Identification of unique sensitizing targets for anti‐inflammatory CDDO‐Me in metastatic melanoma by a large‐scale synthetic lethal RNAi screening

CDDO‐Me has been shown to exert potent anti‐inflammatory activity for chronic kidney disease and antitumor activity for several tumors, including melanoma, in early clinical trials. To improve CDDO‐Me response in melanoma, we utilized a large‐scale synthetic lethal RNAi screen targeting 6000 human druggable genes to identify targets that would sensitize melanoma cells to CDDO‐Me. Based on screening results, five unique genes (GNPAT, SUMO1, SPINT2, FLI1, and SSX1) significantly potentiated the growth inhibitory effects of CDDO‐Me and induced apoptosis in A375, a BRAF mutated melanoma line (P < 0.001). These five genes were then individually validated as targets to potentiate CDDO‐Me activity, and related downstream signaling pathways of these genes were analyzed. In addition, the levels of phosphorylated Erk1/2, Akt, GSK‐2, and PRAS40 were dramatically decreased by downregulating each of these five genes separately, suggesting a set of common mediators. Our findings indicate that GNPAT, SUMO1, SPINT2, FLI1, and SSX1 play critical roles in synergy with inflammation pathways in modulating melanoma cell survival and could serve as sensitizing targets to enhance CDDO‐Me efficacy in melanoma growth control.     

The novel triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid induces apoptosis of human myeloid leukemia cells by a caspase-8-dependent mechanism.

The oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a multifunctional molecule that induces growth inhibition and differentiation of human myeloid leukemia cells. The present studies demonstrate that CDDO treatment results in apoptosis of U-937 and HL-60 myeloid leukemia cells. Similar to 1-beta-D-arabinofuranosylcytosine (ara-C), another agent that inhibits growth and induces apoptosis of these cells, CDDO induced the release of mitochondrial cytochrome c and activation of caspase-3. Overexpression of Bcl-X(L) blocked cytochrome c release, caspase-3 activation, and apoptosis in ara-C-treated cells. By contrast, CDDO-induced release of cytochrome c, and activation of caspase-3 were diminished only in part by Bcl-X(L). In concert with these findings, we demonstrate that CDDO, but not ara-C, activates caspase-8 and thereby caspase-3 by a cytochrome c-independent mechanism. The results also show that CDDO-induced cytochrome c release is mediated by caspase-8-dependent cleavage of Bid. These findings demonstrate that CDDO induces apoptosis of myeloid leukemia cells and that this novel agent activates an apoptotic signaling cascade distinct from that induced by the cytotoxic agent ara-C.     

The novel triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) potently enhances apoptosis induced by tumor necrosis factor in human leukemia cells

Tumor necrosis factor (TNF) is a potent activator of the nuclear factor-kappaB (NF-kappaB) pathway that leads to up-regulation of anti-apoptotic proteins. Hence, TNF induces apoptosis in the presence of inhibitors of protein or RNA synthesis. We report that a novel triterpenoid, 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) inhibits NF-kappaB-mediated gene expression at a step after translocation of activated NF-kappaB to the nucleus. This effect appears specific for the NF-kappaB pathway as CDDO does not inhibit gene expression induced by the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). CDDO in combination with TNF caused a dramatic increase in apoptosis in ML-1 leukemia cells that was associated with activation of caspase-8, cleavage of Bid, translocation of Bax, cytochrome c release, and caspase-3 activation. Experiments with caspase inhibitors demonstrated that caspase-8 was an initiator of this pathway. TNF also induced a transient activation of c-Jun N-terminal kinase (JNK), which upon addition of CDDO was converted to a sustained activation. The activation of JNK was also dependent on caspase-8. Sustained activation of JNK is frequently pro-apoptotic, yet inhibition of JNK did not prevent Bax translocation or cytochrome c release, demonstrating its lack of involvement in CDDO/TNF-induced apoptosis. Apoptosis was acutely induced by CDDO/TNF in every leukemia cell line tested including those that overexpress Bcl-x(L), suggesting that the mitochondrial pathway is not required for apoptosis by this combination. These results suggest that the apoptotic potency of the CDDO/TNF combination occurs through selective inhibition of NF-kappaB-dependent anti-apoptotic proteins, bypassing potential mitochondrial resistance mechanisms, and thus may provide a basis for the development of novel approaches to the treatment of leukemia.     

