The nuclear export protein XPO1 is overexpressed in cancer, leading to the cytoplasmic mislocalization of multiple tumor suppressor proteins. Existing XPO1-targeting agents lack selectivity and have been associated with significant toxicity. Small molecule selective inhibitors of nuclear export (SINEs) were designed that specifically inhibit XPO1. Genetic experiments and X-ray structures demonstrate that SINE covalently bind to a cysteine residue in the cargo-binding groove of

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Protein kinase A (PKA) is an evolutionarily conserved protein which has been studied in model organisms from yeast to man. Although the cAMP-PKA signaling system was the first mammalian second messenger system to be characterized, many aspects of this pathway are still not well understood. Owing to findings over the past decade implicating PKA signaling in endocrine (and other) tumorigenesis, there has been renewed interest in understanding the role of this pathway in physiology, particularly as it pertains to the endocrine system. Because of the availability of genetic tools, mouse modeling has become the pre-eminent system for studying the physiological role of specific genes and gene families as a means to understanding their relationship to human diseases. In this review, we will summarize the current data regarding mouse models that have targeted the PKA signaling system. These data have led to a better understanding of both the complexity and the subtlety of PKA signaling, and point the way for future studies, which may help to modulate this pathway for therapeutic effect.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phosphoAkt and cyclin D1 and subsequent cellcycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagiclysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti- IntroductionMantle cell lymphoma (MCL) is a B-cell malignancy that comprises 3%-8% of non-Hodgkin lymphoma cases diagnosed each year. 1 Whereas the current treatment approach of using combination chemotherapeutic regimens can lead to complete remission, virtually all MCL patients relapse and outcome remains poor, with a median survival of only 3 years. 2 The aggressive clinical behavior of MCL may be because of the complex pathophysiology of the disease, which includes cell-cycle dysregulation driven by cyclin D1 overexpression, alteration in the DNA-damage response, and constitutive activation of key antiapoptotic pathways such as PI3K/Akt and NF-B. [3][4][5][6] Given the absence of curative therapy and the limited number of options for patients with relapsed/refractory MCL, it will be essential to improve our understanding of the complex biology of this disease so that novel treatment approaches can be developed. FTY720 (fingolimod), is a synthetic analog of sphingosine that was developed as an immunosuppressive agent. 7,8 Based on the results of a recent phase 3 clinical trial, FTY720 has been approved by the US Food and Drug Administration (FDA) to treat relapsed multiple sclerosis. 9 We have recently reported that FTY720 has in vitro and in vivo activity in MCL. 10 FTY720 promotes death of MCL cell lines and primary MCL tumor cells via caspaseindependent radical oxygen species (ROS) generation, downmodulation of phospho-Akt and cyclin D1, with accumulation of cells in G 0 /G 1 and G 2 /M phases of the cell cycle. Whereas these data provided information explaining the antitumor activity of FTY720, the effects of this drug on the pathophysiology of MCL required further characterization.In the present study, we show that FTY720 inhibits autophagic flux and induces MCL cell death through lysosomal membrane permeabilization and subsequent translocation of lysosomal hydrolases in the cytosol. Because the autophagy-lysosomal pathway represents an important regulatory mechanism governing the cellular proteome, we hypo...

Flavopiridol is a small molecule inhibitor of cyclin-dependent kinases (CDK) known to impair global transcription via inactivation of positive transcription elongation factor b. It has been demonstrated to have significant activity predominantly in chronic lymphocytic leukemia and acute myeloid leukemia in phase I/II clinical trials while other similar CDK inhibitors are vigorously being pursued in pre-clinical and clinical studies. Although flavopiridol is a potent therapeutic agent against blood diseases, some patients still have primary or acquired resistance throughout their clinical course. Considering the limited knowledge of resistance mechanisms of flavopiridol, we investigated the potential mechanisms of resistance to flavopiridol in a cell line system, which gradually acquired resistance to flavopiridol in vitro, and then confirmed the mechanism in patient samples. Herein, we present that this resistant cell line developed resistance through up-regulation of phosphorylation of RNA polymerase II C-terminal domain, activation of CDK9 kinase activity, and prolonged Mcl-1 stability to counter flavopiridol's drug actions. Further analyses suggest MAPK/ERK activation-mediated Mcl-1 stabilization contributes to the resistance and knockdown of Mcl-1 in part restores sensitivity to flavopiridol-induced cytotoxicity. Altogether, these findings demonstrate that CDK9 is the most relevant target of flavopiridol and provide avenues to improve the therapeutic strategies in blood malignancies.

The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling.

Chronic lymphocytic leukemia (CLL) is a mature B cell malignancy and is the most prevalent type of leukemia in adults. There is no curative therapy for this disease; however, several new agents have shown very promising results. Autophagy has not been studied in CLL and in this study we first sought to determine if autophagy was functional in CLL with classic inducers, and if this contributes to direct cytotoxicity or protection from cell death. While autophagy is activated with all classic stimuli of this process, only unfolded protein endoplasmic reticulum (ER) stress-mediated autophagy protects from cell death. Interestingly, select therapeutic agents (fludarabine, GS-1101, flavopiridol), which are active in CLL, also induce autophagy. Of interest, only the broad cyclin-dependent kinase inhibitor flavopiridol has improved efficacy when autophagy is antagonized biochemically (chloroquine) or by siRNA. This promoted an investigation which demonstrated unexpectedly that flavopiridol mediates ER stress and downstream activation of MAP3K5/ASK1, which ultimately is responsible for cell death. Similarly, autophagy activated in part via ER stress and also CDK5 inhibition is protective against cell death induced by this process. Collectively, our studies demonstrate that in CLL, autophagy is induced by multiple stimuli but only acts as a mechanism of resistance against ER stress-mediating agents. Similarly, flavopiridol mediates ER stress as a primary mechanism of action in CLL, and autophagy serves as a mechanism of resistance to this agent.

Abstract. Lenalidomide is a synthetic derivative of thalidomide exhibiting multiple immunomodulatory activities beneficial in the treatment of several hematological malignancies. Murine pharmacokinetic characterization necessary for translational and further preclinical investigations has not been published. Studies herein define mouse plasma pharmacokinetics and tissue distribution after intravenous (IV) bolus administration and bioavailability after oral and intraperitoneal delivery. Range finding studies used lenalidomide concentrations up to 15 mg/kg IV, 22.5 mg/kg intraperitoneal injections (IP), and 45 mg/kg oral gavage (PO). Pharmacokinetic studies evaluated doses of 0.5, 1.5, 5, and 10 mg/kg IV and 0.5 and 10 mg/kg doses for IP and oral routes. Liquid chromatography-tandem mass spectrometry was used to quantify lenalidomide in plasma, brain, lung, liver, heart, kidney, spleen, and muscle. Pharmacokinetic parameters were estimated using noncompartmental and compartmental methods. Doses of 15 mg/kg IV, 22.5 mg/kg IP, and 45 mg/kg PO lenalidomide caused no observable toxicity up to 24 h postdose. We observed dose-dependent kinetics over the evaluated dosing range. Administration of 0.5 and 10 mg/kg resulted in systemic bioavailability ranges of 90-105% and 60-75% via IP and oral routes, respectively. Lenalidomide was detectable in the brain only after IV dosing of 5 and 10 mg/kg. Dose-dependent distribution was also observed in some tissues. High oral bioavailability of lenalidomide in mice is consistent with oral bioavailability in humans. Atypical lenalidomide tissue distribution was observed in spleen and brain. The observed dose-dependent pharmacokinetics should be taken into consideration in translational and preclinical mouse studies.