Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2's capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD

Interferon-gamma: A Potent Antiviral Agent Targeting Macrophages Infected with LP-BM5 Murine Leukemia Virus, the Causative Agent of 'AIDS' in Mice

Cells of the monocyte/macrophage lineage (MM cells) are known to be infected by retroviruses, including the human immunodeficiency virus (HIV), without cytopathic changes and may serve as a persistent reservoir for the virus during the development of immunodeficiency disease. LP-BM5 murine leukemia virus (MuLV) infection of C57BL/6 mice and cell lines has been used to optimize therapy directed against macrophages. Findings in this murine system may be applicable to HN infection in humans. The effect of recombinant murine interferon-gamma (IFN-γ) and 3' -azido-2',3' -dideoxythymidine (AZT) as single agents or in combination was investigated in both LP-BM5 MuLV de novo infection and chronic infection of macrophages. Results indicate that the therapeutic effects of these single agents were dose-dependent and both agents were similarly effective in reducing the production of infectious virus determined by XC-plaque assay and by measurements of reverse transcriptase activity in culture supernatants; and AZT and IFN-γ reduced the production of virus proteins, quantified by laser densitometry of fluorographs from immunoprecipitated viral proteins using virus-specific antiserum. A combination of IFN-γ and that AZT showed greater antiviral activity in both LP-BM5 MuLV de novo and chronic infection of macrophages than either agent alone, suggesting that IFN-γ and AZT represent a potent combination of antiviral agents targeting macrophages. Further, since a lower concentration of each agent was required for efficacy in combination therapy, toxicity associated with single agent therapy may be avoided

Novel small molecule inhibitors of MDM2/4-p53 interaction, YH264 and its ethyl ester YH263: Preclinical evaluation

Introduction: In p53+/+ cells, expression of MDM2/4 leads to turnover of p53 and inhibition of downstream gene transcription decreasing cell cycle arrest or apoptosis. Prevention of MDM2/4-p53 interaction is a promising therapeutic strategy. Two in-house developed small molecule inhibitors of MDM2/4-p53 binding, YH264 and YH263, had nM activity in protein binding assays, were characterized by co-crystal structures, and had low μM activity in the NCI 60 cell screen. We evaluated in vitro cytotoxicity in HCT 116, human p53+/+ colon cancer cells as well as efficacy, pharmacokinetics, and metabolism in mice bearing HCT 116 xenografts. Methods: Cytotoxicity against HCT 116 cells was assessed by 72 h MTT assay. C.B-17 SCID mice bearing HCT 116 xenografts (5 mice/group) were dosed with either YH264 or YH263 (150 mg/kg IV or PO), or vehicle QDx5 and tumors were measured twice weekly for at least 1 week post dosing. For pharmacokinetic studies, mice bearing HCT 116 xenografts were treated with 150 mg/kg YH264 or YH263 IV and PO. Plasma and tumor were collected between 5 min and 24 h. Urine was collected between 0-6 h and 6-24 h. YH264 and YH263 in cells from the in vitro study and tissues were quantitated with an LC-MS/MS assay. Pharmacokinetic parameters were calculated non-compartmentally. Results: IC50 values of YH264 and YH263 were 18.6 (9 μg/ml) and 8.9 μM (4 μg/ml), respectively. When mice were dosed IV or PO for QDx5 with YH264 or YH263, no decrease in body weights or significant decrease in xenograft volume was observed when compared to vehicle treated mice. Plasma elimination of IV YH264 was biphasic. Plasma and tumor parameters were respectively: Cmax 1,451 μg/ml and 44 μg/g; AUC0-t 125,000 μg·min/ml and 38,000 μg·min/g. Tumor concentrations remained above 17 μg/g out to 24 h. Plasma t1/2 was 147 min and clearance was 1.2 ml/min/kg. Metabolites included hydroxylated and glucuronidated products. After PO dosing, bioavailability was 18% and YH264 was undetectable in tumor. IV plasma elimination of the ethyl-ester YH263 was biphasic as well. Plasma and tumor parameters were respectively: Cmax 995 μg/ml and 21 μg/g; AUC0-t 45,477 μg·min/ml and 18,950 μg·min/g. Tumor concentrations remained above 7 μg/g for 24 h. Plasma t1/2 was 263 min and clearance was 3.3 ml/min/kg. 2% of the YH263 dose was converted to YH264 and there were multiple hydroxylated and glucuronidated metabolites. After PO dosing, bioavailability was 4.8%. In culture YH264 and YH263 cellular concentrations were 60- and 200-fold higher than medium concentrations. Conclusions: At the IC50 medium concentration, the cells accumulated 200-fold higher concentrations: of YH263 than medium. Despite dosing the mice at the maximum soluble dose, we could not achieve tumor concentrations (800 μg/g) equivalent to those required to inhibit cells in vitro. This may explain the absence of efficacy on the current schedule

