Wu-5, a novel USP10 inhibitor, enhances crenolanib-induced FLT3-ITD-positive AML cell death via inhibiting FLT3 and AMPK pathways.

The kinase FLT3 internal tandem duplication (FLT3-ITD) is related to poor clinical outcomes of acute myeloid leukemia (AML). FLT3 inhibitors have provided novel strategies for the treatment of FLT3-ITD-positive AML. But they are limited by rapid development of acquired resistance and refractory in monotherapy. Recent evidence shows that inducing the degradation of FLT3-mutated protein is an attractive strategy for the treatment of FLT3-ITD-positive AML, especially those with FLT3 inhibitor resistance. In this study we identified Wu-5 as a novel USP10 inhibitor inducing the degradation of FLT3-mutated protein. We showed that Wu-5 selectively inhibited the viability of FLT3 inhibitor-sensitive (MV4-11, Molm13) and -resistant (MV4-11R) FLT3-ITD-positive AML cells with IC50 of 3.794, 5.056, and 8.386 µM, respectively. Wu-5 (1-10 µM) dose-dependently induced apoptosis of MV4-11, Molm13, and MV4-11R cells through the proteasome-mediated degradation of FLT3-ITD. We further demonstrated that Wu-5 directly interacted with and inactivated USP10, the deubiquitinase for FLT3-ITD in vitro (IC50 value = 8.3 µM) and in FLT3-ITD-positive AML cells. Overexpression of USP10 abrogated Wu-5-induced FLT3-ITD degradation and cell death. Also, the combined treatment of Wu-5 and crenolanib produced synergistic cell death in FLT3-ITD-positive cells via the reduction of both FLT3 and AMPKα proteins. In support of this, AMPKα inhibitor compound C synergistically enhanced the anti-leukemia effect of crenolanib, while AMPKα activator metformin inhibited the anti-leukemia effect of crenolanib. In summary, we demonstrate that Wu-5, a novel USP10 inhibitor, can overcome FLT3 inhibitor resistance and synergistically enhance the anti-AML effect of crenolanib through targeting FLT3 and AMPKα pathway.

Characterization of naturally occurring pentacyclic triterpenes as novel inhibitors of deubiquitinating protease USP7 with anticancer activity in vitro.

Deubiquitinating protease USP7 is a promising therapeutic target for cancer treatment, and interest in developing USP7 inhibitors has greatly increased. In the present study, we reported a series of natural pentacyclic triterpenes with USP7 inhibitory activity in vitro. Among them, both the ursane triterpenes and oleanane triterpenes were more active than the lupine triterpenes, whereas ursolic acid was the most potent with IC50 of 7.0±1.5 µmol/L. Molecular docking studies showed that ursolic acid might occupy the ubiquitin binding pocket of USP7, with the 17-carboxyl group and 3-hydroxyl group playing a vital role in the USP7-ursolic acid interaction. Using the cellular thermal shift assay, we demonstrated that ursolic acid interacted with USP7 in RPMI8226 human myeloma cells. Ursolic acid dose-dependently inhibited the proliferation of the myeloma cells with IC50 of 6.56 µmol/L, accompanied by reductions in USP7 substrates such as MDM2, UHRF1 and DNMT1. Overexpression of USP7 partially, but significantly attenuated ursolic acid-induced cell death as well as downregulation of MDM2, UHRF1 and DNMT1. In conclusion, we demonstrate for the first time that pentacyclic triterpenes represent a novel scaffold for developing USP7 inhibitors and that USP7 inhibition contributes to the anti-cancer effect of ursolic acid.

Proteomic identification of common SCF ubiquitin ligase FBXO6-interacting glycoproteins in three kinds of cells.

