Inhibition Of Bcl-2 Family Proteins Research Articles

Obatoclax potentiates the cytotoxic effect of cytarabine on acute myeloid leukemia cells by enhancing DNA damage

Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure for acute myeloid leukemia (AML) patients. Overexpression of Bcl-2, Bcl-xL, and/or Mcl-1 has been associated with chemoresistance in AML cell lines and with poor clinical outcome of AML patients. Thus, inhibitors of anti-apoptotic Bcl-2 family proteins could be novel therapeutic agents. In this study, we investigated how clinically achievable concentrations of obatoclax, a pan-Bcl-2 inhibitor, potentiate the antileukemic activity of cytarabine in AML cells. MTT assays in AML cell lines and diagnostic blasts, as well as flow cytometry analyses in AML cell lines revealed synergistic antileukemic activity between cytarabine and obatoclax. Bax activation was detected in the combined, but not the individual, drug treatments. This was accompanied by significantly increased loss of mitochondrial membrane potential. Most importantly, in AML cells treated with the combination, enhanced early induction of DNA double-strand breaks (DSBs) preceded a decrease of Mcl-1 levels, nuclear translocation of Bcl-2, Bcl-xL, and Mcl-1, and apoptosis. These results indicate that obatoclax enhances cytarabine-induced apoptosis by enhancing DNA DSBs. This novel mechanism provides compelling evidence for the clinical use of BH3 mimetics in combination with DNA-damaging agents in AML and possibly a broader range of malignancies.

Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins

Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z′-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach.

Mechanism-based pharmacokinetic/pharmacodynamic meta-analysis of navitoclax (ABT-263) induced thrombocytopenia.

Navitoclax is a first-in-class, orally bioavailable, targeted Bcl-2 family protein inhibitor and promotes apoptosis. Thrombocytopenia is a primary dose-limiting toxicity of navitoclax which exhibited a distinct time profile in circulating platelets from that caused by traditional chemotherapies. A population pharmacokinetic/pharmacodynamic (PK/PD) model was developed to describe the pharmacokinetic of navitoclax as well as the time course of the platelet counts in cancer patients receiving navitoclax. Data from 256 patients who received oral navitoclax (dose range 10-475 mg) as a 14/21-day schedule or a continuous once daily (QD) schedule were used to construct the model using NONMEM. The PK model was a two-compartmental model with a lag-time and a transit compartment in absorption. The PD model was a semi-physiological model that comprised a progenitor cell compartment, three transition compartments representing the maturation chain in the bone marrow and a peripheral blood compartment. Compared with the previously published models, the model established in this analysis applied a different feedback mechanism and introduced a new concept of progenitor cell "pool", which describes a large pool of platelet progenitor cells at the beginning of navitoclax treatment. The PD model was able to describe a slight downward trend of platelet counts over the long-term navitoclax treatment as observed in around 8 % of the patients and the initial drop in platelets seen in our Phase 1/2a studies. We have developed a new semi-physiological platelet model for describing fast drop of platelets after initial navitoclax administration and long-term decline of platelets after continuous administration of navitoclax.

AB0032 Bcl-2:Bim Ratios in Human Peripheral Blood Mononuclear Cell Subsets May Predict Sensitivity to BCL-2 Inhibition

BackgroundThe balance between pro- and anti-apoptotic BCL-2 family proteins plays a critical role in regulation of apoptosis in lymphocytes. Navitoclax is a potent and orally bioavailable BH3 mimetic which inhibits...

Development of 3-phenyl-N-(2-(3-phenylureido)ethyl)-thiophene-2-sulfonamide compounds as inhibitors of antiapoptotic Bcl-2 family proteins.

