Effectiveness of Rhenium(I)-diselenoether Low Doses in a Triple-negative Breast Cancer Chicken Embryo Model.

BACKGROUND/AIM: Rhenium(I)-diselenoether (Re-diSe) is a promising anticancer agent composed of one rhenium and two selenium atoms. Its effectiveness was established in inhibiting cancer cells while maintaining low toxicity toward normal cells at a 5 µM dose for 120 hours in MDA-MB-231 cells. In MDA-MB-231 breast tumor-bearing mice, anti-tumor and anti-metastatic effects were observed at a 10 mg/kg dose. However, contradictory results were observed in the 4T1 breast cancer model, where a dose of 60 mg/kg had a pro-tumor effect. To address these discrepancies, the efficacy of Re-diSe at the effective 10 mg/kg dose was validated in a transplanted MDA-MB-231 breast tumor model using the chicken chorioallantoic membrane assay. MATERIALS AND METHODS: MDA-MB-231 cancer cells were xenografted onto the chicken chorioallantoic membrane (CAM), and daily drug administration was carried out for nine days at doses of 0.1, 1, and 10 mg/kg. At the study's conclusion, a standard histological analysis was conducted. RESULTS: The low dose of 0.1 mg/kg showed a significant reduction in tumor weights compared to controls. The 1 mg/kg dose resulted in an increased inflammation score but did not induce a significant difference in tumor weights compared to the 0.1 mg/kg dose. Notably, at the 10 mg/kg dose, six out of 11 treated embryos displayed no visible signs of tumors. These tumors exhibited extensive tumor necrosis and significant infiltration by inflammatory cells. CONCLUSION: In this particular model, the anticancer efficacy of Re-diSe was achieved at the low dose of 0.1 mg/kg. The higher dose of 10 mg/kg, while eliminating visible tumors, might have immune-mediated effects, as indicated by substantial tumor necrosis and infiltration by inflammatory cells. Overall, this study successfully demonstrated the effectiveness of Re-diSe as an anticancer agent.

Embryonated Chicken Tumor Xenografts Derived from Circulating Tumor Cells as a Relevant Model to Study Metastatic Dissemination: A Proof of Concept.

Patient-Derived Xenografts (PDXs) in the Chorioallantoic Membrane (CAM) are a representative model for studying human tumors. Circulating Tumor Cells (CTCs) are involved in cancer dissemination and treatment resistance mechanisms. To facilitate research and deep analysis of these few cells, significant efforts were made to expand them. We evaluated here whether the isolation of fresh CTCs from patients with metastatic cancers could provide a reliable tumor model after a CAM xenograft. We enrolled 35 patients, with breast, prostate, or lung metastatic cancers. We performed microfluidic-based CTC enrichment. After 48-72 h of culture, the CTCs were engrafted onto the CAM of embryonated chicken eggs at day 9 of embryonic development (EDD9). The tumors were resected 9 days after engraftment and histopathological, immunochemical, and genomic analyses were performed. We obtained in ovo tumors for 61% of the patients. Dedifferentiated small tumors with spindle-shaped cells were observed. The epithelial-to-mesenchymal transition of CTCs could explain this phenotype. Beyond the feasibility of NGS in this model, we have highlighted a genomic concordance between the in ovo tumor and the original patient's tumor for constitutional polymorphism and somatic alteration in one patient. Alu DNA sequences were detected in the chicken embryo's distant organs, supporting the idea of dedifferentiated cells with aggressive behavior. To our knowledge, we performed the first chicken CAM CTC-derived xenografts with NGS analysis and evidence of CTC dissemination in the chicken embryo.

PD-1/PD-L1 Checkpoint Inhibitors Are Active in the Chicken Embryo Model and Show Antitumor Efficacy In Ovo.

