Publications by authors named "Darren C Phillips"

22 Publications

  • Page 1 of 1

Pevonedistat and azacitidine upregulate NOXA () to increase sensitivity to venetoclax in preclinical models of acute myeloid leukemia.

Haematologica 2021 Apr 15. Epub 2021 Apr 15.

Medicine; Program in Cancer Biology, Vanderbilt University School of Medicine; Vanderbilt-Ingram Cancer Center; Center for Immunobiology, Vanderbilt University School of Medicine, Nashville, TN 37232.

Dysregulation of apoptotic machinery is one mechanism by which acute myeloid leukemia (AML) acquires a clonal survival advantage. B-cell lymphoma protein-2 (BCL2) overexpression is a common feature in hematologic malignancies. The selective BCL2 inhibitor, venetoclax (VEN) is used in combination with azacitidine (AZA), a DNA-methyltransferase inhibitor (DNMTi), to treat patients with AML. Despite promising response rates to VEN/AZA, resistance to the agent is common. One identified mechanism of resistance is the upregulation of myeloid cell leukemia-1 protein (MCL1). Pevonedistat (PEV), a novel agent that inhibits NEDD8-activating enzyme, and AZA both upregulate NOXA (PMAIP1), a BCL2 family protein that competes with effector molecules at the BH3 binding site of MCL1. We demonstrate that PEV/AZA combination induces NOXA to a greater degree than either PEV or AZA alone, which enhances VEN-mediated apoptosis. Herein, using AML cell lines and primary AML patient samples ex vivo, including in cells with genetic alterations linked to treatment resistance, we demonstrate robust activity of the PEV/VEN/AZA triplet. These findings were corroborated in preclinical systemic engrafted models of AML. Collectively, these results provide preclinical rational for combining PEV/VEN/AZA as a novel therapeutic approach in overcoming AML resistance current therapies.
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http://dx.doi.org/10.3324/haematol.2020.272609DOI Listing
April 2021

Expanding the repertoire for "large small molecules" : Prodrug ABBV-167 efficiently converts to venetoclax with reduced food effect in healthy volunteers.

Mol Cancer Ther 2021 Mar 30. Epub 2021 Mar 30.

Oncology Discovery, AbbVie, Inc.

Since gaining approval for the treatment of chronic lymphocytic leukemia (CLL), the BCL-2 inhibitor venetoclax has transformed the treatment of this and other blood-related cancers. Reflecting the large and hydrophobic BH3-binding groove within BCL-2, venetoclax has significantly higher molecular weight and lipophilicity than most orally administered drugs, along with negligible water solubility. While a technology-enabled formulation successfully achieves oral absorption in humans, venetoclax tablets have limited drug loading and therefore can present a substantial pill burden for patients in high-dose indications. We therefore generated a phosphate prodrug (3, ABBV-167) that confers significantly increased water solubility to venetoclax and, upon oral administration to healthy volunteers either as a solution or high drug-load immediate release tablet, extensively converts to the parent drug. Additionally, ABBV-167 demonstrated a lower food effect with respect to venetoclax tablets. These data indicate that beyond-rule-of-5 molecules can be successfully delivered to humans via a solubility-enhancing prodrug moiety to afford robust exposures of the parent drug following oral dosing.
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http://dx.doi.org/10.1158/1535-7163.MCT-21-0077DOI Listing
March 2021

Hexavalent TRAIL fusion protein eftozanermin alfa optimally clusters apoptosis-inducing TRAIL receptors to induce on-target antitumor activity in solid tumors.

Cancer Res 2021 Mar 9. Epub 2021 Mar 9.

Oncology Discovery, AbbVie Inc.