Antiapoptotic Bcl-2 family proteins, such as Bcl-xL , Bcl-2, and Mcl-1, are often overexpressed in tumor cells, which contributes to tumor cell resistance to chemotherapies and radiotherapies. Inhibitors of these proteins thus have potential applications in cancer treatment. We discovered, through structure-based virtual screening, a lead compound with micromolar binding affinity to Mcl-1 (inhibition constant (Ki )=3 μM). It contains a phenyltetrazole and a hydrazinecarbothioamide moiety, and it represents a structural scaffold not observed among known Bcl-2 inhibitors. This work presents the structural optimization of this lead compound. By following the scaffold-hopping strategy, we have designed and synthesized a total of 82 compounds in three sets. All of the compounds were evaluated in a fluorescence-polarization binding assay to measure their binding affinities to Bcl-xL , Bcl-2, and Mcl-1. Some of the compounds with a 3-phenylthiophene-2-sulfonamide core moiety showed sub-micromolar binding affinities to Mcl-1 (Ki =0.3-0.4 μM) or Bcl-2 (Ki ≈1 μM). They also showed obvious cytotoxicity on tumor cells (IC50 <10 μM). Two-dimensional heteronuclear single quantum coherence NMR spectra of three selected compounds, that is, YCW-E5, YCW-E10, and YCW-E11, indicated that they bind to the BH3-binding groove on Bcl-xL in a similar mode to ABT-737. Several apoptotic assays conducted on HL-60 cells demonstrated that these compounds are able to induce cell apoptosis through the mitochondrial pathway. We propose that the compounds with the 3-phenylthiophene-2-sulfonamide core moiety are worth further optimization as effective apoptosis inducers with an interesting selectivity towards Mcl-1 and Bcl-2.

Combined inhibition of NF-κB and Bcl-2 triggers synergistic reduction of viability and induces apoptosis in melanoma cells.

Constitutive activation of nuclear factor κB (NF-κB) characterizes melanoma cells. To explore the molecular mechanism of melanoma cell survival by constitutive NF-κB activation, we used the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ), which directly binds to NF-κB. DHMEQ abrogated constitutive NF-κB activity, which included RelA (p65)/p50 in melanoma cell lines G361 and HMV-II; however, the reduction of the viability was marginal. Expression of c-FLIP was not observed in the melanoma cell lines tested, and DHMEQ could not repress the expression of the Bcl-2 family proteins Bcl-2 and Bcl-xL. Concomitant treatment with DHMEQ and the inhibitor of antiapoptotic Bcl-2 family proteins, GX15-070, triggered synergistic reduction of the viability and induced apoptosis of G361 cells. These results indicate that abrogation of the NF-κB pathway alone is not sufficient to suppress the survival of melanoma cells. The NF-κB and the antiapoptotic Bcl-2 pathways cooperatively support the survival, and the dual targeting triggers synergistic reduction of the viability and induces apoptosis of melanoma cells.

Evaluation and critical assessment of putative MCL-1 inhibitors

High levels of BCL-2 family proteins are implicated in a failed/ineffective apoptotic programme, often resulting in diseases, including cancer. Owing to their potential as drug targets in cancer therapy, several inhibitors of BCL-2 family proteins have been developed. These primarily target specific members of the BCL-2 family, particularly BCL-2 and BCL-XL but are ineffective against MCL-1. Major efforts have been invested in developing inhibitors of MCL-1, which is commonly amplified in human tumours and associated with tumour relapse and chemoresistance. In this report, the specificity of several BCL-2 family inhibitors (ABT-263, UCB-1350883, apogossypol and BH3I-1) was investigated and compared with putative MCL-1 inhibitors designed to exhibit improved or selective binding affinities for MCL-1 (TW-37, BI97C1, BI97C10, BI112D1, compounds 6 and 7, and MCL-1 inhibitor molecule (MIM-1)). ABT-263, BI97C1, BI112D1, MIM-1 and TW-37 exhibited specificity in inducing apoptosis in a Bax/Bak- and caspase-9-dependent manner, whereas the other agents showed no killing activity, or little or no specificity. Of these inhibitors, only ABT-263 and UCB-1350883 induced apoptosis in a BCL-2- or BCL-XL-dependent system. In cells that depend on MCL-1 for survival, ABT-263 and TW-37 induced extensive apoptosis, suggesting that at high concentrations these inhibitors have the propensity to inhibit MCL-1 in a cellular context. TW-37 induced apoptosis, assessed by chromatin condensation, caspase processing and phosphatidylserine externalisation, in a BAK-dependent manner and in cells that require MCL-1 for survival. TW-37-mediated apoptosis was also partly dependent on NOXA, suggesting that derivatives of TW-37, if engineered to exhibit better selectivity and efficacy at low nanomolar concentrations, may provide useful lead compounds for further synthetic programmes. Expanded medicinal chemistry iteration, as performed for the ABT series, may likewise improve the potency and specificity of the evaluated MCL-1 inhibitors.