2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride. A novel and highly potent anti-inflammatory and cytoprotective agent

2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride (CDDO anhydride) has been synthesized, which is the first example of an oleanane triterpenoid anhydride. CDDO anhydride shows potency similar to or higher than the corresponding acid (CDDO) in various in vitro and in vivo assays related to inflammation and carcinogenesis. Notably, preliminary phamacokinetics studies show that CDDO anhydride levels are higher than CDDO levels in mouse tissues and blood. Further evaluation of CDDO anhydride is in progress.     

Evidence supporting a role for calcium in apoptosis induction by the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a novel anticancer agent that induces apoptosis in tumor cells. The cytotoxic stress underpinning CDDO-induced apoptosis has not been established. This study compared and contrasted the effects of CDDO on COLO 16 human skin cancer cells and their respiration-deficient (rho(0)) clones to elucidate the stress signal responsible for initiating apoptosis. CDDO promoted apoptosis in COLO 16 cells in a dose- and time-dependent manner. The rho(0) clones appeared to be more sensitive to CDDO-induced apoptosis implying that the disruption of mitochondrial respiration was not directly associated with triggering cell death. After a 4-h exposure to CDDO, mitochondrial inner transmembrane potential-sensitive dyes revealed mitochondrial hyperpolarization in the COLO 16 cells and mitochondrial depolarization in the rho(0) clones. Electron microscopy illustrated that this exposure also promoted mitochondrial condensation, endoplasmic reticulum dilation, and chromatin condensation in the COLO 16 cells. Endoplasmic reticulum dilation and chromatin condensation were also observed in the rho(0) clones, but the mitochondria in these cells were markedly swollen implying that the disruption of intracellular Ca(2+) homeostasis was associated with cell death. A Ca(2+)-sensitive dye confirmed that CDDO increased cytoplasmic free Ca(2+) in the COLO 16 cells, their rho(0) clones, as well as in malignant breast and lung epithelial cells. A cell-permeant Ca(2+) chelator reduced the CDDO-induced increase in cytoplasmic free Ca(2+), and inhibited caspase activation, the development of apoptotic morphology, and DNA fragmentation in the COLO 16 cells, implying that Ca(2+) played a pivotal role in signaling the initiation of apoptosis.     

Triterpenoid CDDO-Me blocks the NF-kappaB pathway by direct inhibition of IKKbeta on Cys-179

The novel oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) and the C-28 methyl ester (CDDO-Me) induce apoptosis of human tumor cells by disruption of redox balance and are currently in clinical trials. The present studies show that CDDO and CDDO-Me block tumor necrosis factoralpha-induced targeting of NF-kappaB p65 to the nucleus. CDDO-Me also blocked tumor necrosis factor alpha-induced phosphorylation of IkappaBalpha. In concert with these results, we found that CDDO-Me inhibits IkappaBalpha kinasebeta (IKKbeta) activity in cells. In support of a direct mechanism, CDDO-Me inhibited recombinant IKKbeta activity in vitro. The results also demonstrate that (i) CDDO and CDDO-Me form adducts with IKKbeta, but not IKKbeta with mutation of Cys-179 to Ala, and (ii) CDDO-Me inhibits IKKbeta by a mechanism dependent on oxidation of Cys-179. These findings indicate that CDDO and CDDO-Me directly block IKKbeta activity and thereby the NF-kappaB pathway by interacting with Cys-179 in the IKKbeta activation loop.     