(1) Purpose: To assess the use of the chicken embryo (in ovo) model as an alternative in vivo model for immuno-oncology (IO) drug development, focusing on programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors. (2) Methods: First, the presence of immune cells in the model was detected through the immunophenotyping of chicken peripheral blood mononuclear cells (PBMCs) based on fluorescence activated cell sorting (FACS) analysis and the immunohistochemistry (IHC) analysis of in ovo tumor-infiltrating lymphocytes. Second, the cross-reactivity between one anti-human PD-1 Ab, pembrolizumab (KEYTRUDA®), and chicken PD-1 was verified through the labelling of chicken splenocytes with pembrolizumab by FACS analysis. Third, the blockade effect of pembrolizumab on chicken PBMCs was assessed in vitro through cytotoxicity assay based on MTT. Fourth, the CAM assay was used to estimate the anti-tumor performance of pembrolizumab through the analyses of tumor growth and chicken immune cell infiltration in tumors. Finally, the efficacy of several PD-1 or PD-L1 inhibitors (nivolumab, atezolizumab and avelumab) on tumor growth was further assessed using the CAM assay. (3) Results: The presence of CD3+, CD4+, CD8+ T lymphocytes and monocytes was confirmed by FACS and IHC analyses. During in vitro assays, pembrolizumab cross-reacted with chicken lymphocytes and induced PD-1/PD-L1 blockade, which permitted the restoration of chicken T-cell's cytotoxicity against human lung cancer H460 tumor cells. All these in vitro results were correlated with in ovo findings based on the CAM assay: pembrolizumab inhibited H460 tumor growth and induced evident chicken immune cell infiltration (with significant chicken CD45, CD3, CD4, CD8 and CD56 markers) in tumors. Furthermore, the potency of the CAM assay was not limited to the application of pembrolizumab. Nivolumab, atezolizumab and avelumab also led to tumor growth inhibition in ovo, on different tumor models. (4) Conclusions: The chicken embryo affords a physiological, immune reactive, in vivo environment for IO research, which allows observation of how the immune system defense against tumor cells, as well as the different immune tolerance mechanisms leading to tumor immune escape. The encouraging results obtained with PD-1/PD-L1 inhibitors in this study reveal the potential use of the chicken embryo model as an alternative, fast, and reliable in vivo model in the different fields of IO drug discovery.

The CAM Model-Q&A with Experts.

The chick chorioallantoic membrane (CAM), as an extraembryonic tissue layer generated by the fusion of the chorion with the vascularized allantoic membrane, is easily accessible for manipulation. Indeed, grafting tumor cells on the CAM lets xenografts/ovografts develop in a few days for further investigations. Thus, the CAM model represents an alternative test system that is a simple, fast, and low-cost tool to study tumor growth, drug response, or angiogenesis in vivo. Recently, a new era for the CAM model in immune-oncology-based drug discovery has been opened up. Although there are many advantages offering extraordinary and unique applications in cancer research, it has also disadvantages and limitations. This review will discuss the pros and cons with experts in the field.

The Chicken Embryo Model: A Novel and Relevant Model for Immune-Based Studies.

Dysregulation of the immune system is associated with many pathologies, including cardiovascular diseases, diabetes, and cancer. To date, the most commonly used models in biomedical research are rodents, and despite the various advantages they offer, their use also raises numerous drawbacks. Recently, another in vivo model, the chicken embryo and its chorioallantoic membrane, has re-emerged for various applications. This model has many benefits compared to other classical models, as it is cost-effective, time-efficient, and easier to use. In this review, we explain how the chicken embryo can be used as a model for immune-based studies, as it gradually develops an embryonic immune system, yet which is functionally similar to humans'. We mainly aim to describe the avian immune system, highlighting the differences and similarities with the human immune system, including the repertoire of lymphoid tissues, immune cells, and other key features. We also describe the general in ovo immune ontogeny. In conclusion, we expect that this review will help future studies better tailor their use of the chicken embryo model for testing specific experimental hypotheses or performing preclinical testing.

Metabolic Signatures of Tumor Responses to Doxorubicin Elucidated by Metabolic Profiling in Ovo.