TNF Receptor Apoptosis-Inducing Ligand (TRAIL) can activate cell surface death receptors resulting in potent tumor cell death via induction of the extrinsic apoptosis pathway. Eftozanermin alfa (ABBV-621) is a second-generation TRAIL receptor agonist engineered as an IgG1-Fc mutant backbone linked to two sets of trimeric native single chain TRAIL receptor binding domain monomers. This hexavalent agonistic fusion protein binds to the death-inducing DR4 and DR5 receptors with nanomolar affinity to drive on-target biological activity with enhanced caspase-8 aggregation and DISC formation independent of FcγR-mediated cross-linking, and without clinical signs or pathological evidence of toxicity in non-rodent species. ABBV-621 induced cell death in approximately 36% (45/126) of solid cancer cell lines in vitro at sub-nanomolar concentrations. An in vivo patient-derived xenograft (PDX) screen of ABBV-621 activity across 15 different tumor indications resulted in an overall response (OR) of 29% (47/162). Although DR4 (TNFSFR10A) and/or DR5 (TNFSFR10B) expression levels did not predict the level of response to ABBV-621 activity in vivo, KRAS mutations were associated with elevated TNFSFR10A and TNFSFR10B and were enriched in ABBV-621 responsive colorectal carcinoma (CRC) PDX models. To build upon the OR of ABBV-621 monotherapy in CRC (45%; 10/22) and pancreatic cancer (35%; 7/20), we subsequently demonstrated that inherent resistance to ABBV-621 treatment could be overcome in combination with chemotherapeutics or with selective inhibitors of BCL-XL. In summary, these data provide a pre-clinical rationale for the ongoing Phase-1 clinical trial (NCT03082209) evaluating the activity of ABBV-621 in cancer patients.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2178DOI Listing
March 2021

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-X Inhibitor.

ACS Med Chem Lett 2020 Oct 30;11(10):1829-1836. Epub 2020 Mar 30.

AbbVie Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, United States.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-X inhibitor that selectively and potently induces apoptosis in BCL-X-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-X inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-X. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549103PMC
October 2020

5-Azacitidine Induces NOXA to Prime AML Cells for Venetoclax-Mediated Apoptosis.

Clin Cancer Res 2020 07 13;26(13):3371-3383. Epub 2020 Feb 13.

Oncology Discovery, AbbVie Inc., North Chicago, Illinois.

Purpose: Patients with acute myeloid leukemia (AML) frequently do not respond to conventional therapies. Leukemic cell survival and treatment resistance have been attributed to the overexpression of B-cell lymphoma 2 (BCL-2) and aberrant DNA hypermethylation. In a phase Ib study in elderly patients with AML, combining the BCL-2 selective inhibitor venetoclax with hypomethylating agents 5-azacitidine (5-Aza) or decitabine resulted in 67% overall response rate; however, the underlying mechanism for this activity is unknown.

Experimental Design: We studied the consequences of combining two therapeutic agents, venetoclax and 5-Aza, in AML preclinical models and primary patient samples. We measured expression changes in the integrated stress response (ISR) and the BCL-2 family by Western blot and qPCR. Subsequently, we engineered (NOXA)- and (PUMA)-deficient AML cell lines using CRISPR- methods to understand their respective roles in driving the venetoclax/5-Aza combinatorial activity.

Results: In this study, we demonstrate that venetoclax and 5-Aza act synergistically to kill AML cells and display combinatorial antitumor activity . We uncover a novel nonepigenetic mechanism for 5-Aza-induced apoptosis in AML cells through transcriptional induction of the proapoptotic BH3-only protein NOXA. This induction occurred within hours of treatment and was mediated by the ISR pathway. NOXA was detected in complex with antiapoptotic proteins, suggesting that 5-Aza may be "priming" the AML cells for venetoclax-induced apoptosis. knockout confirmed its major role in driving venetoclax and 5-Aza synergy.

Conclusions: These data provide a novel nonepigenetic mechanism of action for 5-Aza and its combinatorial activity with venetoclax through the ISR-mediated induction of .
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1900DOI Listing
July 2020

A novel CDK9 inhibitor increases the efficacy of venetoclax (ABT-199) in multiple models of hematologic malignancies.

Leukemia 2020 06 11;34(6):1646-1657. Epub 2019 Dec 11.

Oncology-Discovery, AbbVie Inc., 1 North Waukegan Road, North Chicago, IL, 60064, USA.