Characterization of the Stereochemical Structures of 2H‐Thiazolo[3,2‐a]pyrimidine Compounds and Their Binding Affinities for Anti‐apoptotic Bcl‐2 Family Proteins

In a previous study we reported a class of compounds with a 2H-thiazolo[3,2-a]pyrimidine core structure as general inhibitors of anti-apoptotic Bcl-2 family proteins. However, the absolute stereochemical configuration of one carbon atom on the core structure remained unsolved, and its potential impact on the binding affinities of compounds in this class was unknown. In this study, we obtained pure R and S enantiomers of four selected compounds by HPLC separation and chiral synthesis. The absolute configurations of these enantiomers were determined by comparing their circular dichroism spectra to that of an appropriate reference compound. In addition, a crystal structure of one selected compound revealed the exocyclic double bond in these compounds to be in the Z configuration. The binding affinities of all four pairs of enantiomers to Bcl-xL , Bcl-2, and Mcl-1 proteins were measured in a fluorescence-polarization-based binding assay, yielding inhibition constants (Ki values) ranging from 0.24 to 2.20 μM. Interestingly, our results indicate that most R and S enantiomers exhibit similar binding affinities for the three tested proteins. A binding mode for this compound class was derived by molecular docking and molecular dynamics simulations to provide a reasonable interpretation of this observation.

ApoG2 induces ER stress-dependent apoptosis in gastric cancer cells in vitro and its real-time evaluation by bioluminescence imaging in vivo

Apogossypolone (ApoG2), a potent small molecular inhibitor of Bcl-2 family proteins, is reported to have a significant anti-cancer effect in several types of cancers, but it has not been investigated in gastric cancer. In this study, we demonstrate in vitro and in vivo that ApoG2 inhibits human gastric cancer. Gastric carcinoma cell growth and proliferation was significantly hampered in vitro, as measured by MTT and colony formation assays. Real-time bioluminescence imaging indicated that ApoG2 causes tumor growth delay in a murine xenograft model. Further studies revealed that the ApoG2 induced apoptosis in gastric cancer cells was associated with the endoplasmic reticulum stress-induced apoptosis pathway. Conclusively, our results indicate that ApoG2 may be a promising agent for gastric cancer therapy.

Microarray determination of Bcl-2 family protein inhibition sensitivity in breast cancer cells

This study tests the hypothesis that reverse transcription polymerase chain reaction (RT-PCR) microarrays can be used to predict the relative sensitivity to induction of apoptosis in breast cancer cells exposed to inhibitors of antiapoptotic Bcl-2 family proteins. Four cell lines, MDA-MB-231 (MDA-231) and MDA-MB-468 (MDA-468), BT-20 and T47-D were screened for relative expression of Bcl-2 family members A1, Mcl-1, Bcl-2, Bcl-xL and Bcl-w mRNA by RT-PCR microarrays and Western analysis. The four cell lines were treated with 1 μmol/L obatoclax (GX15-070) and/or 2 Gy radiation (RT) and monitored for apoptosis after 48 h. Cell lines showing the highest total fold-increase of Bcl-2 family member mRNA, MDA-231 and MDA-468, also showed the highest levels of apoptosis induction (approximately 70% with obatoclax alone and 82% with obatoclax plus RT). Cell lines with little or no increase in Bcl-2 family mRNA (BT-20 and T47-D) showed less apoptosis (30% following treatment with obatoclax and 42% with obatoclax plus RT). RT-PCR arrays can predict the relative apoptosis response of breast cancer cells to the pan Bcl-2 inhibitor obatoclax alone or when combined with radiation.

A threshold mechanism mediates p53 cell fate decision between growth arrest and apoptosis

The p53 tumor suppressor responds to certain cellular stresses by inducing transcriptional programs that can lead to growth arrest or apoptosis. However, the molecular mechanisms responsible for choosing between these two cell fates are not well understood. Previous studies have suggested that p53 selectively activates proarrest target genes, due to the higher affinity of p53 for their promoters compared with proapoptotic genes. Here we show using microarray and chromatin immunoprecipitation that p53 binds to and transcriptionally activates both its proarrest and proapoptotic target genes proportionally to induced p53 expression levels. Further, we provide evidence that to trigger apoptosis, cells must overcome an apoptotic threshold, whose height is determined by expression levels of p53 and its targets, the duration of their expression and the cellular context. We demonstrate in multiple cells lines that below this threshold, expression levels of p53 and its targets were sufficient to induce arrest but not apoptosis. Above this threshold, p53 and its targets triggered extensive apoptosis. Moreover, lowering this threshold with inhibitors of antiapoptotic Bcl-2 family proteins sensitized cells to p53-induced apoptosis. These findings argue that agents that lower the apoptotic threshold should increase the efficacy of p53-mediated cancer therapy.

