Darren C. Phillips

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

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.

Sphingosine-Induced Apoptosis in Rhabdomyosarcoma Cell Lines Is Dependent on Pre-Mitochondrial Bax Activation and Post-Mitochondrial Caspases

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.

A caspase-8-independent component in TRAIL/Apo-2L-induced cell death in human rhabdomyosarcoma cells.

AbstractTumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to the Tumor necrosis factor (TNF) family of death-inducing ligands, and signaling downstream of TRAIL ligation to its receptor(s) remains to be fully elucidated. Components of the death-inducing signaling complex (DISC) and TRAIL signaling downstream of receptor activation were examined in TRAIL - sensitive and -resistant models of human rhabdomyosarcoma (RMS). TRAIL ligation induced DISC formation in TRAIL-sensitive (RD, Rh18, Rh30) and TRAIL-resistant RMS (Rh28, Rh36, Rh41), with recruitment of FADD and procaspase-8. In RD cells, overexpression of dominant-negative FADD (DNFADD) completely abolished TRAIL-induced cell death in contrast to dominant-negative caspase- 8 (DNC8), which only partially inhibited TRAIL-induced apoptosis, growth inhibition, or loss in clonogenic survival. DNC8 did not inhibit the cleavage of Bid or the activation of Bax. Overexpression of Bcl-2 or Bcl-xL inhibited TRAIL-induced apoptosis, growth inhibition, and loss in clonogenic survival. Bcl-2 and Bcl-xL, but not DNC8, inhibited TRAIL-induced Bax activation. Bcl-xL did not inhibit the early activation of caspase-8 (<4 h) but inhibited cleavage of Bid, suggesting that Bid is cleaved downstream of the mitochondria, independent of caspase-8. Exogenous addition of sphingosine also induced activation of Bax via a caspase-8-and Bid-independent mechanism. Further, inhibition of sphingosine kinase completely protected cells from TRAIL-induced apoptosis. Data demonstrate that in RMS cells, the TRAIL signaling pathway circumvents caspase-8 activation of Bid upstream of the mitochondria and that TRAIL acts at the level of the mitochondria via a mechanism that may involve components of the sphingomyelin cycle.

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

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.

Abstract 6: Quantitative assessment of BCL-2:BIM complexes as a pharmacodynamic marker for venetoclax (ABT-199)

The BCL-2-selective inhibitor venetoclax (ABT-199) binds with high affinity to the BH3-binding groove of BCL-2, thereby competing for binding with the BH3-only protein BIM (Souers et al., 2013). Venetoclax is currently being evaluated in clinical trials for CLL, AML, multiple myeloma and NHL. To facilitate these studies, we developed and validated a 384-well electrochemiluminescent ELISA (MSD, Gaithersburg, MD,USA) that quantifies expression of BCL-2, BCL-XL, and MCL-1protein alone or in complex with BIM. We subsequently quantified expression of BCL-2 and BCL-2:BIM complexes in 16 hematologic tumor cell lines. We found the EC50 of venetoclax in these tumor cell lines to correlate strongly with baseline BCL-2:BIM complex levels. This correlation was superior to the correlation between venetoclax EC50 and absolute BCL-2 expression. We also applied the assay to measure disruption of BCL-2:BIM complexes in vivo. Treatment of the Non-Hodgkin9s Lymphoma (NHL) xenograft model SU-DHL-4 with a BCL-2-selective inhibitor resulted in disruption of tumor BCL-2:BIM complexes that aligned with serum and tumor concentrations of inhibitor. Collectively, these data demonstrate that quantifying BCL-2:BIM complexes offers an accurate means of assessing target engagement by venetoclax and, potentially, predicting its efficacy. The utility of this assay is currently being assessed in clinical trials. Citation Format: Sha Jin, Paul Tapang, Donald J. Osterling, Wenqing Gao, Daniel H. Albert, Andrew J. Souers, Joel D. Leverson, Darren C. Phillips, Jun Chen. Quantitative assessment of BCL-2:BIM complexes as a pharmacodynamic marker for venetoclax (ABT-199). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 6. doi:10.1158/1538-7445.AM2015-6