MCL-1 is one of the most frequently amplified genes in cancer, facilitating tumor initiation and maintenance and enabling resistance to anti-tumorigenic agents including the BCL-2 selective inhibitor venetoclax. The expression of MCL-1 is maintained via P-TEFb-mediated transcription, where the kinase CDK9 is a critical component. Consequently, we developed a series of potent small-molecule inhibitors of CDK9, exemplified by the orally active A-1592668, with CDK selectivity profiles that are distinct from related molecules that have been extensively studied clinically. Short-term treatment with A-1592668 rapidly downregulates RNA pol-II (Ser 2) phosphorylation resulting in the loss of MCL-1 protein and apoptosis in MCL-1-dependent hematologic tumor cell lines. This cell death could be attenuated by either inhibiting caspases or overexpressing BCL-2 protein. Synergistic cell killing was also observed between A-1592668 or the related analog A-1467729, and venetoclax in a number of hematologic cell lines and primary NHL patient samples. Importantly, the CDK9 inhibitor plus venetoclax combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in mouse models of lymphoma and AML. These data indicate that CDK9 inhibitors could be highly efficacious in tumors that depend on MCL-1 for survival or when used in combination with venetoclax in malignancies dependent on MCL-1 and BCL-2.
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http://dx.doi.org/10.1038/s41375-019-0652-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266741PMC
June 2020

Concomitant targeting of BCL2 with venetoclax and MAPK signaling with cobimetinib in acute myeloid leukemia models.

Haematologica 2020 03 23;105(3):697-707. Epub 2019 May 23.

Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

The pathogenesis of acute myeloid leukemia (AML) involves serial acquisition of mutations controlling several cellular processes, requiring combination therapies affecting key downstream survival nodes in order to treat the disease effectively. The BCL2 selective inhibitor venetoclax has potent anti-leukemia efficacy; however, resistance can occur due to its inability to inhibit MCL1, which is stabilized by the MAPK pathway. In this study, we aimed to determine the anti-leukemia efficacy of concomitant targeting of the BCL2 and MAPK pathways by venetoclax and the MEK1/2 inhibitor cobimetinib, respectively. The combination demonstrated synergy in seven of 11 AML cell lines, including those resistant to single agents, and showed growth-inhibitory activity in over 60% of primary samples from patients with diverse genetic alterations. The combination markedly impaired leukemia progenitor functions, while maintaining normal progenitors. Mass cytometry data revealed that BCL2 protein is enriched in leukemia stem/progenitor cells, primarily in venetoclax-sensitive samples, and that cobimetinib suppressed cytokine-induced pERK and pS6 signaling pathways. Through proteomic profiling studies, we identified several pathways inhibited downstream of MAPK that contribute to the synergy of the combination. In OCI-AML3 cells, the combination downregulated MCL1 protein levels and disrupted both BCL2:BIM and MCL1:BIM complexes, releasing BIM to induce cell death. RNA sequencing identified several enriched pathways, including MYC, mTORC1, and p53 in cells sensitive to the drug combination. , the venetoclax-cobimetinib combination reduced leukemia burden in xenograft models using genetically engineered OCI-AML3 and MOLM13 cells. Our data thus provide a rationale for combinatorial blockade of MEK and BCL2 pathways in AML.
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http://dx.doi.org/10.3324/haematol.2018.205534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049339PMC
March 2020

Acquired resistance to venetoclax (ABT-199) in t(14;18) positive lymphoma cells.

Oncotarget 2016 Oct;7(43):70000-70010

Department of Laboratory Medicine, Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, USA.

The chromosomal translocation t(14;18) in follicular lymphoma (FL) is a primary oncogenic event resulting in BCL-2 over-expression. This study investigates activity of the BH3 mimetic venetoclax (ABT-199), which targets BCL-2, and mechanisms of acquired resistance in FL.The sensitivity of FL cells to venetoclax treatment correlated with BCL-2/BIM ratio. Cells with similar expression of anti-apoptotic proteins, but with higher levels of BIM were more sensitive to the treatment. Venetoclax induced dissociation of BCL-2/ BIM complex and a decrease in mitochondrial potential. Interestingly the population of cells that survived venetoclax treatment showed increased p-ERK1/2 and p-BIM (S69), as well as a decrease in total BIM levels. Venetoclax resistant cells initially showed elevated levels of p-AKT and p-Foxo1/3a, a dissociation of BIM/BCL-2/BECLIN1 complex, and a decrease in SQSTM1/p62 level (indicating increased autophagy) together with a slight decline in BIM expression. After stable resistant cell lines were established, a significant reduction of BCL-2 levels and almost total absence of BIM was observed.The acquisition of these resistance phenotypes could be prevented via selective ERK/AKT inhibition or anti-CD20 antibody treatment, thus highlighting possible combination therapies for FL patients.
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http://dx.doi.org/10.18632/oncotarget.12132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342530PMC
October 2016