2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) directly targets mitochondrial glutathione to induce apoptosis in pancreatic cancer

Surgical resection is the only curative strategy for pancreatic cancer (PC). Unfortunately, >80% of pancreatic cancer patients bear inoperable, locally advanced, chemoresistant tumors demonstrating the urgent need for development of novel therapeutic approaches to treat this disease. Here we report that the synthetic triterpenoid 2-cyano-3,12 dioxooleana-1,9 dien-28-imidazolide (CDDO-Im) antagonizes PC cell growth by inducing apoptosis at submicromolar concentrations. Notably, we demonstrate for the first time that the cytotoxicity of CDDO-Im is accompanied by the rapid and selective depletion of mitochondrial glutathione that results in accumulation of reactive oxygen species, oxidation of the cellular glutathione pool, loss of mitochondrial membrane potential, and phosphatidylserine externalization. The parent compound CDDO as well as the methyl ester of CDDO also depleted mitochondrial glutathione, demonstrating that this effect is mediated by the triterpenoid nucleus of these agents. Co-treatment with sulfhydryl nucleophiles completely prevented apoptosis and loss of viability induced by CDDO-Im, whereas alkylation of intracellular thiols by diethylmaleate or co-treatment with dithiothreitol decreased the accumulation of a biotinylated derivative of CDDO, TP-301, in PC cells, suggesting that intracellular reduced thiols are functional targets of the electrophilic triterpenoid nucleus of CDDO and its derivatives. In conclusion, our report is the first to identify mitochondrial glutathione as a target of CDDO and its derivatives and demonstrates that depletion of this antioxidant in the mitochondria is an effective strategy to induce cell death in PC cells. These results suggest that CDDO and its derivatives may offer a clinical benefit for the treatment of PC.     

Inhibition of mitochondrial LonP1 protease by allosteric blockade of ATP binding and hydrolysis via CDDO and its derivatives

The mitochondrial protein LonP1 is an ATP-dependent protease that mitigates cell stress and calibrates mitochondrial metabolism and energetics. Biallelic mutations in the LONP1 gene are known to cause a broad spectrum of diseases, and LonP1 dysregulation is also implicated in cancer and age-related disorders. Despite the importance of LonP1 in health and disease, specific inhibitors of this protease are unknown. Here, we demonstrate that 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its -methyl and -imidazole derivatives reversibly inhibit LonP1 by a noncompetitive mechanism, blocking ATP-hydrolysis and thus proteolysis. By contrast, we found that CDDO-anhydride inhibits the LonP1 ATPase competitively. Docking of CDDO derivatives in the cryo-EM structure of LonP1 shows these compounds bind a hydrophobic pocket adjacent to the ATP-binding site. The binding site of CDDO derivatives was validated by amino acid substitutions that increased LonP1 inhibition and also by a pathogenic mutation that causes cerebral, ocular, dental, auricular and skeletal (CODAS) syndrome, which ablated inhibition. CDDO failed to inhibit the ATPase activity of the purified 26S proteasome, which like LonP1 belongs to the AAA⁺ superfamily of ATPases Associated with diverse cellular Activities, suggesting that CDDO shows selectivity within this family of ATPases. Furthermore, we show that noncytotoxic concentrations of CDDO derivatives in cultured cells inhibited LonP1, but not the 26S proteasome. Taken together, these findings provide insights for future development of LonP1-specific inhibitors with chemotherapeutic potential.     

A novel dicyanotriterpenoid, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile, active at picomolar concentrations for inhibition of nitric oxide production

New oleanane triterpenoids with various substituents at the C-17 position of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and methyl 2-carboxy-3,12-dioxooleana-1,9(11)-dien-28-oate were synthesized. Among them, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-onitrile shows extremely high inhibitory activity (IC(50)=1 pM level) against production of nitric oxide induced by interferon-gamma in mouse macrophages. This potency is about 100 times and 30 times more potent than CDDO and dexamethasone, respectively.     