BACKGROUND: Dysregulated cancer metabolism is associated with acquired resistance to chemotherapeutic treatment and contributes to the activation of cancer survival mechanisms. However, which metabolic pathways are activated following treatment often remains elusive. The combination of chicken embryo tumor models (in ovo) with metabolomics phenotyping could offer a robust platform for drug testing. Here, we assess the potential of this approach in the treatment of an in ovo triple negative breast cancer with doxorubicin. METHODS: MB-MDA-231 cells were grafted in ovo. The resulting tumors were then treated with doxorubicin or dimethyl sulfoxide (DMSO) for six days. Tumors were collected and analyzed using a global untargeted metabolomics and comprehensive lipidomics. RESULTS: We observed a significant suppression of tumor growth in the doxorubicin treated group. The metabolic profiles of doxorubicin and DMSO-treated tumors were clearly separated in a principle component analysis. Inhibition of glycolysis, nucleotide synthesis, and glycerophospholipid metabolism appear to be triggered by doxorubicin treatment, which could explain the observed suppressed tumor growth. In addition, metabolic cancer survival mechanisms could be supported by an acceleration of antioxidative pathways. CONCLUSIONS: Metabolomics in combination with in ovo tumor models provide a robust platform for drug testing to reveal tumor specific treatment targets such as the antioxidative tumor capacity.

Addressing current challenges and future directions in immuno-oncology: expert perspectives from the 2017 NIBIT Foundation Think Tank, Siena, Italy.

A collaborative think tank involving panellists from immuno-oncology networks, clinical/translational investigators and the pharmaceutical industry was held in Siena, Italy, in October 2017 to discuss the evolving immune-oncology landscape, identify selected key challenges, and provide a perspective on the next steps required in the translation of current research and knowledge to clinical reality. While there is a trend of combining new agents (e.g., co-stimulator agonists) with a PD-1/PD-L1 treatment backbone, use of alternative combination therapy approaches should also be considered. While the rapid evolution in systems biology provides a deeper understanding of tumor and tumor microenvironment heterogeneity, there remains the need to identify and define genuinely predictive biomarkers to guide treatment and patient selection. Cross-specialty and cross-sector collaboration, along with a broader collective data-sharing approach are key to optimizing immuno-oncology therapy in clinical practice. Continued support of younger research-clinicians is essential for future success in clinical, translational and basic science investigations.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

Rhus coriaria suppresses angiogenesis, metastasis and tumor growth of breast cancer through inhibition of STAT3, NFκB and nitric oxide pathways.

Recently, we reported that Rhus coriaria exhibits anticancer activities by promoting cell cycle arrest and autophagic cell death of the metastatic triple negative MDA-MB-231 breast cancer cells. Here, we investigated the effect of Rhus coriaria on the migration, invasion, metastasis and tumor growth of TNBC cells. Our current study revealed that non-cytotoxic concentrations of Rhus coriaria significantly inhibited migration and invasion, blocked adhesion to fibronectin and downregulated MMP-9 and prostaglandin E2 (PgE2). Not only did Rhus coriaria decrease their adhesion to HUVECs and to lung microvascular endothelial (HMVEC-L) cells, but it also inhibited the transendothelial migration of MDA-MB-231 cells through TNF-α-activated HUVECs. Furthermore, we found that Rhus coriaria inhibited angiogenesis, reduced VEGF production in both MDA-MB-231 and HUVECs and downregulated the inflammatory cytokines TNF-α, IL-6 and IL-8. The underlying mechanism for Rhus coriaria effects appears to be through inhibiting NFκB, STAT3 and nitric oxide (NO) pathways. Most importantly, by using chick embryo tumor growth assay, we showed that Rhus coriaria suppressed tumor growth and metastasis in vivo. The results described in the present study identify Rhus coriaria as a promising chemopreventive and therapeutic candidate that modulate triple negative breast cancer growth and metastasis.

Genetic and pharmacologic inhibition of mTORC1 promotes EMT by a TGF-ß-independent mechanism.

Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process that converts epithelial cells into highly motile mesenchymal cells. This physiologic process occurs largely during embryonic development but is aberrantly reactivated in different pathologic situations, including fibrosis and cancer. We conducted a siRNA screening targeted to the human kinome with the aim of discovering new EMT effectors. With this approach, we have identified mTOR complex 1 (mTORC1), a nutrient sensor that controls protein and lipid synthesis, as a key regulator of epithelial integrity. Using a combination of RNAi and pharmacologic approaches, we report here that inhibition of either mTOR or RPTOR triggers EMT in mammary epithelial cells. This EMT was characterized by the induction of the mesenchymal markers such as fibronectin, vimentin, and PAI-1, together with the repression of epithelial markers such as E-cadherin and ZO-3. In addition, mTORC1 blockade enhanced in vivo migratory properties of mammary cells and induced EMT independent of the TGF-ß pathway. Finally, among the transcription factors known to activate EMT, both ZEB1 and ZEB2 were upregulated following mTOR repression. Their increased expression correlated with a marked reduction in miR-200b and miR-200c mRNA levels, two microRNAs known to downregulate ZEB1 and ZEB2 expression. Taken together, our findings unravel a novel function for mTORC1 in maintaining the epithelial phenotype and further indicate that this effect is mediated through the opposite regulation of ZEB1/ZEB2 and miR-200b and miR-200c. Furthermore, these results suggest a plausible etiologic explanation for the progressive pulmonary fibrosis, a frequent adverse condition associated with the therapeutic use of mTOR inhibitors.

Anti-metastatic and anti-tumor growth effects of Origanum majorana on highly metastatic human breast cancer cells: inhibition of NFκB signaling and reduction of nitric oxide production.

BACKGROUND: We have recently reported that Origanummajorana exhibits anticancer activity by promoting cell cycle arrest and apoptosis of the metastatic MDA-MB-231 breast cancer cell line. Here, we extended our study by investigating the effect of O. majorana on the migration, invasion and tumor growth of these cells. RESULTS: We demonstrate that non-cytotoxic concentrations of O. majorana significantly inhibited the migration and invasion of the MDA-MB-231 cells as shown by wound-healing and matrigel invasion assays. We also show that O. majorana induce homotypic aggregation of MDA-MB-231 associated with an upregulation of E-cadherin protein and promoter activity. Furthermore, we show that O. majorana decrease the adhesion of MDA-MB-231 to HUVECs and inhibits transendothelial migration of MDA-MB-231 through TNF-α-activated HUVECs. Gelatin zymography assay shows that O. majorana suppresses the activities of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9). ELISA, RT-PCR and Western blot results revealed that O. majorana decreases the expression of MMP-2, MMP-9, urokinase plasminogen activator receptor (uPAR), ICAM-1 and VEGF. Further investigation revealed that O. majorana suppresses the phosphorylation of IκB, downregulates the nuclear level of NFκB and reduces Nitric Oxide (NO) production in MDA-MB-231 cells. Most importantly, by using chick embryo tumor growth assay, we also show that O. majorana promotes inhibition of tumor growth and metastasis in vivo. CONCLUSION: Our findings identify Origanummajorana as a promising chemopreventive and therapeutic candidate that modulate breast cancer growth and metastasis.

Azaindole derivatives are inhibitors of microtubule dynamics, with anti-cancer and anti-angiogenic activities.

