Overexpression Of Bcl-2 Family Members Research Articles

Abstract 2961: deBouganin conjugated to trastuzumab overcomes multiple mechanisms of T-DM1 drug resistance

Abstract DeBouganin is a T-cell epitope-depleted variant of the type I Ribosome Inactivating Protein (RIP) plant toxin Bouganin. DeBouganin binds and deadenylates the sarcin/ricin loop of the 28S subunit of the ribosomal RNA leading to protein synthesis inhibition and apoptosis. To demonstrate the potential advantages of deBouganin over current small molecule payloads, deBouganin was randomly chemically conjugated to trastuzumab with a DAR of approximately 2 and compared to T-DM1 both in vitro and in vivo. The trastuzumab-deBouganin conjugate (T-deB) demonstrated a tighter IC50 range of killing and an overall greater potency in vitro against most cells lines with high levels of Her2 expression as compared to T-DM1. In addition, unlike T-DM1, T-deB was unaffected by inhibitors of multidrug resistance (MDR) and overexpression of Bcl-2 family members. Moreover, T-deB potency was unchanged by Her2-Her3 dimerization in the presence of heregulin. Contrary to T-DM1 which showed only minimal cytotoxicity, T-deB was highly potent in vitro against tumor cells with cancer stem cell (CSC) properties by preventing the formation of tumorspheres. Furthermore, in a BT-474 xenograft study, T-deB was more efficacious than T-DM1 resulting in increased survival of the T-deB treated mice. Overall, the results demonstrate the potency and efficacy of deBouganin and emphasize the importance of using payloads with different MOAs. The data suggest that deBouganin used alone or in combination with other payloads could be a highly effective cancer therapeutic that would provide prolonged clinical benefit. Citation Format: Rachelle L. Dillon, Shilpa Chooniedass, Arjune Premsukh, Gregory P. Adams, Joycelyn Entwistle, Glen C. MacDonald, Jeannick Cizeau. deBouganin conjugated to trastuzumab overcomes multiple mechanisms of T-DM1 drug resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2961.

Abstract 2011: A novel bcl-2/xL inhibitor induces tumor cell apoptosis and potentiates the activity of standard chemotherapeutics

Abstract Tumor cells often evade apoptosis by tipping the balance of Bcl-2 family and BH3-only protein expression towards a more anti-apoptotic environment, thus providing a survival advantage as well as a means of resistance to anti-cancer therapies. Indeed, over-expression of Bcl-2 family members has been shown to confer chemo-resistance in multiple tumor types. One potential result of this survival adaptation, however, is that the tumor cells become dependent on said Bcl-2 family member and hence increasingly vulnerable to targeted pro-apoptotic BH3 mimetics. Clinical validation of this concept has been demonstrated with the Bcl-2/xL inhibitor Navitoclax, where as a single agent in phase I/II trials in relapsed, refractory CLL patients, a 20% response rate was observed. Due to the potential that directly targeting the apoptotic machinery holds in treating cancer, developing BH3 mimetics that easily and effectively combine with standard chemotherapies is an attractive proposition. With this in mind, we have discovered and characterized a novel small molecule BH3 mimetic with nanomolar affinity for Bcl-2 and Bcl-xL. Here we characterize the in vitro and in vivo activity of this inhibitor in a variety of cell types. In addition to exhibiting potent single agent anti-cancer activity, it also potentiates the effectiveness of standard chemotherapeutic agents. Importantly, it also induces regression of sensitive tumors in vivo after a single IV bolus dose. Overall, this novel Bcl-2/xL inhibitor represents an exciting tool to further explore the mechanistic rationale for using potent BH3 mimetics as a foundation therapy across a broad range of tumor types where resistance to standard therapies arises from deregulation of the Bcl-2 family of anti-apoptotic proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2011. doi:1538-7445.AM2012-2011

Abstract C14: Pim kinase inhibitors sensitize prostate cancer cells to apoptosis triggered by Bcl-2 family inhibitor ABT-737

Abstract Pim serine/threonine kinases contribute to prostate tumorigenesis and therapeutic resistance, yet Pim kinase inhibitors appear to have only limited effects on prostate cancer cell survival. Since overexpression of Bcl-2 family members are implicated in chemotherapeutic resistance in prostate cancer, we investigated the cooperative effects of Pim kinase inhibition with ABT-737, a small molecule antagonist of Bcl-2 family members. Strikingly, the addition of ABT-737 to Pim inhibitors triggered a robust apoptosis of prostate cancer cells in vitro and in vivo. Pim inhibitors decreased levels of the Bcl-2 family member Mcl-1, both by blocking 5′- cap dependent translation and decreasing protein half life. Additionally, Pim inhibition transcriptionally increased levels of the BH3 protein Noxa by activating the unfolded protein response (UPR), lead to eIF-2α phosphorylation and increased expression of CHOP. Increased levels of Noxa also inactivated the remaining levels of Mcl-1 protein activity. Notably, these specific protein changes were essential to the apoptotic process because ABT-737 did not inhibit Mcl-1 protein activity and Mcl-1 overexpression blocked the apoptotic activity of ABT-737. Our results therefore suggest that this combination treatment could be developed as a potential therapy for human prostate cancer where overexpression of Pim kinases and antiapoptotic Bcl-2 family members drives tumor cell resistance to current anticancer therapies. Citation Format: Jin H. Song, Andrew S. Kraft. Pim kinase inhibitors sensitize prostate cancer cells to apoptosis triggered by Bcl-2 family inhibitor ABT-737 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C14.

Pim kinase inhibitors sensitize prostate cancer cells to apoptosis triggered by Bcl-2 family inhibitor ABT-737.

Pim serine/threonine kinases contribute to prostate tumorigenesis and therapeutic resistance, yet Pim kinase inhibitors seem to have only limited effects on prostate cancer cell survival. Because overexpression of Bcl-2 family members are implicated in chemotherapeutic resistance in prostate cancer, we investigated the cooperative effects of Pim kinase inhibition with ABT-737, a small molecule antagonist of Bcl-2 family members. Strikingly, the addition of ABT-737 to Pim inhibitors triggered a robust apoptosis of prostate cancer cells in vitro and in vivo. Pim inhibitors decreased levels of the Bcl-2 family member Mcl-1, both by blocking 5'-cap dependent translation and decreasing protein half life. In addition, Pim inhibition transcriptionally increased levels of the BH3 protein Noxa by activating the unfolded protein response (UPR), lead to eIF-2α phosphorylation and increased expression of CHOP. Increased levels of Noxa also inactivated the remaining levels of Mcl-1 protein activity. Notably, these specific protein changes were essential to the apoptotic process because ABT-737 did not inhibit Mcl-1 protein activity and Mcl-1 overexpression blocked the apoptotic activity of ABT-737. Our results therefore suggest that this combination treatment could be developed as a potential therapy for human prostate cancer where overexpression of Pim kinases and antiapoptotic Bcl-2 family members drives tumor cell resistance to current anticancer therapies.

A Phase I Trial of the Small Molecule Pan-Bcl-2 Family Inhibitor Obatoclax Mesylate (GX15-070) Administered by Continuous Infusion for up to Four Days to Patients with Hematological Malignancies.

Obatoclax antagonizes the BH3-binding groove of the bcl-2 family of anti apoptotic proteins. It is active in chronic lymphocytic leukemia(CLL; O’Brien et al, ASH 2005) with a recommended phase II dose of 28 mg/m2 given over 3 h every 3 weeks with DLT of grade 3 infusional CNS toxicities. We evaluated prolonged infusions to minimize these toxicities while maintaining clinical activity. An initial trial established a Phase II dose of 28 mg/m2 over 24 h every 2 weeks with dose limiting toxicities (DLTs) at 40 mg/m2 and responses in 4/8 MDS patients (Borthakur et al., ASH 2006). We report on 21 patients who received obatoclax 20–28 mg/m2/24 h weekly (n=9) or 20 mg/m2/24 h for 2, 3 or 4 consecutive days every 2–3 weeks (n=12). The final dose level of 28 mg/m2/24 h for 4 days is now open. Median age was 61 (range 26–75) and 11 patients were male. Diagnoses included: acute myelogenous leukemia (AML; 13), myelodysplastic syndromes (MDS; 6), acute lymphocytic leukemia (ALL; 1) and CLL (1). A total of 180 infusions of 24 h were administered. The most common adverse events (AE) were euphoric mood (57%), fatigue (57%), febrile neutropenia (43%), gait disturbance (43%), chills (33%), diarrhea (33%), nausea (33%), somnolence (33%), dizziness (29%). All were of Grades 1–2 with the exception of 7 grade 3 AEs of febrile neutropenia (unrelated to obatoclax administration) and 1 each of diarrhea, fatigue and gait disturbance. There were no DLTs. Multiple day infusions were well tolerated; for the 20 mg/m2/24 h x 4days dose level, 4/6 patients received ≥ 4 cycles, with two ongoing at this time. Plasma concentrations of obatoclax reached a steady state before end of infusion. Mean Cmax values at 20 mg/m2/24 h during1x24, 2x24, 3x24,4x24 h, and 28 mg/m2/24 h during 1x24 h infusions was 15.3, 8.4, 10.1, 15.7 and 10.8 ng/mL, respectively. The mean AUC(0–tlast) values at 20 mg/m2/24 h associated with 1x24, 2x24, 3x24, 4x24 h, and 28 mg/m2/24 h associated with 1x24 h infusions were 264.3, 396.9, 624.3, 1109.3 and 211.1 ng · hr/mL, respectively, increasing as expected with the duration of the infusion. One patient with treatment-related AML with a t(9;11)(p22;q23) translocation achieved a cytogenetic complete response (CR) with complete hematological recovery and transfusion independence on Day 9 following the start of weekly 24 h infusions of obatoclax. This CR was sustained > 8 months while the patient received a total of 35 weekly infusions of 20 mg/m2 without cumulative toxicities. Conclusions: obatoclax can be administered by prolonged infusions without eliciting novel or cumulative toxicities. The dramatic response observed in a patient with AML with immediate recovery of peripheral blood counts supports that the cytotoxic effects of obatoclax are specific to malignant cells while sparing normal bone marrow, as did our earlier reports of improvement in cytopenias in patients with MDS and CLL. The infusional schedules described here will be attractive to combine with standard chemotherapy regimens for AML and other indications to improve treatment outcomes where they may be limited by overexpression of Bcl-2 family members.

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

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. 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. 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. 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.

Saturated Fatty Acid-induced Apoptosis in MDA-MB-231 Breast Cancer Cells: A ROLE FOR CARDIOLIPIN

Little is known about the biochemical basis of the action of free fatty acids (FFA) on breast cancer cell proliferation and apoptosis. Here we report that unsaturated FFAs stimulated the proliferation of human MDA-MB-231 breast cancer cells, whereas saturated FFAs inhibited it and caused apoptosis. Saturated FFA palmitate decreased the mitochondrial membrane potential and caused cytochrome c release. Palmitate-induced apoptosis was enhanced by the fat oxidation inhibitor etomoxir, whereas it was reduced by fatty-acyl CoA synthase inhibitor triacsin C. The non-metabolizable analog 2-bromopalmitate was not cytotoxic. This indicates that palmitate must be metabolized to exert its toxic effect but that its action does not involve fat oxidation. Pharmacological studies showed that the action of palmitate is not mediated via ceramides, reactive oxygen species, or changes in phosphatidylinositol 3-kinase activity. Palmitate caused early enhancement of cardiolipin turnover and decreased the levels of this mitochondrial phospholipid, which is necessary for cytochrome c retention. Cosupplementation of oleate, or increasing beta-oxidation with the AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside, both restored cardiolipin levels and blocked palmitate-induced apoptosis. Oleate was preferentially metabolized to triglycerides, and oleate cosupplementation channeled palmitate esterification processes to triglycerides. Overexpression of Bcl-2 family members blocked palmitate-induced apoptosis. The results provide evidence that a decrease in cardiolipin levels and altered mitochondrial function are involved in palmitate-induced breast cancer cell death. They also suggest that the antiapoptotic action of oleate on palmitate-induced cell death involves both restoration of cardiolipin levels and redirection of palmitate esterification processes to triglycerides.

Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults.

A number of bioreactor configurations have been developed for the manufacture of products from mammalian cell hosts. Even in the most efficient of these, however, problems such as nutrient exhaustion, growth factor deprivation, and toxin accumulations may arise. Consequently, the current effort focused on the feasibility of overexpressing anti-apoptosis genes in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells as a means of limiting cell death upon exposure to three such insults. Extended periods of glucose deprivation, serum withdrawal, and treatment with ammonium chloride each caused significant damage, often apoptotic in nature, to BHK and CHO cells, typically rendering cultures completely nonviable. The overexpression of bcl-2 and bcl-x(L), however, was able to abrogate the cell death in BHK cultures, though to varying degrees. For instance, the presence of Bcl-2, which did little to suppress apoptosis upon glucose deprivation, significantly improved the viabilities of these cells during serum withdrawal. In contrast, bcl-x(L) overexpression provided BHK cells with enhanced protection in the absence of glucose, allowing cultures to remain viable throughout the entire three week study. CHO cultures, on the other hand, displayed similar trends in survival in response to both glucose and serum deprivation. During these studies, Bcl-x(L) was consistently able to afford cells the highest degree of protection, though Bcl-2 also enhanced culture viabilities and viable numbers. Death suppression following exposure to 50 mM ammonium chloride was observed to a limited extent in both BHK and CHO cells overexpressing bcl-2 and bcl-x(L). However, even during such harsh treatment, Bcl-x(L) was able to enhance the survival of both cultures, providing CHO cells with viable numbers that were nearly 20-fold that of the controls after five days of exposure. Furthermore, the extensions in cell survival provided by the anti-apoptosis gene products enabled the recovery of many of the cultures during rescue attempts in which the death-inducing stimulus was removed. Clearly, engineering cells to better withstand and recover from the insults common during the large scale cultivation of mammalian cells has a number of potential applications in the biopharmaceutical industries where cell death can limit culture productivities.

Part I. Bcl‐2 and bcl‐x L limit apoptosis upon infection with alphavirus vectors

Viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. In particular, alphavirus constructs based on Sindbis virus (SV) and Semliki Forest virus (SFV) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. Two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO); the first was a fully competent SV derivative carrying the gene for chloramphenicol acetyltransferase (dsSV-CAT), while the second was a replication deficient SFV construct containing the human interleukin-12 (IL-12) p35 and p40 genes (SFV-IL-12). Since infection with these vectors induced apoptosis in both cell lines, the present effort was dedicated to determining the ability of anti-apoptosis genes to limit the cell death associated with these virus constructs. Infection with the dsSV-CAT vector resulted in the rapid death of BHK and CHO cells within 4 days, a phenomenon which was considerably delayed by stably overexpressing bcl-2 or bcl-x(L). In fact, cellular lifespans were doubled in both BHK-bcl2 and CHO-bclx(L) cells relative to the parental cell lines. Furthermore, the presence of these gene products provided increases of up to 2-fold in recombinant CAT production. Overexpression of bcl-2 and bcl-x(L) also altered the response of these cells upon infection with SFV-IL-12. While the parental cell lines were completely nonviable within 1 week, the BHK-bcl2, BHK-bclx(L), and CHO-bclx(L) cells each recovered from the infection, resuming exponential growth and regaining viabilities of over 90% by 9 days post-infection. Total IL-12 productivities were nearly doubled by Bcl-2 and Bcl-x(L) in the CHO cells, although this effect was apparently cell-line specific, as the native BHK cells were able to secrete more IL-12 than either of its transfected derivatives. Regardless, the presence of the anti-apoptosis genes allowed the production of IL-12 to be maintained, albeit at low levels, from each of the cell lines for the duration of the culture process. Therefore, overexpression of bcl-2 family members can have a significant impact on culture viabilities and recombinant protein production during alphavirus infections of mammalian cells.

Differential role of caspase-8 and BID activation during radiation- and CD95-induced apoptosis.

Activation of the CD95 death receptor as well as ionizing radiation induces apoptotic cell death in human lymphoma cells. The activation of caspases is a hallmark of apoptosis induction irrespective of the apoptotic trigger. In contrast to death receptor signaling, the exact mechanisms of radiation-induced caspase activation are not well understood. We provide evidence that both, radiation and CD95 stimulation, induce the rapid activation of caspase-8 and BID followed by apoptosis in Jurkat T-cells. To analyse the relative position of caspase-8 within the apoptotic cascade we studied caspase activation and apoptosis in Jurkat cells overexpressing Bcl-2 or Bcl-xL. Caspase-8 activation, pro-apoptotic BID cleavage and apoptosis in response to radiation were abrogated in these cells, while the responses to CD95 stimulation were only partially attenuated by overexpression of Bcl-2 family members. In parallel, the breakdown of the mitochondrial transmembrane potential (DeltaPsim) in response to radiation was inhibited by overexpression of Bcl-2/Bcl-xL Jurkat cells genetically deficient for caspase-8 were found to be completely resistant towards CD95. However, radiation-induced apoptotic responses in caspase-8-negative cells displayed only a modest reduction. We conclude that ionizing radiation activates caspase-8 and BID downstream of mitochondrial damage suggesting that, in contrast to CD95, both events function as executioners rather than initiators of the apoptotic process.