MCL-1 Is a Key Determinant of Breast Cancer Cell Survival: Validation of MCL-1 Dependency Utilizing a Highly Selective Small Molecule Inhibitor.

Mol Cancer Ther 2015 Aug 26;14(8):1837-47. Epub 2015 May 26.

Oncology Discovery, AbbVie Inc., North Chicago, Illinois.

Hyperexpression of antiapoptotic BCL-2 family proteins allows cells to survive despite the receipt of signals that would ordinarily induce their deletion, a facet frequently exploited by tumors. Tumors addicted to the BCL-2 family proteins for survival are now being targeted therapeutically. For example, navitoclax, a BCL-2/BCL-XL/BCL-W inhibitor, is currently in phase I/II clinical trials in numerous malignancies. However, the related family member, MCL-1, limits the efficacy of navitoclax and other chemotherapeutic agents. In the present study, we identify breast cancer cell lines that depend upon MCL-1 for survival and subsequently determine the mechanism of apoptosis mediated by the MCL-1 selective inhibitor A-1210477. We demonstrate that apoptosis resulting from a loss in MCL-1 function requires expression of the proapoptotic protein BAK. However, expression of BCL-XL can limit apoptosis resulting from loss in MCL-1 function through sequestration of free BIM. Finally, we demonstrate substantial synergy between navitoclax and MCL-1 siRNA, the direct MCL-1 inhibitor A-1210477, or the indirect MCL-1 inhibitor flavopiridol, highlighting the therapeutic potential for inhibiting BCL-XL and MCL-1 in breast cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-14-0928DOI Listing
August 2015

Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy.

Sci Transl Med 2015 Mar;7(279):279ra40

AbbVie Inc., North Chicago, IL 60064, USA.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.
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http://dx.doi.org/10.1126/scitranslmed.aaa4642DOI Listing
March 2015

Structure-guided design of a series of MCL-1 inhibitors with high affinity and selectivity.

J Med Chem 2015 Mar 26;58(5):2180-94. Epub 2015 Feb 26.

AbbVie Inc. , 1 North Waukegan Road, North Chicago, Illinois 60064, United States.

Myeloid cell leukemia 1 (MCL-1) is a BCL-2 family protein that has been implicated in the progression and survival of multiple tumor types. Herein we report a series of MCL-1 inhibitors that emanated from a high throughput screening (HTS) hit and progressed via iterative cycles of structure-guided design. Advanced compounds from this series exhibited subnanomolar affinity for MCL-1 and excellent selectivity over other BCL-2 family proteins as well as multiple kinases and GPCRs. In a MCL-1 dependent human tumor cell line, administration of compound 30b rapidly induced caspase activation with associated loss in cell viability. The small molecules described herein thus comprise effective tools for studying MCL-1 biology.
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http://dx.doi.org/10.1021/jm501258mDOI Listing
March 2015

Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity.

ACS Med Chem Lett 2014 Oct 26;5(10):1088-93. Epub 2014 Aug 26.

AbbVie, Inc. , 1 North Waukegan Road, North Chicago, Illinois 60064 United States.

A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.
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http://dx.doi.org/10.1021/ml5001867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190639PMC
October 2014

ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets.

Nat Med 2013 Feb 6;19(2):202-8. Epub 2013 Jan 6.

AbbVie Inc., North Chicago, Illinois, USA.