Systemic administration of a cyclic signal transducer and activator of transcription 3 (STAT3) decoy oligonucleotide inhibits tumor growth without inducing toxicological effects.

Hyperactivation of signal transducer and activator of transcription 3 (STAT3) has been linked to tumorigenesis in most malignancies, including head and neck squamous cell carcinoma. Intravenous delivery of a chemically modified cyclic STAT3 decoy oligonucleotide with improved serum and thermal stability demonstrated antitumor efficacy in conjunction with downmodulation of STAT3 target gene expression such as cyclin D1 and Bcl-X(L) in a mouse model of head and neck squamous cell carcinoma. The purpose of the present study was to determine the toxicity and dose-dependent antitumor efficacy of the cyclic STAT3 decoy after multiple intravenous doses in Foxn1 nu mice in anticipation of clinical translation. The two doses (5 and 10 mg/kg) of cyclic STAT3 decoy demonstrated a significant decrease in tumor volume compared with the control groups (mutant cyclic STAT3 decoy or saline) in conjunction with downmodulation of STAT3 target gene expression. There was no dose-dependent effect of cyclic STAT3 decoy on tumor volume or STAT3 target gene expression. There were no significant changes in body weights between the groups during the dosing period, after the dosing interval or on the day of euthanasia. No hematology or clinical chemistry parameters suggested toxicity of the cyclic STAT3 decoy compared with saline control. No gross or histological pathological abnormalities were noted at necropsy in any of the animals. These findings suggest a lack of toxicity of intravenous administration of a cyclic STAT3 decoy oligonucleotide. In addition, comparable antitumor effects indicate a lack of dose response at the two dose levels investigated

In vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of pyrazole-based small molecule inhibitors of Mdm2/4-p53 interaction

PURPOSE: The interaction of p53 with its negative regulators Mdm2/4 has been widely studied (Khoury and Domling in Curr Pharm Des 18(30):4668-4678, 2012). In p53(+/+) cells, expression of Mdm2/4 leads to p53 turnover, inhibition of downstream transcription, decreasing cell cycle arrest, or apoptosis. We report in vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of YH264, YH263, and WW751, three proposed small molecule inhibitors of the Mdm2/4-p53 interaction. METHODS: MTT cytotoxicity assays were performed, and alterations in proteins were examined using western blots. Mice were dosed 150 mg/kg YH264 or YH263 IV or PO QDx5. Mice were IV dosed 88, 57, or 39 mg/kg WW751 for 3, 5, or 5 days. YH264, YH263, and WW751 and metabolites were quantitated by LC-MS/MS. RESULTS: IC50 values for YH264, YH263, and WW751 against p53 wild-type HCT 116 cells after 72 h of incubation were 18.3 ± 2.3, 8.9 ± 0.6, and 3.1 ± 0.2 μM, respectively. Only YH264 appeared to affect p53 expression in vitro. None of the compounds affected the growth of HCT 116 xenografts in C.B-17 SCID mice. YH264 plasma half-life was 147 min; YH263 plasma half-life was 263 min; and WW751 plasma half-life was less than 120 min. CONCLUSIONS: Despite dosing the mice at the maximum soluble doses, we could not achieve tumor concentrations equivalent to the intracellular concentrations required to inhibit cell growth in vitro. YH263 and WW751 do not appear to affect p53/Mdm2, and none of the three were active in a subcutaneous HCT 116 p53(+/+) xenograft model