FBOX6 ubiquitin ligase complex is involved in the endoplasmic reticulum-associated degradation pathway by mediating the ubiquitination of glycoproteins. FBXO6 interacts with the chitobiose in unfolded N-glycoprotein, pointing glycoproteins toward E2 for ubiquitination. Although the glycoprotein-recognizing mechanism of FBXO6 is well documented, its bona fide interacting glycoproteins are largely unknown. Here we utilized a protein purification approach combined with LC-MS to systematically identify the FBXO6-interacting glycoproteins. Following identification of 39 proteins that specifically interact with FBXO6 in all three different cell lines, 293T, HeLa and Jurkat cells, we compared the protein complex organization between wild-type FBXO6 and its mutant, which fails to recognize glycoproteins. Combining these databases, 29 highly confident glycoproteins that interact with FBXO6 in an N-glycan dependent manner are identified. Our data provide valuable information for the discovery of the interacting glycoproteins of FBXO6 and also demonstrate the potential of these approaches as general platforms for the global discovery of interacting glycoproteins of other FBAs (F-box associated regions) containing F-box proteins.

Pharicin B stabilizes retinoic acid receptor-α and presents synergistic differentiation induction with ATRA in myeloid leukemic cells.

All-trans retinoic acid (ATRA), a natural ligand for the retinoic acid receptors (RARs), induces clinical remission in most acute promyelocytic leukemia (APL) patients through the induction of differentiation and/or eradication of leukemia-initiating cells. Here, we identify a novel natural ent-kaurene diterpenoid derived from Isodon pharicus leaves, called pharicin B, that can rapidly stabilize RAR-α protein in various acute myeloid leukemic (AML) cell lines and primary leukemic cells from AML patients, even in the presence of ATRA, which is known to induce the loss of RAR-α protein. Pharicin B also enhances ATRA-dependent the transcriptional activity of RAR-α protein in the promyelocytic leukemia-RARα-positive APL cell line NB4 cells. We also showed that pharicin B presents a synergistic or additive differentiation-enhancing effect when used in combination with ATRA in several AML cell lines and, especially, some primary leukemic cells from APL patients. In addition, pharicin B can overcome retinoid resistance in 2 of 3 NB4-derived ATRA-resistant subclones. These findings provide a good example for chemical biology-based investigations of pathophysiological and therapeutic significances of RAR-α and PML-RAR-α proteins. The effectiveness of the ATRA/pharicin B combination warrants further investigation on their use as a therapeutic strategy for AML patients.

Ikaros is degraded by proteasome-dependent mechanism in the early phase of apoptosis induction.

Ikaros is an important transcription factor involved in the development and differentiation of hematopoietic cells. In this work, we found that chemotherapeutic drugs or ultraviolet radiation (UV) treatment could reduce the expression of full-length Ikaros (IK1) protein in less than 3h in leukemic NB4, Kasumi-1 and Jurkat cells, prior to the activation of caspase-3. Etoposide treatment could not alter the mRNA level of IK1 but it could shorten the half-life of IK1. Co-treatment with the proteasome inhibitor MG132 or epoxomicin but not calpain inhibitor calpeptin inhibited etoposide-induced Ikaros downregulation. Overexpression of IK1 could accelerate etoposide-induced apoptosis in NB4 cells, as evidenced by the increase of Annexin V positive cells and the more early activation of caspase 3. To our knowledge, this is the first report to show that upon chemotherapy drugs or UV treatment, IK1 could be degraded via the proteasome system in the early phase of apoptosis induction. These data might shed new insight on the role of IK1 in apoptosis and the post-translational regulation of IK1.

Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia.

Identifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin-Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

Overexpression of Fbxo6 inactivates spindle checkpoint by interacting with Mad2 and BubR1.

The spindle assembly checkpoint prevents chromosome mis-segregation during mitosis by delaying sister chromatid separation. Several F-box protein members play critical roles in maintaining genome stability and regulating cell cycle progress via ubiquitin-mediated protein degradation. Here, we showed that Fbxo6 critically regulated spindle checkpoint and chromosome segregation. Fbxo6 was phosphorylated during mitosis. Overexpression of Fbxo6 lead to faster exit from nocodazole-induced mitosis arrest through premature sister chromatid separation. Moreover, we found substantially more binuclear and multilobed nuclei cells accompanied with impaired cell viability in Fbxo6-overexpressed HeLa cells. Mechanistically, Fbxo6 interacted with spindle checkpoint proteins including Mad2 and BubR1 leading to the premature exit from mitosis. Overall, we revealed a novel role of Fbxo6 in regulating spindle checkpoint, which may shed light on the regulation of genome instability of cancer cells.