Proteins in the B cell CLL/lymphoma 2 (BCL-2) family are key regulators of the apoptotic process. This family comprises proapoptotic and prosurvival proteins, and shifting the balance toward the latter is an established mechanism whereby cancer cells evade apoptosis. The therapeutic potential of directly inhibiting prosurvival proteins was unveiled with the development of navitoclax, a selective inhibitor of both BCL-2 and BCL-2-like 1 (BCL-X(L)), which has shown clinical efficacy in some BCL-2-dependent hematological cancers. However, concomitant on-target thrombocytopenia caused by BCL-X(L) inhibition limits the efficacy achievable with this agent. Here we report the re-engineering of navitoclax to create a highly potent, orally bioavailable and BCL-2-selective inhibitor, ABT-199. This compound inhibits the growth of BCL-2-dependent tumors in vivo and spares human platelets. A single dose of ABT-199 in three patients with refractory chronic lymphocytic leukemia resulted in tumor lysis within 24 h. These data indicate that selective pharmacological inhibition of BCL-2 shows promise for the treatment of BCL-2-dependent hematological cancers.
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http://dx.doi.org/10.1038/nm.3048DOI Listing
February 2013

Free radicals and redox signalling in T-cells during chronic inflammation and ageing.

Biochem Soc Trans 2011 Oct;39(5):1273-8

School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK.

During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2',7'-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.
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http://dx.doi.org/10.1042/BST0391273DOI Listing
October 2011

Aberrant reactive oxygen and nitrogen species generation in rheumatoid arthritis (RA): causes and consequences for immune function, cell survival, and therapeutic intervention.

Antioxid Redox Signal 2010 Mar;12(6):743-85

Life and Health Sciences, Aston University, Birmingham B4 7ET, West Midlands, United Kingdom.

The infiltration and persistence of hematopoietic immune cells within the rheumatoid arthritis (RA) joint results in elevated levels of pro-inflammatory cytokines, increased reactive oxygen (ROS) and -nitrogen (RNS) species generation, that feeds a continuous self-perpetuating cycle of inflammation and destruction. Meanwhile, the controlled production of ROS is required for signaling within the normal physiological reaction to perceived "foreign matter" and for effective apoptosis. This review focuses on the signaling pathways responsible for the induction of the normal immune response and the contribution of ROS to this process. Evidence for defects in the ability of immune cells in RA to regulate the generation of ROS and the consequence for their immune function and for RA progression is considered. As the hypercellularity of the rheumatoid joint and the associated persistence of hematopoietic cells within the rheumatoid joint are symptomatic of unresponsiveness to apoptotic stimuli, the role of apoptotic signaling proteins (specifically Bcl-2 family members and the tumor suppressor p53) as regulators of ROS generation and apoptosis are considered, evaluating evidence for their aberrant expression and function in RA. We postulate that ROS generation is required for effective therapeutic intervention.
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http://dx.doi.org/10.1089/ars.2009.2607DOI Listing
March 2010

Assays to measure p53-dependent and -independent apoptosis.

Methods Mol Biol 2009 ;559:143-59

Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN, USA.

Paramount to the maintenance of normal tissue homeostasis is the induction of programmed cell death, otherwise known as apoptosis. Several disease states, including cancer, are characterized by an inability to remove unwanted cells due to a failure to commit to apoptosis. What is more, apoptosis is the central functional response behind many agents utilized in the treatment of cancer. Many of these antitumorigenic agents rely on the activation of the tumor suppressor p53. As the physiological "guardian of the genome," p53's normal function is to sense stressed or damaged cells and arrest proliferation, allowing time for cellular repair. However, if the damage is excessive, cells are removed prior to the onset of malignancy through apoptosis. Current chemotherapeutic strategies manipulate this property by damaging cells and turning on p53's transcriptional function, which consequently upregulates the expression of proapoptotic proteins such as Puma. We have also demonstrated that Puma is capable of inducing apoptosis independent of p53. In this regard, defects in the apoptotic machinery or in p53 function itself lead to a resistant phenotype that in cancer results in chemotherapeutic failure, and more often than not, poor prognosis. This chapter describes protocols for the determination of p53-dependent and -independent apoptosis utilizing primary cells from genetically altered mice.
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http://dx.doi.org/10.1007/978-1-60327-017-5_11DOI Listing
October 2009

Induction of apoptosis promoted by Bang52; a small molecule that downregulates Bcl-x(L).

