Dual-inhibition of NAMPT and PAK4 induces anti-tumor effects in 3D-spheroids model of platinum-resistant ovarian cancer.

Ovarian cancer follows a characteristic progression pattern, forming multiple tumor masses enriched with cancer stem cells (CSCs) within the abdomen. Most patients develop resistance to standard platinum-based drugs, necessitating better treatment approaches. Targeting CSCs by inhibiting NAD+ synthesis has been previously explored. Nicotinamide phosphoribosyltransferase (NAMPT), which is the rate limiting enzyme in the salvage pathway for NAD+ synthesis is an attractive drug target in this pathway. KPT-9274 is an innovative drug targeting both NAMPT and p21 activated kinase 4 (PAK4). However, its effectiveness against ovarian cancer has not been validated. Here, we show the efficacy and mechanisms of KPT-9274 in treating 3D-cultured spheroids that are resistant to platinum-based drugs. In these spheroids, KPT-9274 not only inhibited NAD+ production in NAMPT-dependent cell lines, but also suppressed NADPH and ATP production, indicating reduced mitochondrial function. It also downregulated of inflammation and DNA repair-related genes. Moreover, the compound reduced PAK4 activity by altering its mostly cytoplasmic localization, leading to NAD+-dependent decreases in phosphorylation of S6 Ribosomal protein, AKT, and ß-Catenin in the cytoplasm. These findings suggest that KPT-9274 could be a promising treatment for ovarian cancer patients who are resistant to platinum drugs, emphasizing the need for precision medicine to identify the specific NAD+ producing pathway that a tumor relies upon before treatment.

A Single-Arm, Open-Label Phase II Study of ONC201 in Recurrent/Refractory Metastatic Breast Cancer and Advanced Endometrial Carcinoma.

BACKGROUND: ONC201 is a small molecule that can cause nonapoptotic cell death through loss of mitochondrial function. Results from the phase I/II trials of ONC201 in patients with refractory solid tumors demonstrated tumor responses and prolonged stable disease in some patients. METHODS: This single-arm, open-label, phase II clinical trial evaluated the efficacy of ONC201 at the recommended phase II dose (RP2D) in patients with recurrent or refractory metastatic breast or endometrial cancer. Fresh tissue biopsies and blood were collected at baseline and at cycle 2 day 2 for correlative studies. RESULTS: Twenty-two patients were enrolled; 10 patients with endometrial cancer, 7 patients with hormone receptor-positive breast cancer, and 5 patients with triple-negative breast cancer. The overall response rate was 0%, and the clinical benefit rate, defined by complete response (CR) + partial response (PR) + stable disease (SD), was 27% (n = 3/11). All patients experienced an adverse event (AE), which was primarily low grade. Grade 3 AEs occurred in 4 patients; no grade 4 AEs occurred. Tumor biopsies did not show that ONC201 consistently induced mitochondrial damage or alterations in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the TRAIL death receptors. ONC201 treatment caused alterations in peripheral immune cell subsets. CONCLUSION: ONC201 monotherapy did not induce objective responses in recurrent or refractory metastatic breast or endometrial cancer at the RP2D dose of 625 mg weekly but had an acceptable safety profile (ClinicalTrials.gov Identifier: NCT03394027).

Targeting Mitochondria with ClpP Agonists as a Novel Therapeutic Opportunity in Breast Cancer.

Breast cancer is the most frequently diagnosed malignancy worldwide and the leading cause of cancer mortality in women. Despite the recent development of new therapeutics including targeted therapies and immunotherapy, triple-negative breast cancer remains an aggressive form of breast cancer, and thus improved treatments are needed. In recent decades, it has become increasingly clear that breast cancers harbor metabolic plasticity that is controlled by mitochondria. A myriad of studies provide evidence that mitochondria are essential to breast cancer progression. Mitochondria in breast cancers are widely reprogrammed to enhance energy production and biosynthesis of macromolecules required for tumor growth. In this review, we will discuss the current understanding of mitochondrial roles in breast cancers and elucidate why mitochondria are a rational therapeutic target. We will then outline the status of the use of mitochondria-targeting drugs in breast cancers, and highlight ClpP agonists as emerging mitochondria-targeting drugs with a unique mechanism of action. We also illustrate possible drug combination strategies and challenges in the future breast cancer clinic.

Targeting TRAIL Death Receptors in Triple-Negative Breast Cancers: Challenges and Strategies for Cancer Therapy.

The tumor necrosis factor (TNF) superfamily member TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer cells via death receptor (DR) activation with little toxicity to normal cells or tissues. The selectivity for activating apoptosis in cancer cells confers an ideal therapeutic characteristic to TRAIL, which has led to the development and clinical testing of many DR agonists. However, TRAIL/DR targeting therapies have been widely ineffective in clinical trials of various malignancies for reasons that remain poorly understood. Triple negative breast cancer (TNBC) has the worst prognosis among breast cancers. Targeting the TRAIL DR pathway has shown notable efficacy in a subset of TNBC in preclinical models but again has not shown appreciable activity in clinical trials. In this review, we will discuss the signaling components and mechanisms governing TRAIL pathway activation and clinical trial findings discussed with a focus on TNBC. Challenges and potential solutions for using DR agonists in the clinic are also discussed, including consideration of the pharmacokinetic and pharmacodynamic properties of DR agonists, patient selection by predictive biomarkers, and potential combination therapies. Moreover, recent findings on the impact of TRAIL treatment on the immune response, as well as novel strategies to address those challenges, are discussed.

Mitochondrial Matrix Protease ClpP Agonists Inhibit Cancer Stem Cell Function in Breast Cancer Cells by Disrupting Mitochondrial Homeostasis.

Mitochondria are multifaceted organelles which are important for bioenergetics, biosynthesis and signaling in metazoans. Mitochondrial functions are frequently altered in cancer to promote both the energy and the necessary metabolic intermediates for biosynthesis required for tumor growth. Cancer stem cells (CSCs) contribute to chemotherapy resistance, relapse, and metastasis. Recent studies have shown that while non-stem, bulk cancer cells utilize glycolysis, breast CSCs are more dependent on oxidative phosphorylation (OxPhos) and therefore targeting mitochondria may inhibit CSC function. We previously reported that small molecule ONC201, which is an agonist for the mitochondrial caseinolytic protease (ClpP), induces mitochondrial dysfunction in breast cancer cells. In this study, we report that ClpP agonists inhibit breast cancer cell proliferation and CSC function in vitro and in vivo. Mechanistically, we found that OxPhos inhibition downregulates multiple pathways required for CSC function, such as the mevalonate pathway, YAP, Myc, and the HIF pathway. ClpP agonists showed significantly greater inhibitory effect on CSC functions compared with other mitochondria-targeting drugs. Further studies showed that ClpP agonists deplete NAD(P)+ and NAD(P)H, induce redox imbalance, dysregulate one-carbon metabolism and proline biosynthesis. Downregulation of these pathways by ClpP agonists further contribute to the inhibition of CSC function. In conclusion, ClpP agonists inhibit breast CSC functions by disrupting mitochondrial homeostasis in breast cancer cells and inhibiting multiple pathways critical to CSC function. Significance: ClpP agonists disrupt mitochondrial homeostasis by activating mitochondrial matrix protease ClpP. We report that ClpP agonists inhibit cell growth and cancer stem cell functions in breast cancer models by modulating multiple metabolic pathways essential to cancer stem cell function.

Characterization of TR-107, a novel chemical activator of the human mitochondrial protease ClpP.

We recently described the identification of a new class of small-molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small-molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. We selected one compound (TR-107) with excellent potency, specificity, and drug-like properties for further evaluation. TR-107 showed ClpP-dependent growth inhibition in the low nanomolar range that was equipotent to paclitaxel in triple-negative breast cancer (TNBC) cell models. TR-107 also reduced specific mitochondrial proteins, including OXPHOS and TCA cycle components, in a time-, dose-, and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed the inactivation of OXPHOS and increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared with other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. Using human TNBC MDA-MB-231 xenografts, the antitumor response to TR-107 was investigated. Oral administration of TR-107 resulted in a reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. ClpP activation in vivo was validated by immunoblotting for TFAM and other mitochondrial proteins. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.

MEDI3039, a novel highly potent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor 2 agonist, causes regression of orthotopic tumors and inhibits outgrowth of metastatic triple-negative breast cancer.

BACKGROUND: TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists are attractive anti-tumor agents because of their capability to induce apoptosis in cancer cells by activating death receptors (DR) 4 and 5 with little toxicity against normal cells. Despite an attractive mechanism of action, previous clinical efforts to use TRAIL receptor agonists have been unsuccessful. In this study, we examined MEDI3039, a highly potent multivalent DR5 agonist, in breast cancer cell lines and in vivo models. METHODS: As in vitro model systems, we used 19 breast cancer cell lines that are categorized into four subtypes: ER+, HER2 amplified, basal A (triple-negative breast cancer) TNBC, and basal B TNBC. Cell viability was analyzed by MTS and RealTime live/dead assays. As in vivo model systems, MDA-MB231T orthotopic primary tumor growth in the mammary fat pad (MFP) and two experimental lung metastasis models were used. The effect of MEDI3039 on MFP tumors was assessed with immunohistochemical analysis. Lung metastases were analyzed with Bouin's and H&E staining. RESULTS: MEDI3039 killed multiple breast cancer cell lines, but the sensitivity varied among different subtypes. Sensitivity was basal B TNBC >> basal A TNBC > HER2 amplified > ER+ (average IC50 = 1.4, 203, 314, 403 pM, respectively). While the pattern of relative sensitivity was similar to GST-TRAIL in most cell lines, MEDI3039 was at least two orders of magnitude more potent compared with GST-TRAIL. In the MFP model, weekly treatment with 0.1 or 0.3 mg/kg MEDI3039 for 5 weeks inhibited tumor growth by 99.05% or 100% (median), respectively, compared with the control group, and extended animal survival (p = 0.08 or p = 0.0032 at 0.1 or 0.3 mg/kg, respectively). MEDI3039-induced caspase activation was confirmed in tumors grown in MFP (p < 0.05). In an experimental pulmonary metastasis model, MEDI3039 significantly suppressed outgrowth of surface (p < 0.0001) and microscopic metastases (p < 0.05). In an established lung metastasis model, MEDI3039 significantly inhibited growth of metastases (p < 0.01 in surface [> 4 mm], p < 0.01 in tumor percentage) and extended animal survival (p < 0.0001). CONCLUSION: MEDI3039 is a potent DR5 agonist in breast cancer cells in vitro and in vivo and has potential as a cancer drug in breast cancer patients, especially those with basal B TNBC.

ONC201 kills breast cancer cells in vitro by targeting mitochondria.

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

Lack of Casein Kinase 1 Delta Promotes Genomic Instability - The Accumulation of DNA Damage and Down-Regulation of Checkpoint Kinase 1.

Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes. Mice lacking CK1δ have a perinatal lethal phenotype and typically weigh 30% less than their wild type littermates. However, the causes of death and small size are unknown. We observed cells with abnormally large nuclei in tissue from Csnk1d null embryos, and multiple centrosomes in mouse embryo fibroblasts (MEFs) deficient in CK1δ (MEFCsnk1d null). Results from γ-H2AX staining and the comet assay demonstrated significant DNA damage in MEFCsnk1d null cells. These cells often contain micronuclei, an indicator of genomic instability. Similarly, abrogation of CK1δ expression in control MEFs stimulated micronuclei formation after doxorubicin treatment, suggesting that CK1δ loss increases vulnerability to genotoxic stress. Cellular levels of total and activated checkpoint kinase 1 (Chk1), which functions in the DNA damage response and mitotic checkpoints, and its downstream effector, Cdc2/CDK1 kinase, were often decreased in MEFCsnk1d null cells as well as in control MEFs transfected with CK1δ siRNA. Hydroxyurea-induced Chk1 activation, as measured by Ser345 phosphorylation, and nuclear localization also were impaired in MEF cells following siRNA knockdown of CK1δ. Similar results were observed in the MCF7 human breast cancer cell line. The decreases in phosphorylated Chk1 were rescued by concomitant expression of siRNA-resistant CK1δ. Experiments with cycloheximide demonstrated that the stability of Chk1 protein was diminished in cells subjected to CK1δ knockdown. Together, these findings suggest that CK1δ contributes to the efficient repair of DNA damage and the proper functioning of mitotic checkpoints by maintaining appropriate levels of Chk1.

Casein kinase 1δ functions at the centrosome and Golgi to promote ciliogenesis.

Inhibition of casein kinase 1 delta (CK1δ) blocks primary ciliogenesis in human telomerase reverse transcriptase immortalized retinal pigmented epithelial and mouse inner medullary collecting duct cells-3. Mouse embryonic fibroblasts (MEFs) and retinal cells from Csnk1d (CK1δ)-null mice also exhibit ciliogenesis defects. CK1δ catalytic activity and centrosomal localization signal (CLS) are required to rescue cilia formation in MEFs(Csnk1d null). Furthermore, expression of a truncated derivative containing the CLS displaces full-length CK1δ from the centrosome and decreases ciliary length in control MEFs, suggesting that centrosomal CK1δ has a role in ciliogenesis. CK1δ inhibition also alters pericentrosomal or ciliary distribution of several proteins involved in ciliary transport, including Ras-like in rat brain-11A, Ras-like in rat brain-8A, centrosomal protein of 290 kDa, pericentriolar material protein 1, and polycystin-2, as well as the Golgi distribution of its binding partner, A-kinase anchor protein 450 (AKAP450). As reported for AKAP450, CK1δ was required for microtubule nucleation at the Golgi and maintenance of Golgi integrity. Overexpression of an AKAP450 fragment containing the CK1δ-binding site inhibits Golgi-derived microtubule nucleation, Golgi distribution of intraflagellar transport protein 20 homologue, and ciliogenesis. Our results suggest that CK1δ mediates primary ciliogenesis by multiple mechanisms, one involving its centrosomal function and another dependent on its interaction with AKAP450 at the Golgi, where it is important for maintaining Golgi organization and polarized trafficking of multiple factors that mediate ciliary transport.

Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling.

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594-609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/ß-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ε as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser(594), Thr(595), and Ser(597) of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates ß-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.

Atypical protein kinase Cι is required for Wnt3a-dependent neurite outgrowth and binds to phosphorylated dishevelled 2.

Previously we reported that Wnt3a-dependent neurite outgrowth in Ewing sarcoma family tumor cell lines was mediated by Frizzled3, Dishevelled (Dvl), and c-Jun N-terminal kinase (Endo, Y., Beauchamp, E., Woods, D., Taylor, W. G., Toretsky, J. A., Uren, A., and Rubin, J. S. (2008) Mol. Cell. Biol. 28, 2368-2379). Subsequently, we observed that Dvl2/3 phosphorylation correlated with neurite outgrowth and that casein kinase 1δ, one of the enzymes that mediate Wnt3a-dependent Dvl phosphorylation, was required for neurite extension (Greer, Y. E., and Rubin, J. S. (2011) J. Cell Biol. 192, 993-1004). However, the functional relevance of Dvl phosphorylation in neurite outgrowth was not established. Dvl1 has been shown by others to be important for axon specification in hippocampal neurons via an interaction with atypical PKCζ, but the role of Dvl phosphorylation was not evaluated. Here we report that Ewing sarcoma family tumor cells express PKCι but not PKCζ. Wnt3a stimulated PKCι activation and caused a punctate distribution of pPKCι in the neurites and cytoplasm, with a particularly intense signal at the centrosome. Knockdown of PKCι expression with siRNA reagents blocked neurite formation in response to Wnt3a. Aurothiomalate, a specific inhibitor of PKCι/Par6 binding, also suppressed neurite extension. Wnt3a enhanced the co-immunoprecipitation of endogenous PKCι and Dvl2. Although FLAG-tagged wild-type Dvl2 immunoprecipitated with PKCι, a phosphorylation-deficient Dvl2 derivative did not. This derivative also was unable to rescue neurite outgrowth when endogenous Dvl2/3 was suppressed by siRNA (González-Sancho, J. M., Greer, Y. E., Abrahams, C. L., Takigawa, Y., Baljinnyam, B., Lee, K. H., Lee, K. S., Rubin, J. S., and Brown, A. M. (2013) J. Biol. Chem. 288, 9428-9437). Taken together, these results suggest that site-specific Dvl2 phosphorylation is required for Dvl2 association with PKCι. This interaction is likely to be one of the mechanisms essential for Wnt3a-dependent neurite outgrowth.

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells.

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co-expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth-promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial-mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co-transfectants exhibited invasive properties in three-dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf-1, a specific antagonist of the Wnt/ß-catenin pathway, or short hairpin RNA targeting ß-catenin expression demonstrated that the invasive activity was not mediated by ß-catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context-dependent. While Rspo2 and Wnt1 act synergistically in the ß-catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures.

Casein kinase 1 delta functions at the centrosome to mediate Wnt-3a-dependent neurite outgrowth.

Previously we determined that Dishevelled-2/3 (Dvl) mediate Wnt-3a-dependent neurite outgrowth in Ewing sarcoma family tumor cells. Here we report that neurite extension was associated with Dvl phosphorylation and that both were inhibited by the casein kinase 1 (CK1) δ/ε inhibitor IC261. Small interfering RNAs targeting either CK1δ or CK1ε decreased Dvl phosphorylation, but only knockdown of CK1δ blocked neurite outgrowth. CK1δ but not CK1ε was detected at the centrosome, an organelle associated with neurite formation. Deletion analysis mapped the centrosomal localization signal (CLS) of CK1δ to its C-terminal domain. A fusion protein containing the CLS and EGFP displaced full-length CK1δ from the centrosome and inhibited Wnt-3a-dependent neurite outgrowth. In contrast to wild-type CK1ε, a chimera comprised of the kinase domain of CK1ε and the CLS of CK1δ localized to the centrosome and rescued Wnt-3a-dependent neurite outgrowth suppressed by CK1δ knockdown. These results provide strong evidence that the centrosomal localization of CK1δ is required for Wnt-3a-dependent neuritogenesis.