Hsp90 Is a Novel Target Molecule of CDDO-Me in Inhibiting Proliferation of Ovarian Cancer Cells.

Synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oate (CDDO-Me) has been shown as a promising agent against ovarian cancer. However, the underlying mechanism is not well understood. Here, we demonstrate that CDDO-Me directly interacts with Hsp90 in cells by cellular thermal shift assay. CDDO-Me treatment leads to upregulation of Hsp70 and degradation of Hsp90 clients (ErbB2 and Akt), indicating the inhibition of Hsp90 by CDDO-Me in cells. Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells. Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition. This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90. This study identifies Hsp90 as a novel target protein of CDDO-Me, and provides a novel insight into the mechanism of action of CDDO-Me in ovarian cancer cells.

Targeting catalase but not peroxiredoxins enhances arsenic trioxide-induced apoptosis in K562 cells.

Despite considerable efficacy of arsenic trioxide (As2O3) in acute promyelocytic leukemia (APL) treatment, other non-APL leukemias, such as chronic myeloid leukemia (CML), are less sensitive to As2O3 treatment. However, the underlying mechanism is not well understood. Here we show that relative As2O3-resistant K562 cells have significantly lower ROS levels than As2O3-sensitive NB4 cells. We compared the expression of several antioxidant enzymes in these two cell lines and found that peroxiredoxin 1/2/6 and catalase are expressed at high levels in K562 cells. We further investigated the possible role of peroxirdoxin 1/2/6 and catalase in determining the cellular sensitivity to As2O3. Interestingly, knockdown of peroxiredoxin 1/2/6 did not increase the susceptibility of K562 cells to As2O3. On the contrary, knockdown of catalase markedly enhanced As2O3-induced apoptosis. In addition, we provide evidence that overexpression of BCR/ABL cannot increase the expression of PRDX 1/2/6 and catalase. The current study reveals that the functional role of antioxidant enzymes is cellular context and treatment agents dependent; targeting catalase may represent a novel strategy to improve the efficacy of As2O3 in CML treatment.

Ikaros inhibits proliferation and, through upregulation of Slug, increases metastatic ability of ovarian serous adenocarcinoma cells.

The transcription factor Ikaros was originally found to function as a key regulator of lymphocyte differentiation. In this study, we provide the first evidence that Ikaros is expressed at higher levels in ovarian cancer tissues compared with normal ovarian tissues and is significantly associated with high FIGO stage and low differentiation state in ovarian serous adenocarcinoma. To this end, we transfected IK1 (full length of Ikaros) into the SKOV3 ovarian cancer cell line and examined cell biological behaviors including proliferation, migration and invasion. We found that overexpression of IK1 inhibited cell proliferation by inducing G1 arrest, accompanied by the upregulation of P27 and P21 and downregulation of cyclin D1 and D2. On the other hand, IK1 increased the migration and invasion of ovarian cancer cells, as assessed by scratch-wound assay, transwell migration assay, and invasion assay. Overexpression of IK1 significantly increased Slug but not Snail1 expression at both mRNA and protein levels. It also downregulated and upregulated E-cadherin and MMP-2, two target genes of Slug involved in migration, respectively. Furthermore, knocking down Slug abrogated IK1-mediated increase in migration and invasion. These data suggest that Slug plays an important role in IK1-induced migration and invasion. In conclusion, we show for the first time that IK1 plays a dual role in the proliferation, migration and invasion of ovarian cancer cells, providing new insights into their metastasis.

PU.1 directly regulates retinoic acid-induced expression of RIG-G in leukemia cells.