BACKGROUND AND PURPOSE: Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell-permeable microtubule-targeting agents. EXPERIMENTAL APPROACH: Using a cell-based assay designed to probe for microtubule polymerization status, we screened a chemical library and identified two azaindole derivatives, CM01 and CM02, as cell-permeable microtubule-depolymerizing agents. The mechanism of the anti-tumour effects of these two compounds was further investigated both in vivo and in vitro. KEY RESULTS: CM01 and CM02 induced G2/M cell cycle arrest and exerted potent cytostatic effects on several cancer cell lines including multidrug-resistant (MDR) cell lines. In vitro experiments revealed that the azaindole derivatives inhibited tubulin polymerization and competed with colchicines for this effect, strongly indicating that tubulin is the cellular target of these azaindole derivatives. In vivo experiments, using a chicken chorioallantoic xenograft tumour assay, established that these compounds exert a potent anti-tumour effect. Furthermore, an assay probing the growth of vessels out of endothelial cell spheroids showed that CM01 and CM02 exert anti-angiogenic activities. CONCLUSIONS AND IMPLICATIONS: CM01 and CM02 are reversible microtubule-depolymerizing agents that exert potent cytostatic effects on human cancer cells of diverse origins, including MDR cells. They were also shown to inhibit angiogenesis and tumour growth in chorioallantoic breast cancer xenografts. Hence, these azaindole derivatives are attractive candidates for further preclinical investigations.

The combination of a histone deacetylase inhibitor with the BH3-mimetic GX15-070 has synergistic antileukemia activity by activating both apoptosis and autophagy.

We analyzed the cellular and molecular effects of two different histone deacetylase inhibitors (HDACi), MGCD0103 and vorinostat, in combination with GX15-070, a BH3-mimetic, in acute myeloid leukemia (AML) cell lines and primary AML cells, and demonstrated that the combination has a synergistic antileukemia effect. We observed that in addition to apoptosis, autophagy also accounts for the observed nonapoptotic decrease of cell viability. Mechanistically, we established a role for calpain activity and ER-located caspase signaling in the induction of both autophagy and apoptosis following this combination of drugs. These findings reveal that for this specific combination, autophagy plays a positive role in inducing cytotoxicity, and that the involved ER signaling networks, as well as their clinical relevance, should be further studied in both preclinical and clinical trials of leukemia and other malignancies.

Phase I dose finding studies of obatoclax (GX15-070), a small molecule pan-BCL-2 family antagonist, in patients with advanced solid tumors or lymphoma.

PURPOSE: Two phase I, single-agent studies were conducted to determine the dose and regimen of obatoclax, an antagonist of all BCL-2 antiapoptotic proteins, for evaluation in phase II trials. The two studies, GX001 and GX005, evaluated the safety and tolerability of weekly 1-hour and 3-hour infusions of obatoclax, respectively. EXPERIMENTAL DESIGN: Eligible patients in both studies were adults with solid tumor or lymphoma and performance status 0-1 for whom standard therapies were not appropriate. In the GX001 study an accelerated dose titration design was initially used with subsequent cohorts of three to six patients with 40% dose increments between levels. In the GX005 study three to six patients entered at each dose level with 40% dose increments between levels. RESULTS: Thirty-five patients were enrolled in studies GX001 (n = 8) and GX005 (n = 27). Clinically significant central nervous system (CNS) toxicity was observed using the 1-hour infusion schedule. The obatoclax maximum tolerated dose (MTD) in GX001 was 1.25 mg/m(2) due to these infusional CNS events. The 3-hour infusion schedule studied in GX005 had improved tolerability, and the obatoclax MTD was 20 mg/m(2). One patient in GX005 with relapsed non-Hodgkin's lymphoma achieved partial response of 2 months' duration, and one patient with relapsed non-Hodgkin's lymphoma had stable disease for 18 months. CONCLUSIONS: The 1-hour infusion schedule of obatoclax was associated with neuropsychiatric dose-limiting toxicities at relatively low doses (MTD, 1.25 mg/m(2)). The 3-hour i.v. infusion of obatoclax administered once weekly to patients with solid tumors was better tolerated (MTD, 20 mg/m(2)), and evidence of clinical activity was observed.

The combination of a histone deacetylase inhibitor with the Bcl-2 homology domain-3 mimetic GX15-070 has synergistic antileukemia activity by activating both apoptosis and autophagy.