ApoG2 as the most potent gossypol derivatives inhibits cell growth and induces apoptosis on gastric cancer cells

Gastric cancer is one of the most common types of malignancies and proteins from the Bcl-2 family are highly expressed in human gastric cancer. Apogossypolone (ApoG2), the most potent gossypol derivative, has been defined as a novel small-molecule inhibitor of anti-apoptotic Bcl-2 family proteins. However, whether or not it can inhibit the growth and proliferation of gastric cancer cell lines has not been demonstrated to date. Here, we assessed the effects of anti-growth of ApoG2 on gastric cancer cell lines in vitro and explored the possible molecular mechanisms of ApoG2. Using the MTT assay and flow cytometry, we found that ApoG2 has the significant anti-growth effect on MKN28, MKN45 and AGS cell lines in a time- and dose-dependent manner. Compared to (-)-gossypol, MTT assay and flow cytometry results showed that anti-growth effect of ApoG2 is inferior, but the colony formation ability of ApoG2 is superior. Furthermore, western blot results revealed that ApoG2 inhibits the growth and proliferation of gastric cancer cells by down-regulating of Bcl-2 protein expression, up-regulating of Bax and activating of Caspase-3. Taken together, albeit the ApoG2 inferior to (-)-gossypol in many ways on gastric cancer in vitro, our results suggest that ApoG2 could effectively inhibit the growth and proliferation of gastric cancer cell lines through the mitochondrial pathway of apoptosis.

Sabutoclax, a Novel Pan BCL2 Family Inhibitor, Sensitizes Dormant Blast Crisis Chronic Myeloid Leukemia Stem Cells to Dasatinib

AbstractAbstract 3739Leukemia stem cells (LSC) represent a frequently dormant self-renewing population integral to the initiation, maintenance, and progression of human chromic myeloid leukemia (CML). The current standard of care dasatinib, a BCR-ABL targeted tyrosine kinase inhibitor (TKI), effectively eradicates the bulk of CML cells but frequently fails to affect the LSC population that is thought to drive CML relapse. Members in the BCL2 family are proteins that regulate apoptosis, 6 of which regulate cell survival. Each of these 6 members has a long and short isoform with opposing functions; generally, long isoforms promote cell survival while the short isoforms promote apoptosis. Previously, we demonstrated that upregulation of pro-survival BCL2 proteins in CML LSC contributes to chemotherapy resistance and LSC quiescence in protective hematopoietic niches. LSC found in different hematopoietic niches differ in their response to TKI treatment. Niche affects LSC cell cycle, either by maintaining quiescence or by promoting rapid cell cycling. Quiescent cells are a hurdle for traditional chemotherapy, which usually targets rapidly cycling cells, leaving the quiescent LSC untouched. We hypothesize that the inhibition of pro-survival BCL2 protein family members will sensitize LSC to dasatinib therapy and therefore prevent CML relapse.We tested a novel pan pro-survival BCL2 family protein inhibitor, sabutoclax, delivered by intravenous injection either alone or in combination with oral dasatinib in immunodeficient RAG2−/-gc−/- mice engrafted with BC CML patient samples. After treatment, LSC burden, self-renewal, and cell cycle status were quantified using FACS. Our results showed a reduction in the LSC burden in combination treated mice when compared to mice that received either drug alone. Mice treated with the combination regimen were found to have fewer quiescent human leukemic cells than their counterparts that received single agent treatments. Immunofluorescence staining confirmed the reduction of quiescent cells in the bone marrow after combination treatment when compared to single agent or vehicle treatments. We validated the molecular targets by using human specific splice isoform primers to perform RT-qPCR on FACS sorted LSC and showed a reduction in the BCL2 long to short isoform ratio in sabutoclax versus vehicle treated animals, indicating a skewing towards the pro-apoptotic splice variant. Together, these results indicate that the combination strategy with a pan pro-survival BCL2 family inhibitor and a tyrosine kinase inhibitor may be the foundation for a promising clinical strategy to effectively eliminate LSC and prevent cancer progression and relapse. Disclosures:No relevant conflicts of interest to declare.

Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.

