT excretion in female mice and largely protected against ANIT-induced liver injury. In summary, the current study showed that, at least in mice, HFD feeding can differentially regulate Mrp2 expression and function and depending upon the chemical exposure may enhance or reduce susceptibility to toxicity. Taken together, these data provide a novel interaction between diet and gender in regulating hepatobiliary excretion and susceptibility to injury.

Impact of HuR inhibition by the small molecule MS-444 on colorectal cancer cell tumorigenesis

Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality. Observed during CRC tumorigenesis is loss of post-transcriptional regulation of tumor-promoting genes such as COX-2, TNFα and VEGF. Overexpression of the RNA-binding protein HuR (ELAVL1) occurs during colon tumorigenesis and is abnormally present within the cytoplasm, where it post-transcriptionally regulates genes through its interaction with 3′UTR AU-rich elements (AREs). Here, we examine the therapeutic potential of targeting HuR using MS-444, a small molecule HuR inhibitor. Treatment of CRC cells with MS-444 resulted in growth inhibition and increased apoptotic gene expression, while similar treatment doses in non-transformed intestinal cells had no appreciable effects. Mechanistically, MS-444 disrupted HuR cytoplasmic trafficking and released ARE-mRNAs for localization to P-bodies, but did not affect total HuR expression levels. This resulted in MS-444-mediated inhibition of COX-2 and other ARE-mRNA expression levels. Importantly, MS-444 was well tolerated and inhibited xenograft CRC tumor growth through enhanced apoptosis and decreased angiogenesis upon intraperitoneal administration. In vivo treatment of MS-444 inhibited HuR cytoplasmic localization and decreased COX-2 expression in tumors. These findings provide evidence that therapeutic strategies to target HuR in CRC warrant further investigation in an effort to move this approach to the clinic.