RIG-G is a retinoic acid- or interferon-induced gene with potential anti-proliferation function. However, the mechanism underlying ATRA-induced RIG-G induction is not completely understood. Here, we demonstrate that ATRA up-regulates the expression of PU.1, which in turn directly binds to the promoter and increases the expression of RIG-G gene. Luciferase reporter assay and electrophoretic mobility shift assay reveal that PU.1 preferentially binds to one of the two putative binding sites on the RIG-G promoter. Moreover, silencing of PU.1 by shRNA markedly inhibited ATRA- but not IFNα-induced expression of RIG-G. These data provide new insight into the mechanism of ATRA-induced RIG-G expression.

Synergistic mitosis-arresting effects of arsenic trioxide and paclitaxel on human malignant lymphocytes.

The treatment outcome of acute lymphoblastic leukemia (ALL) has improved steadily over the last 50 years. However, the cure rates are unlikely to be raised further with current therapies. Since increasing the dosage of chemotherapeutic agents could also elevate toxicity, a solution to how one could achieve maximum therapeutic effect with the minimum dosage possible is imminent. One possibility is the employment of combination drug therapies. Arsenic trioxide (ATO) is a widely used drug for acute promyelocytic leukemia (APL). Its combination with other drugs presented therapeutic activities in malignant cancers other than APL. Considering the fact that ATO induces mitotic arrest prior to apoptosis induction, we attempted to investigate the potential anti-cancer effects of ATO in combination with the microtubule-stabilizing agent, paclitaxel (PTX), using malignant lymphocytes as in vitro models. Three malignant lymphocytic cell lines and primary cells were treated with ATO and/or PTX. Using the Chou-Talalay analysis for evaluation of combined effect of ATO and PTX, we found a synergistic effect of the two drugs in the inhibition of cell growth. We also found that the combination of ATO and PTX at low concentrations synergistically induced mitotic arrest followed by apoptosis in malignant lymphocytes, which increased phosphorylated cyclin-dependent kinase 1 (Cdk1) on Thr(161) and promoted the dysregulated activation of Cdk1. The ATO/PTX combination also significantly enhanced the activation of spindle checkpoint by inducing the formation of the inhibitory checkpoint complex BubR1/Cdc20. Our study provided the first in vitro demonstration that low concentrations of ATO and PTX synergistically induce mitotic arrest in malignant lymphocytes.

Pharicin A, a novel natural ent-kaurene diterpenoid, induces mitotic arrest and mitotic catastrophe of cancer cells by interfering with BubR1 function.

In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in leukemia and solid tumor-derived cells identified by their morphology, DNA content and mitotic marker analyses. Pharicin A-induced mitotic arrest is associated with unaligned chromosomes, aberrant BubR1 localization and deregulated spindle checkpoint activation. Pharicin A directly binds to BubR1 in vitro, which is correlated with premature sister chromatid separation in vivo. Pharicin A also induces mitotic arrest in paclitaxel-resistant Jurkat and U2OS cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing mitotic progression and initiating mitotic catastrophe, which merits further preclinical and clinical investigations for cancer drug development.

Defects in chromosome congression and mitotic progression in KIF18A-deficient cells are partly mediated through impaired functions of CENP-E.

KIF18A, a molecular motor, is an essential component in the regulation of orderly chromosome congression by attenuation of the kinetochore oscillation amplitude at the midzone during mitosis in vertebrate cells. Here we report that KIF18A depletion resulted in mitotic arrest which was accompanied by the presence of unaligned chromosomes in HeLa cells. This resembles the phenotype induced by an impaired function of CENP-E, also a mitotic kinesin essential for the formation of the mitotic spindles. Our further analysis showed that KIF18A depletion caused specific downregulation of CENP-E. Downregulation of CENP-E as the result of KIF18A silencing was not due to reduced transcription but primarily due to the enhanced protein degradation. Co-immunoprecipitation revealed that KIF18A physically interacted with CENP-E and BubR1 during mitosis. Ectopic expression of the wild-type tail domain of CENP-E, but not a corresponding mutant, significantly suppressed chromosome congression defects in mitotic cells. Together, our studies strongly suggest that chromosome congression defects as the result of KIF18A depletion is at least in part mediated through destabilizing kinetochore CENP-E.