Bioorg Med Chem Lett 2009 May 21;19(9):2429-34. Epub 2009 Mar 21.

Laboratory of Cell Biology, NCI, NIH, Bethesda, MD 20892, USA.

Cancer cells evade death by over-producing specific proteins that inhibit apoptosis. One such group of proteins is the Bcl-2 family, of which Bcl-x(L) is an important member. This protein binds and inhibits BAK, another protein that promotes apoptosis. While the development of chemical inhibitors that block Bcl-x(L)-BAK association have been the focus of intense research efforts, we demonstrate in this manuscript an alternative strategy to downregulate Bcl-x(L). We have identified a small molecule (Bang52) that induces apoptosis in a lymphoblast-derived cell line by lowering levels of Bcl-x(L). Since Bang52 bears no resemblance to any chemical binder of Bcl-x(L) we believe that degradation of the protein is stimulated by a new type of pathway. These findings highlight a novel approach to the development of small molecules that promote apoptosis.
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http://dx.doi.org/10.1016/j.bmcl.2009.03.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808702PMC
May 2009

Sphingosine-induced apoptosis in rhabdomyosarcoma cell lines is dependent on pre-mitochondrial Bax activation and post-mitochondrial caspases.

Cancer Res 2007 Jan;67(2):756-64

Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA, and UMR 7175-LC1, Pharmacologie et Physicochimie, Faculté de Pharmacie, Université Louis Pasteur, Illkirch, France.

Sphingolipids is the collective term ascribed to components of the sphingomyelin cycle. Modulation of the cellular levels of individual sphingolipids can induce a diverse range of cellular responses including apoptosis, proliferation, and cell cycle arrest. We present data showing that rhabdomyosarcoma cell lines, independent of lineage (alveolar rhabdomyosarcoma and embryonal rhabdomyosarcoma), are particularly sensitive to the induction of apoptosis as a result of an elevation in the cellular levels of sphingosine (D-erythro-sphingosine). Sphingosine-mediated apoptosis does not require its metabolism to the related proapoptotic molecule ceramide and is stereospecific because exposure of the rhabdomyosarcoma cell line RD to the L-erythro and DL-threo isoforms of sphingosine did not induce apoptosis. Importantly, for efficient induction of apoptosis, sphingosine required Bax activation and consequential translocation to the mitochondria. This resulted in selective mitochondrial release of cytochrome c and Smac/Diablo but not other mitochondrial related factors (apoptosis-inducing factor, endonuclease G, and HtrA2/Omi). Using small interfering RNA, reduced Bax expression conferred the impaired release of mitochondrial cytochrome c to the cytoplasm following sphingosine exposure, inhibiting the induction of apoptosis. Furthermore, dissipation of the inner mitochondrial membrane potential and enhanced production of reactive oxygen species were not observed. Bax activation and cytochrome c release were independent of caspases; however, caspase-3 and caspase-9 activity distal to the mitochondria was essential for the execution of apoptosis.
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http://dx.doi.org/10.1158/0008-5472.CAN-06-2374DOI Listing
January 2007

Cyclooxygenase-2 inhibition sensitizes human colon carcinoma cells to TRAIL-induced apoptosis through clustering of DR5 and concentrating death-inducing signaling complex components into ceramide-enriched caveolae.

Cancer Res 2005 Dec;65(24):11447-58

Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Cyclooxygenase-2 (COX-2) is up-regulated in human colon carcinomas, and its inhibition is associated with a reduction in tumorigenesis and a promotion of apoptosis. However, the mechanisms responsible for the antitumor effects of COX-2 inhibitors and how COX-2 modulates apoptotic signaling have not been clearly defined. We have shown that COX-2 inhibition sensitizes human colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by inducing clustering of the TRAIL receptor DR5 at the cell surface and the redistribution of the death-inducing signaling complex components (DR5, FADD, and procaspase-8) into cholesterol-rich and ceramide-rich domains known as caveolae. This process requires the accumulation of arachidonic acid and sequential activation of acid sphingomyelinase for the generation of ceramide within the plasma membrane outer leaflet. The current study highlights a novel mechanism to circumvent colorectal carcinoma cell resistance to TRAIL-mediated apoptosis using COX-2 inhibitors to manipulate the lipid metabolism within the plasma membrane.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-1494DOI Listing
December 2005

Ceramide induces a loss in cytosolic peroxide levels in mononuclear cells.