Synthesis and biological evaluation of amino acid methyl ester conjugates of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid against the production of nitric oxide (NO)

2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, 2) was condensed with various amino acid methyl esters at the C-28 carboxylic acid. The new amide conjugates were evaluated for their inhibition of nitric oxide (NO) production in RAW264.7 cells stimulated with interferon-γ (IFNγ). Of these new compounds, CDDO conjugates with alanine, valine, and serine are nearly equipotent to CDDO-ethyl amide (4), a triterpenoid with promising biological activity in numerous disease models. Some of these conjugates also induce the in vitro expression of heme oxygenase-1, and inhibit the proliferation of Panc-1343 pancreatic cells.     

Novel synthetic oleanane triterpenoids: a series of highly active inhibitors of nitric oxide production in mouse macrophages

Novel oleanane triterpenoids with modified rings A and C were designed and synthesized. Among them, methyl 2-carboxy-3,12-dioxooleana-1,9-dien-28-oate showed similar high inhibitory activity (IC₅₀ = 0.8 nM) to 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), which we have synthesized previously, against production of nitric oxide induced by interferon-γ in mouse macrophages.     

Triterpenoid CDDO-Im downregulates PML/RARalpha expression in acute promyelocytic leukemia cells

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of diverse human tumor cells. In the present study, we examined the effects of the CDDO imidazolide imide (CDDO-Im) on the NB4 acute promyelocytic leukemia (APL) cell line and primary APL cells. The results show that CDDO-Im selectively downregulates expression of the PML/retinoic receptor alpha fusion protein by a caspase-dependent mechanism and sensitizes APL cells to the differentiating effects of all-trans retinoic acid (ATRA). CDDO-Im treatment of APL cells was also associated with disruption of redox balance and activation of the extrinsic apoptotic pathway. In concert with these results, CDDO-Im sensitizes APL cells to arsenic trioxide (ATO)-induced apoptosis. Our findings indicate that CDDO-Im may be effective in the treatment of APL by: (i) downregulation of PML/RARalpha; (ii) enhancement of ATRA-induced differentiation; and (iii) sensitization of ATO-induced APL cell death.     

Neuroprotective effect of Nrf2/ARE activators, CDDO ethylamide and CDDO trifluoroethylamide, in a mouse model of amyotrophic lateral sclerosis

Oxidative damage, neuroinflammation, and mitochondrial dysfunction contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS), and these pathologic processes are tightly regulated by the Nrf2/ARE (NF-E2-related factor 2/antioxidant response element) signaling program. Therefore, modulation of the Nrf2/ARE pathway is an attractive therapeutic target for neurodegenerative diseases such as ALS. We examined two triterpenoids, CDDO (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) ethylamide and CDDO trifluoroethylamide (CDDO-TFEA), that potently activate Nrf2/ARE in a cell culture model of ALS and in the G93A SOD1 mouse model of ALS. Treatment of NSC-34 cells stably expressing mutant G93A SOD1 with CDDO-TFEA upregulated Nrf2 expression and resulted in translocation of Nrf2 into the nucleus. Western blot analysis showed an increase in the expression of Nrf2/ARE-regulated proteins. When treatment started at a “presymptomatic age” of 30 days, both of these compounds significantly attenuated weight loss, enhanced motor performance, and extended the survival of G93A SOD1 mice. Treatment started at a “symptomatic age,” as assessed by impaired motor performance, was neuroprotective and slowed disease progression. These findings provide further evidence that compounds that activate the Nrf2/ARE signaling pathway may be useful in the treatment of ALS.     

Synthesis and biological evaluation of 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4-ethynylimidazole. A novel and highly potent anti-inflammatory and cytoprotective agent

To explore more potent N-acylimidazole analogues of CDDO than CDDO-Im, which is one of the most potent compounds in several widely used bioassays related to protection against inflammation and carcinogenesis; we have synthesized and evaluated five new N-acyl(acetylenic)imidazole analogues. Among them, 4-ethynylimidazole 4 is nearly equivalent to CDDO-Im in potency in these bioassays. Remarkably, the solid form of 4 is more stable than that of CDDO-Im. These findings suggest that 4 is a very promising anti-inflammatory and cytoprotective agent and its further preclinical evaluation is warranted.