PURPOSE: Single-agent histone deacetylase inhibitors (HDACi) have limited clinical activity in human leukemia. Although the way HDACi exert their antileukemia effect is not fully understood, it is accepted that induction of apoptosis is important. We hypothesized, therefore, that combination of an HDACi with a proapoptotic agent, such as the Bcl-2 homology domain-3 mimetic GX15-070, could result in enhanced antileukemia activity. EXPERIMENTAL DESIGN: We analyzed the cellular and molecular effects of two different HDACi (MGCD0103 and vorinostat) in combination with GX15-070 in leukemia cell lines and primary acute myelogenous leukemia cells. RESULTS: We showed that the combination had synergistic antileukemia effect both in leukemia cell lines and in primary acute myelogenous leukemia cells. Using molecular markers and electron microscopy, we observed that in addition to apoptosis, autophagy accounts for the nonapoptotic decrease in cell viability, an effect that could be inhibited by chloroquine, an inhibitor of autophagy. Finally, we established a role for calpain activity in the induction of both autophagy and apoptosis by this combination. CONCLUSIONS: The combination of an HDACi and GX15-070 has synergistic antileukemia activity, and the effect is mediated by induction of apoptosis and autophagy. The combination should be studied in clinical trials of leukemia and the role of autophagy in leukemia therapy needs to be better understood.

Phase I study of obatoclax mesylate (GX15-070), a small molecule pan-Bcl-2 family antagonist, in patients with advanced chronic lymphocytic leukemia.

Obatoclax mesylate is a small molecule pan-Bcl-2 antagonist with in vitro activity against chronic lymphocytic leukemia (CLL) cells. Obatoclax was administered to patients with advanced CLL at doses ranging from 3.5 to 14 mg/m(2) as a 1-hour infusion and from 20 to 40 mg/m(2) as a 3-hour infusion every 3 weeks. Twenty-six patients received a total of 74 cycles. Dose-limiting reactions were neurologic (somnolence, euphoria, ataxia) and associated with the infusion. The maximum tolerated dose (MTD) was 28 mg/m(2) over 3 hours every 3 weeks. One (4%) of 26 patients achieved a partial response. Patients with anemia (3/11) or thrombocytopenia (4/14) experienced improvements in hemoglobin and platelet counts. Circulating lymphocyte counts were reduced in 18 of 26 patients with a median reduction of 24%. Overall, the maximum plasma concentration (C(max)) and area under the curve (AUC) values of obatoclax were dose proportional. Activation of Bax and Bak was demonstrated in peripheral blood mononuclear cells, and induction of apoptosis was related to overall obatoclax exposure, as monitored by the plasma concentration of oligonucleosomal DNA/histone complexes. Obatoclax mesylate has biologic activity and modest single-agent activity in heavily pretreated patients with advanced CLL. Further evaluation in less heavily pretreated patients and in combination with other therapeutic agents is warranted. This trial has been registered with http://clinicaltrials.gov under identifier NCT00600964.

A phase I study of the pan bcl-2 family inhibitor obatoclax mesylate in patients with advanced hematologic malignancies.

PURPOSE: The outcome of patients with refractory leukemia and myelodysplasia is poor, and new therapies are needed. The antiapoptotic proteins of the Bcl-2 family are overexpressed in these malignancies and are potential therapeutic targets. Therefore, we conducted a phase I clinical trial of the small-molecule pan-Bcl-2 inhibitor, obatoclax mesylate, in patients with refractory leukemia and myelodysplasia to assess its safety and define its optimal dose. EXPERIMENTAL DESIGN: Forty-four patients with refractory leukemia or myelodysplasia were treated with obatoclax mesylate by continuous intravenous infusion at increasing doses and frequencies. RESULTS: A total of 306 infusions of obatoclax mesylate were administered with a median of 5 infusions per patient. The study drug was well tolerated up to the highest dose planned without dose-limiting toxicity. Grade 1/2 central nervous system symptoms were the most common adverse events attributable to the study drug. One patient with acute myeloid leukemia with mixed lineage leukemia t(9;11) rearrangement achieved a complete remission, which lasted 8 months. Three of 14 patients with myelodysplasia showed hematologic improvement with RBC or platelet transfusion independence. CONCLUSIONS: Obatoclax mesylate is well tolerated and these results support its further investigation in patients with leukemia and myelodysplasia.