Biochem J 2003 Nov;375(Pt 3):567-79

Molecular Biosciences Group, School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK.

Ceramide (a sphingolipid) and reactive oxygen species are each partly responsible for intracellular signal transduction in response to a variety of agents. It has been reported that ceramide and reactive oxygen species are intimately linked and show reciprocal regulation [Liu, Andreieu-Abadie, Levade, Zhang, Obeid and Hannun (1998) J. Biol. Chem. 273, 11313-11320]. Utilizing synthetic, short-chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide formation or using stimulation of CD95 to induce ceramide formation, we found that the principal redox-altering property of ceramide is to lower the [peroxide](cyt) (cytosolic peroxide concentration). Apoptosis of Jurkat T-cells, primary resting and phytohaemagglutinin-activated human peripheral blood T-lymphocytes was preceded by a loss in [peroxide](cyt), as measured by the peroxide-sensitive probe 2',7'-dichlorofluorescein diacetate (also reflected in a lower rate of superoxide dismutase-inhibitable cytochrome c reduction), and this was not associated with a loss of membrane integrity. Where growth arrest of U937 monocytes was observed without a loss of membrane integrity, the decrease in [peroxide](cyt) was of a lower magnitude when compared with that preceding the onset of apoptosis in T-cells. Furthermore, decreasing the cytosolic peroxide level in U937 monocytes before the application of synthetic ceramide by pretreatment with either of the antioxidants N -acetyl cysteine or glutathione conferred apoptosis. However, N -acetyl cysteine or glutathione did not affect the kinetics or magnitude of ceramide-induced apoptosis of Jurkat T-cells. Therefore the primary redox effect of cellular ceramide accumulation is to lower the [peroxide](cyt) of both primary and immortalized cells, the magnitude of which dictates the cellular response.
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http://dx.doi.org/10.1042/BJ20030693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223707PMC
November 2003

The anti-inflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species.

Br J Pharmacol 2003 Feb;138(3):501-11

Molecular Biosciences Group, Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET.

1 The mechanism of action by which methotrexate (MTX) exerts its anti-inflammatory and immunosuppressive effects remains unclear. The aim of this study is to investigate the hypothesis that MTX exerts these effects via the production of reactive oxygen species (ROS). 2 Addition of MTX (100 nM-10 micro M) to U937 monocytes induced a time and dose dependent increase in cytosolic peroxide [peroxide](cyt) from 6-16 h. MTX also caused corresponding monocyte growth arrest, which was inhibited (P<0.05) by pre-treatment with N-acetylcysteine (NAC; 10 mM) or glutathione (GSH; 10 mM). In contrast, MTX induction of [peroxide](cyt) in Jurkat T cells was more rapid (4 h; P<0.05), but was associated with significant apoptosis at 16 h at all doses tested (P<0.05) and was significantly inhibited by NAC or GSH (P<0.05). 3 MTX treatment of monocytes (10 nM-10 micro M) for 16 h significantly reduced total GSH levels (P<0.05) independently of dose (P>0.05). However, in T-cells, GSH levels were significantly elevated following 30 nM MTX treatment (P<0.05) but reduced by doses exceeding 1 micro M compared to controls (P<0.05). 4 MTX treatment significantly reduced monocyte adhesion to 5 h and 24 h LPS (1 micro g ml(-1)) activated human umbilical vein endothelial cells (HUVEC; P<0.05) but not to resting HUVEC. Pre-treatment with GSH prevented MTX-induced reduction in adhesion. 5 In conclusion, ROS generation by MTX is important for cytostasis in monocytes and cytotoxicity T-cells. Furthermore, MTX caused a reduction in monocyte adhesion to endothelial cells, where the mechanism of MTX action requires the production of ROS. Therefore its clinical efficacy can be attributed to multiple targets.
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http://dx.doi.org/10.1038/sj.bjp.0705054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1573681PMC
February 2003