Methylseleninic acid potentiates multiple types of cancer cells to ABT-737-induced apoptosis by targeting Mcl-1 and Bad

ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins, holds great promise to complement current cancer therapies. However many types of solid cancer cells are resistant to ABT-737. One practical approach to improve its therapeutic efficacy is to combine with the agents that can overcome such resistance to restore the sensitivity. In the present study, a second-generation selenium compound methylseleninic acid (MSeA) synergistically sensitized MDA-MB-231 human breast cancer cells, HT-29 human colon cancer cells and DU145 human prostate cancer cells to apoptosis induction by ABT-737, as evidenced by greater than additive enhancement of Annexin V/FITC positive (apoptotic) cells and activation of multiple caspases and PARP cleavage. Mechanistic investigation demonstrated that MSeA significantly decreased basal Mcl-1 expression and ABT-737-induced Mcl-1 expression. Knocking down of Mcl-1 with RNAi approach supported the functional significance of this molecular target. More importantly, we identified inactivation of Bad by phosphorylation on ser-136 and ser-112 as a novel mechanism involved in ABT-737 resistance, which can be overcome by combining with MSeA. In addition, we found that expression of Bax was required for the efficient execution of synergistic sensitization. Our findings, for the first time, provide a strong mechanistic rationale for developing MSeA as a novel sensitizing agent of ABT-737.

Design, synthesis, and activity evaluation of broad-spectrum small-molecule inhibitors of anti-apoptotic Bcl-2 family proteins: Characteristics of broad-spectrum protein binding and its effects on anti-tumor activity

On the basis of the comparison of the structure of the Bim BH3: Bcl-x(L) complex and that of the ABT-737: Bcl-x(L) complex, a series of class A compounds were designed. These compounds had the basic skeleton of ABT-737 and the h2 residues of Bim BH3. These residues had shown themselves to be relevant to Bim BH3's broad-spectrum binding properties in saturation mutagenesis assays. Unlike ABT-737, which is a selective inhibitor of anti-apoptotic members of the Bcl-2 protein family, the class A compounds showed broad-spectrum binding activity to target proteins similar to those of Bim BH3 peptide. Then class B compounds were synthesized by modifying the structure of the most effective class A compound, A-4. Most of these class B compounds showed better binding affinity to the target proteins than the class A compounds had. They also showed themselves more effective than ABT-737 at inhibiting growth in multiple tumor cell lines known to express Bcl-x(L), Bcl-2, and Mcl-1 proteins at high levels. Compounds B-11 and B-12 had the strongest anti-tumor activity of any compounds we produced. This study suggests that it is feasible to design small-molecule inhibitors based on the structure of Bim BH3, which shows broad-spectrum binding to Bcl-x(L), Bcl-2, and Mcl-1 proteins. Our results also suggest that the broad-spectrum properties of small-molecule inhibitors binding to target proteins play a critical role in inhibiting the growth of many tumor cells. Finally, our study provides a series of lead compounds that merit further research into anti-cancer therapeutics.

Synergistic Activity of Fenretinide and the Bcl-2 Family Protein Inhibitor ABT-737 against Human Neuroblastoma

Fenretinide (4-HPR) is a cytotoxic retinoid with minimal systemic toxicity that has shown clinical activity against recurrent high-risk neuroblastoma. To identify possible synergistic drug combinations for future clinical trials, we determined whether ABT-737, a small-molecule BH3-mimetic that inhibits most proteins of the antiapoptotic Bcl-2 family, could enhance 4-HPR activity in neuroblastoma. Eleven neuroblastoma cell lines were tested for the cytotoxic activity of 4-HPR and ABT-737 as single agents and in combination using the DIMSCAN fluorescence digital imaging cytotoxicity assay. The effect of these agents alone and in combination on mitochondrial membrane depolarization and apoptosis (by flow cytometry), cytochrome c release, caspases, Bax-α, t-Bid, and Bak activation, and subcutaneous xenografts in nu/nu mice was also determined. Multilog synergistic cytotoxicity was observed for the drug combination in all of the 11 neuroblastoma cell lines tested, including MDR lines and those insensitive to either drug as single agents. 4-HPR + ABT-737 induced greater mitochondrial membrane depolarization and mitochondrial cytochrome c release, greater activation of caspases, Bax-α, t-Bid, and Bak, and a higher level of apoptosis than either drug alone. In vivo, 4-HPR + ABT-737 increased the event-free survival of the MDR human neuroblastoma line CHLA-119 implanted subcutaneously in nu/nu mice (194.5 days for the combination vs. 68 days for ABT-737 and 99 days for 4-HPR). Thus, the combination of 4-HPR with a BH3-mimetic drug warrants clinical trials in recurrent neuroblastoma.

Inhibition of Rac GTPase signaling and downstream prosurvival Bcl-2 proteins as combination targeted therapy in MLL-AF9 leukemia

AbstractThe Rac family of small Rho GTPases coordinates diverse cellular functions in hematopoietic cells including adhesion, migration, cytoskeleton rearrangements, gene transcription, proliferation, and survival. The integrity of Rac signaling has also been found to critically regulate cellular functions in the initiation and maintenance of hematopoietic malignancies. Using an in vivo gene targeting approach, we demonstrate that Rac2, but not Rac1, is critical to the initiation of acute myeloid leukemia in a retroviral expression model of MLL-AF9 leukemogenesis. However, loss of either Rac1 or Rac2 is sufficient to impair survival and growth of the transformed MLL-AF9 leukemia. Rac2 is known to positively regulate expression of Bcl-2 family proteins toward a prosurvival balance. We demonstrate that disruption of downstream survival signaling through antiapoptotic Bcl-2 proteins is implicated in mediating the effects of Rac2 deficiency in MLL-AF9 leukemia. Indeed, overexpression of Bcl-xL is able to rescue the effects of Rac2 deficiency and MLL-AF9 cells are exquisitely sensitive to direct inhibition of Bcl-2 family proteins by the BH3-mimetic, ABT-737. Furthermore, concurrent exposure to NSC23766, a small-molecule inhibitor of Rac activation, increases the apoptotic effect of ABT-737, indicating the Rac/Bcl-2 survival pathway may be targeted synergistically.

High-Throughput Screen for the Chemical Inhibitors of Antiapoptotic Bcl-2 Family Proteins by Multiplex Flow Cytometry

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

Abstract 4105: Hedgehog inhibition promotes type I TRAIL cytotoxicity in human cholangiocarcinoma cells

Abstract [Background and Aim] TRAIL is a promising therapeutic agent for human malignancies that can activate apoptosis via mitochondria-independent (Type I) or mitochodria-dependent (Type II) death receptor signaling. However, many malignant cells are resistant to TRAIL cytotoxicity and fail to trigger Type I or Type II killing. Using cholangiocarcinoma cells, we identified a role for Hedgehog signaling via GLI transcription factors in the regulation of TRAIL cytotoxicity, and provide new mechanistic insights whereby Hedghog inhibition increases apoptosis. [Materials and Methods] The human cholangiocarcinoma cell lines KMCH, HuCCT-1, and Mz-ChA-1 were employed. Hedgehog signaling was inhibited with cyclopamine or an shRNA construct targeting smootherned (SMO). Type II TRAIL signaling was inhibited with shRNA constructs for Bid or Bim and siRNA for Bax and Bak. Antiapoptotic mediators including Inhibitor of Apoptosis Proteins (IAPs) and Bcl-2 family proteins were examined by immunoblot analysis and/or real time RT-PCR. Apoptosis was quantified morphologically by DAPI and fluorescence microscopy, and biochemically by caspase-3/7 activity. GLI transcription factor binding to the XIAP promoter was examined by EMSA and chromatin immuneprecipitation (ChIP) assay. [Results] Pharmacologic or genetic inhibition of Hedgehog signaling down-regulates both XIAP and cIAP-1 mRNA and protein expression. Knockdown of XIAP, but not cIAP-1, by small-interfering RNA markedly sensitized cells to TRAIL-induced apoptosis, suggesting XIAP is the functional target. Putative GLI-binding sites were identified in the XIAP promoter, and EMSA and ChIP assays demonstrated direct binding by GLI proteins. Sensitization to TRAIL-induced apoptosis was not altered by knockdown of the BH3-only proteins Bim and Bid, or by knockdown of Bax plus Bak, implicating a mitochondria-independent Type I pathway of apoptosis. [Conclusion] These data provide evidence for up-regulation of XIAP by an oncogenic Hedgehog pathway and demonstrate that Hedgehog inhibition sensitizes cells to TRAIL via decreasing XIAP levels. Hedgehog inhibition thereby promotes Type I death receptor signaling and sensitizes cells to apoptosis, which suggests Hedgehog and XIAP inhibition may be a therapeutic approach for TRAIL-resistant neoplasms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2011-4105