Mechanisms of antileukemic activity of the novel Bcl-2 homology domain-3 mimetic GX15-070 (obatoclax).

In this study, we investigated the mechanism of apoptosis induction of obatoclax (GX15-070), a novel Bcl-2 homology domain-3 (BH3) mimetic, in acute myeloid leukemia (AML) cell lines and primary AML samples. Obatoclax inhibited cell growth of HL-60, U937, OCI-AML3, and KG-1 cell lines. Apoptosis induction contributed to the observed antiproliferative effects at concentrations of this agent that mirror its affinity for antiapoptotic Bcl-2 proteins. We show that obatoclax can promote the release of cytochrome c from isolated leukemia cell mitochondria and that apoptosis induced by this agent is preceded by the release of Bak from Mcl-1, liberation of Bim from both Bcl-2 and Mcl-1, and the formation of an active Bak/Bax complex. Notably, apoptosis was diminished, but not fully prevented, in the absence of Bak/Bax or Bim, suggesting that obatoclax has additional targets that contribute to its cytotoxicity. At growth inhibitory doses that did not induce apoptosis or decrease viability, obatoclax induced an S-G(2) cell-cycle block. Obatoclax induced apoptosis in AML CD34+ progenitor cells with an average IC(50) of 3.59 +/- 1.23 micromol/L although clonogenicity was inhibited at concentrations of 75 to 100 nmol/L. Obatoclax synergized with the novel BH3 mimetic ABT-737 to induce apoptosis in OCI-AML3 cells and synergistically induced apoptosis in combination with AraC in leukemic cell lines and in primary AML samples. In conclusion, we show that obatoclax potently induces apoptosis and decreases leukemia cell proliferation and may be used in a novel therapeutic strategy for AML alone and in combination with other targeted agents and chemotherapeutics.

A small molecule pan-Bcl-2 family inhibitor, GX15-070, induces apoptosis and enhances cisplatin-induced apoptosis in non-small cell lung cancer cells.

PURPOSE: Overexpression of Bcl-2 family members as well as deregulated apoptosis pathways are known hallmarks of lung cancer. Non-small cell lung cancer (NSCLC) cells are typically resistant to cytotoxic chemotherapy and approaches that alter the balance between pro-survival and pro-death Bcl-2 family members have shown promise in preclinical models of NSCLC. METHODS: Here we evaluated the effects of a novel pan-Bcl-2 inhibitor GX15-070 on NSCLC survival and when combined with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors as well as traditional cytotoxic agents. GX15-070 is a small molecule agent that binds anti-apoptotic Bcl-2 proteins and interferes with their ability to interact with pro-apoptotic proteins. We evaluated the effect of GX15-070 and correlated the effect on EGFR status as well as Bcl-2 family protein expression. RESULTS: We show that GX15-070 can disrupt Mcl-1:Bak interactions in lung cancer cells. We identified differential sensitivity of a panel of lung cancer cells to GX15-070 and no clear relationship existed between EGFR status or Bcl-2 family protein expression and sensitivity to GX15-070. GX15-070 could induce apoptosis in a subset of lung cancer cell lines and this correlated with the effects on cell viability. GX15-070 combined with gefitinib was synergistic in a cell line dependent on EGFR for survival but GX15-070 could not reverse resistance to gefitinib in cell lines not dependent on EGFR for survival. Finally, we observed synergy between GX15-070 and cisplatin in NSCLC cells. CONCLUSIONS: Based on these results, GX15-070 can trigger apoptosis in NSCLC cells and can enhance chemotherapy-induced death. These data suggest that clinical trials with GX15-070 in combination with cytotoxic chemotherapy are indicated.

Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1-mediated resistance to apoptosis.

Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing.