Guodong Chen

A deuterated analog of dasatinib disrupts cell cycle progression and displays anti-non-small cell lung cancer activity in vitro and in vivo.

The pan‐Src family kinase inhibitor dasatinib has been approved for chronic myeloid leukemia treatment but displays limited activity in lung cancer patients. In this study, we used a deuterium substitution strategy to develop a class of novel chemicals based on dasatinib and found that these compounds maintain inhibition on c‐Src activity and display anti‐non‐small cell lung cancer activity in vitro and in vivo. BRP800, one of these compounds, was chosen for further studies. BRP800 mainly displayed antiproliferative but not proapoptotic activity. Molecularly, BRP800 did not show significant effects on the expression of antiapoptotic genes, such as Bcl‐2 and Mcl1, or on the activation of apoptotic enzymes, such as caspase‐3, ‐8 or 9. However, BRP800 decreased expression of cell cycle promoting genes such as cyclins D1, D3, E, A and CDK4 and 6, and increased the expression of cell cycle negative regulators including p21, p27 and p53. Consistent with these findings, BRP800 arrested cells at the G0/G1 phase in a concentration‐dependent manner, and the G0/G1 fraction was increased from 64% in control to 85% in BRP800‐treated cells. We also evaluated the effects of BRP800 on NSCLC xenografts using H460 as a model in nude mice. Compared with the known NSCLC drug docetaxel, BRP800 displayed potent and similar antitumor activity but with less toxicity. These findings suggest that the deuterated analog of dasatinib is antiproliferative by inhibiting c‐Src and disrupting cell cycle progression, and could be further developed as a novel drug for non‐small lung cancer treatment.

The ubiquitin ligase HERC4 mediates c-Maf ubiquitination and delays the growth of multiple myeloma xenografts in nude mice

The transcription factor c-Maf is extensively involved in the pathophysiology of multiple myeloma (MM), a fatal malignancy of plasma cells. In the present study, affinity chromatography and mass spectrometry were used to identify c-Maf ubiquitination-associated proteins, from which the E3 ligase HERC4 was found to interact with c-Maf and catalyzed its polyubiquitination and subsequent proteasome-mediated degradation. HERC4 mediated polyubiquitination at K85 and K297 in c-Maf, and this polyubiquitination could be prevented by the isopeptidase USP5. Further analysis on the NCI-60 cell line collection revealed that RPMI 8226, a MM-derived cell line, expressed the lowest level of HERC4. Primary bone marrow analysis revealed HERC4 expression was high in normal bone marrow, but was steadily decreased during myelomagenesis. These findings suggested HERC4 played an important role in MM progression. Moreover, ectopic HERC4 expression decreased MM proliferation in vitro, and delayed xenograft tumor growth in vivo. Therefore, modulation of c-Maf ubiquitination by targeting HERC4 may represent a new therapeutic modality for MM.

A novel PI3K inhibitor displays potent preclinical activity against an androgen-independent and PTEN-deficient prostate cancer model established from the cell line PC3

Recent studies demonstrated that targeting the phosphatidylinositide 3-kinase (PI3K)/AKT signaling pathway is a major strategy for the treatment of androgen-independent prostate cancer. In the present study, we developed an analog BENC-511 from a recently reported PI3K inhibitor S14161 by structural optimization. Using PC3 and DU145 as the model cell lines, we found PTEN-deficient PC3 cells were more sensitive than PTEN-expressing DU145 ones in terms of cell proliferation, apoptosis, and caspase-3 activation. These findings were consistent with the inhibition on PI3K/AKT signals. BENC-511 preferably suppressed AKT activation in PC3 over DU145 cells. Notably, PTEN restoration attenuated BENC-511 induced apoptosis. Moreover, BENC-511 displayed great therapeutic efficacy in a PC3-derived prostate cancer model in nude mice. With an oral dosage of 50mg/kg, BENC-511 decreased tumor growth more than 50% in 27 days, which was accompanied with PARP cleavage, but did not show overt toxicity. This study lays a solid rationale for the development of BENC-511 as a drug for the treatment of PTEN-deficient and androgen-independent prostate cancers.

SC06, a novel small molecule compound, displays preclinical activity against multiple myeloma by disrupting the mTOR signaling pathway.

The mammalian target of rapamycin (mTOR) is extensively involved in multiple myeloma (MM) pathophysiology. In the present study, we reported a novel small molecule SC06 that induced MM cell apoptosis and delayed MM xenograft growth in vivo. Oral administration of SC06 to mice bearing human MM xenografts resulted in significant inhibition of tumor growth at doses that were well tolerated. Mechanistic studies revealed that SC06 selectively inhibited the mTOR signaling pathway but had no effects on other associated kinases, such as AKT, ERK, p38, c-Src and JNK. Further studies showed that SC06-decreased mTOR activation was associated with the downregulation of Raptor, a key component of the mTORC1 complex. SC06 also suppressed the phosphorylation of 4E-BP1 and P70S6K, two typical substrates in the mTORC1 signaling pathway. Notably, expression of Raptor, phosphorylation of mTOR and phosphorylated 4E-BP1 was also decreased in the tumor tissues from SC06-treated mice, which was consistent with the cellular studies. Therefore, given the potency and low toxicity, SC06 could be developed as a potential anti-MM drug candidate by disrupting the mTOR signaling.

Ubiquitination of the transcription factor c-MAF is mediated by multiple lysine residues

The transcription factor c-MAF could be polyubiquitinated and subsequently degraded in the proteasomes. Theoretically, any lysine residues in c-MAF could be ubiquitinated. In the present study, we tried to find out the specific lysine residue(s) mediating c-MAF ubiquitination. Through a series of mutational screens from lysine (K) to arginine (R), we found that any single lysine mutation (K to R) failed to prevent c-MAF ubiquitination, and any single lysine residue alone could not mediate c-MAF ubiquitination, which indicated that multiple lysine residues were required for c-MAF ubiquitination. Bioinformatics and computing analyses revealed that K85 and K350 could mediate c-MAF ubiquitination, which was confirmed by the cell-based assays. However, this duo was not the only pair because the K85R/K350R mutant could also be ubiquitinated. Functionally, both M12 (K85/K350) and W12 (K85R/K350R) mutants increased cyclin D2 promoter-driven luciferase activity, but they were less potent than the lysine-free counterpart (M14). In addition, M14 induced a higher level of expression of cyclin D2 at both mRNA and protein levels. Therefore, we demonstrated that c-MAF ubiquitination is mediated by multiple lysine residues, of which K85 and K350 were sufficient but not the only residues in mediating c-MAF ubiquitination. Moreover, c-MAF was found to be degraded by lysosomes. This study added a novel insight for c-MAF ubiquitination and degradation, suggesting that c-MAF stability is strictly regulated.

Abstract A230: An anti-leishmanial thiadiazine agent induces multiple myeloma cell apoptosis by suppressing the nuclear factor kappaB signaling pathway.

Nuclear factor kappaB (NF-κB) is a nuclear transcription factor that regulates expression of a large number of genes critical for cell proliferation, survival, inflammation, and tumorigenesis. Recent evidence indicates that NF-κB and its signaling pathways are constitutively activated both in myeloma cell lines and in primary myeloma cells. Downregulated NF-κB signaling by bortezomib and other compounds has demonstrated that the NF-κB signaling could be a promising therapeutic target of MM. In the present study, we found that one of the tetrahydro-2H-1,3,5-thiadiazine-2-thione (THTT) derivatives displayed potent anti-myeloma activity by inhibiting the NF-κB signaling pathway. DETT downregulated IKKα, β, p65, and p50 expression and inhibited phosphorylation of p65 (Ser536) and IBα. Simultaneously, DETT increased IκBα, an inhibitor of the p65/p50 heterodimer, even in the presence of stimulants lipopolysacharride, tumor necrosis factor alpha or interleukin-6. DETT inhibited NF-κB transcription activity and down-regulated NF-κB targeted genes including Bcl-2, Bcl-XL, and XIAP. Deregulation of NF-κB signaling by DETT resulted in MM cell apoptosis characterized by cleavage of caspase-3, caspase-8, and PARP. Notably, this apoptosis was partly blocked by activation of NF-κB signaling in the presence of TNFα and IL-6. Moreover, DETT delayed myeloma tumor growth in nude mice without overt toxicity. DETT is therefore a potent candidate for MM therapy as an inhibitor of the NF-κB signaling pathway. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A230. Citation Format: Guodong Chen, Kunkun Han, Xin Xu, Xiaolin Du, Zubin Zhang, Juan Tang, Min Shi, Man Wang, Jie Li, Biyin Cao, Xinliang Mao. An anti-leishmanial thiadiazine agent induces multiple myeloma cell apoptosis by suppressing the nuclear factor kappaB signaling pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A230.

Abstract A225: Identification of a promising PI3K inhibitor for the treatment of multiple myeloma through structural optimization.

Multiple myeloma (MM) is an incurable malignancy of plasma cells, which is associated with the overactivated phosphatidylinositol 3-kinases (PI3Ks)/AKT signaling pathway. Stimulated by interleukin 6 and or insulin-like growth factor 1, PI3K/AKT plays a key role in MM survival and chemoresistance, while genetic knockdown of these PI3-kinases by small interfering RNA (siRNA) leads to MM cell apoptosis and decreased proliferation. Therefore, PI3K is granted as a promising therapeutic target for MM. We previously identified the small molecule compound S14161 an inhibitor of pan-Class I PI3K isoforms by a high throughput screening strategy (Mao et al., 2011). S14161 displayed potent preclinical activity against MM and leukemia in vitro and in vivo. In the present study, we synthesized a panel of S14161 analogs based on the structure-activity relationship, of which BENC-511 was demonstrated to be more potent than S14161 in suppressing PI3K activity and inducing MM cell death. BENC-511 was derived from S14161 by removing 4-fluorophenyl group at the 2-position of the chromene core. Different from S14161, BENC-511 presented as a specific inhibitor for PI3K α and δ, but had no inhibitory effects on PI3K β, γ, AKT, or mTOR in the cell-free enzymatic assays. BENC-511 inhibited phosphorylation of AKT and its downstream signals mTOR, P70S6K, and 4E-BP-1 at low micromolar concentrations. Consistent with its effects on PI3K activity and cancer cell proliferation, BENC-511 induced MM cell apoptosis. Notably, addition of insulin-like growth factor 1 and interleukin-6, two important triggers for PI3K activation in MM cells, partly blocked BENC-511-induced MM cell death. BENC-511 also showed potent anti-myeloma activity in vivo. Oral administration of BENC-511 decreased tumor growth up to 80% within 3 weeks in two independent MM xenograft models, but presented minimal toxicity. Suppression of BENC-511 on MM tumor growth was associated with decreased PI3K/AKT activity and increased cell apoptosis. Because of its robust potential and low toxicity (LD50 oral >1.5 g/kg), BENC-511 could be developed as a promising agent for the treatment of multiple myeloma by targeting the PI3K/AKT signaling pathway. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A225. Citation Format: Kunkun Han, Xin Xu, Guodong Chen, Yuanying Zeng, Jingyu Zhu, Xiaolin Du, Zubin Zhang, Biyin Cao, Zhaopeng Liu, Xinliang Mao. Identification of a promising PI3K inhibitor for the treatment of multiple myeloma through structural optimization. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A225.