Bcl-2 Antisense Oligonucleotide Research Articles

NIR light-controlled DNA nanodevice for amplified mRNA imaging and precise gene therapy

Accurately delivering antisense oligonucleotides (ASOs) to tumor cells for gene therapy poses a significant challenge. To address this issue, we develop a NIR light-activable DNA nanodevice to target survivin mRNA and simultaneously release ASOs in tumor cells, which allows high-precision spatiotemporal imaging and efficient gene therapy. The concept is based on upconversion nanoparticles, which can convert NIR light into UV emissions to activate an entropy-driven DNA walking system for mRNA recognition in tumor cells. The intramolecular toehold-mediated entropy-driven catalytic reaction generates a large amount of Bcl-2 ASOs, which allows accurate delivery of ASOs for gene therapy due to the specific targeting of mRNA in tumor cells. The results from in vitro and in vivo experiments show that the NIR light-activated DNA nanodevice can detect mRNA with high sensitivity by DNA walking amplification and high precision in gene delivery. Moreover, the released ASOs from DNA walking system down-regulate the expression of Bcl-2 anti-apoptosis protein and induce tumor cell apoptosis, thereby suppressing tumor growth without a transfection reagent. The NIR-light-controlled DNA nanodevice holds great potential for precise gene therapy in clinical applications.

Theranostic DNA nanostructure based on phenotype-specific activation of antisense oligonucleotides

Antisense oligonucleotide (ASO) is a powerful agent for gene therapy, designed to form complementary pairs with specific mRNA to inhibit gene expression. However, low specificity limits its potential. To overcome this challenge, we developed a Y-shape DNA nanostructure that enhances the specificity in ASO-based treatment by introducing a detection trigger. The design incorporates the phenotype-specific miR21 activation and the sequential release of Bcl2 ASO. As a result, our Y-shape DNA nanostructure downregulates >50 % Bcl2 mRNA expression and induces >60 % cell death in breast cancer cells. Meanwhile, this approach shows no obvious damage to the non-cancerous cells, indicating the therapeutic potential as a theranostics agent in precision medicine with the combination of biomarker sensing and treatment. Overall, our Y-shape DNA nanostructure serves as a promising strategy providing potential in customized conformation design with specific target sequences in gene therapy.

Cancer-cell-membrane-camouflaged supramolecular self-assembly of antisense oligonucleotide and chemodrug for targeted combination therapy.

The anti-apoptotic B-cell lymphoma-2 (Bcl-2) family of proteins are critical regulators of cell death that are overexpressed in many cancer cells, especially in multi-drug resistant cancer cells. Combinatorial gene- and chemotherapies using antisense oligonucleotides (ASOs) to suppress the expression of Bcl-2-family mRNA and restore the sensitivity of the cell to chemodrugs provide a promising pathway for anticancer treatment. However, intrinsic differences between macromolecular ASOs and small molecular chemodrugs make their co-delivery challenging. Moreover, extraneous carriers may induce immunogenicity and inflammation problems. Herein, we develop a targeted nanodrug delivery system using the cationic amphiphilic chemodrug mitoxantrone (Mito), which interacts with Bcl-2 ASO through electrostatic interaction and self-assembles into nanoparticles (NP[Bcl-2/Mito]), whose size can be controlled by regulating the ratio of ASO and Mito. NP[Bcl-2/Mito] can protect the ASO from degradation during delivery and combine gene- and chemotherapies to improve the anticancer effect. Furthermore, cancer cell membranes (CCMs) derived from homologous tumors were used to camouflage NP[Bcl-2/Mito] (NP[Bcl-2/Mito]@CCM) to achieve immune escape and tumor targeting. Both in vitro and in vivo assessments demonstrate the excellent performance of NP[Bcl-2/Mito]@CCM for drug-resistant breast tumor therapy. This CCM-camouflaged ASO/chemodrug nanoplatform provides a promising pathway for the targeted delivery of ASOs and chemodrugs for tumor combination therapy.

Targeted co-delivery of a photosensitizer and an antisense oligonucleotide based on an activatable hyaluronic acid nanosystem with endogenous oxygen generation for enhanced photodynamic therapy of hypoxic tumors

Photodynamic therapy (PDT) is a promising cancer treatment modality with advantages of minimal invasiveness, repeatable therapy, and mild systemic toxicity. However, the limited bioavailability of photosensitizer (PS), tumor hypoxia, and the presence of antiapoptotic proteins in cancer cells, has hampered the efficiency of PDT. To address these limitations, herein, we developed a hyaluronic acid (HA) based nanosystem (HA-Ce6-Hemin@DNA-Protamine NPs, HCH@DP) loaded with chlorin e6 (Ce6, as PS), hemin (as mimetic catalase) and antisense oligonucleotide (ASO) of B-cell lymphoma 2 (Bcl-2) anti-apoptosis protein via a simple electrostatic self-assembly method for enhanced PDT of hypoxic solid tumors. The HCH@DP can target deliver the PS and ASO to tumor cells via cancer cell overexpressed HA receptors (i.e., CD44 or RHAMM). The Ce6 was released from HA-ss-Ce6 (HSC conjugates) after the reaction of cleavable disulfide bond with glutathione (GSH), which recovered the fluorescence and phototoxicity of Ce6 upon laser irradiation. Meanwhile, the catalase-mimicking hemin (degradation of HA-eda-hemin by hyaluronidase) decomposed the tumor overdressed endogenous H2O2 to oxygen, which relieved tumor hypoxia and further overcome hypoxia-associated resistance of PDT. Furthermore, the inhibition of Bcl-2 expression by Bcl-2 ASO also greatly improved the cellular sensitivity to PDT. Both in vitro and in vivo results showed the tumor cell targeting ability, hypoxia relief and significantly enhanced antitumor PDT efficacy of HCH@DP for hypoxic tumor cells upon laser irradiation. Thus, by improving the target delivery of PS and ASO, relieving tumor hypoxia, and down-regulation of anti-apoptotic proteins, this HCH@DP nanosystem achieved enhanced PDT efficiency against hypoxic tumors. In general, our work provided a promising strategy to increase the utilization of key components (PS and oxygen) of PDT and the cell sensitivity to PDT by targeting co-delivery PS and oligonucleotides to tumor cells via a biocompatible HA based carrier, thereby achieving efficiently PDT treatment of hypoxic solid tumors with potential translation possibility. Statement of significanceThe efficiency of PDT against solid tumor is severely restricted by the limited bioavailability of photosensitizer, tumor hypoxia, and the presence of antiapoptotic proteins in cancer cells. Herein, we have developed an activatable hyaluronic acid (HA) based nanosystem (HA-Ce6-Hemin@DNA-Protamine NPs, HCH@DP) via a simple electrostatic self-assembly method for PDT treatment of hypoxic solid tumors. The HCH@DP enabled to target co-delivery of photosensitizer and antisense oligonucleotide to tumor cells, overcoming tumor hypoxia through in situ oxygen production and improving cellular sensitivity by efficiently reducing anti-apoptosis effect of cancer cells for synergistically enhancing PDT efficiency. This work suggests a promising strategy to develop small molecule drug and oligonucleotides co-delivery nanoplatforms for efficiently PDT treatment of hypoxic solid tumor.

Structure and Gating Behavior of the Human Integral Membrane Protein VDAC1 in a Lipid Bilayer.

The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, is responsible for the transport of all ions and metabolites into and out of mitochondria. Larger than any of the β-barrel structures determined to date by magic-angle spinning (MAS) NMR, but smaller than the size limit of cryo-electron microscopy (cryo-EM), VDAC1’s 31 kDa size has long been a bottleneck in determining its structure in a near-native lipid bilayer environment. Using a single two-dimensional (2D) crystalline sample of human VDAC1 in lipids, we applied proton-detected fast magic-angle spinning NMR spectroscopy to determine the arrangement of β strands. Combining these data with long-range restraints from a spin-labeled sample, chemical shift-based secondary structure prediction, and previous MAS NMR and atomic force microscopy (AFM) data, we determined the channel’s structure at a 2.2 Å root-mean-square deviation (RMSD). The structure, a 19-stranded β-barrel, with an N-terminal α-helix in the pore is in agreement with previous data in detergent, which was questioned due to the potential for the detergent to perturb the protein’s functional structure. Using a quintuple mutant implementing the channel’s closed state, we found that dynamics are a key element in the protein’s gating behavior, as channel closure leads to the destabilization of not only the C-terminal barrel residues but also the α2 helix. We showed that cholesterol, previously shown to reduce the frequency of channel closure, stabilizes the barrel relative to the N-terminal helix. Furthermore, we observed channel closure through steric blockage by a drug shown to selectively bind to the channel, the Bcl2-antisense oligonucleotide G3139.

Making Sense of Antisense Oligonucleotide Therapeutics Targeting Bcl-2.

The B-cell lymphoma 2 (Bcl-2) family, comprised of pro- and anti-apoptotic proteins, regulates the delicate balance between programmed cell death and cell survival. The Bcl-2 family is essential in the maintenance of tissue homeostasis, but also a key culprit in tumorigenesis. Anti-apoptotic Bcl-2, the founding member of this family, was discovered due to its dysregulated expression in non-Hodgkin’s lymphoma. Bcl-2 is a central protagonist in a wide range of human cancers, promoting cell survival, angiogenesis and chemotherapy resistance; this has prompted the development of Bcl-2-targeting drugs. Antisense oligonucleotides (ASO) are highly specific nucleic acid polymers used to modulate target gene expression. Over the past 25 years several Bcl-2 ASO have been developed in preclinical studies and explored in clinical trials. This review will describe the history and development of Bcl-2-targeted ASO; from initial attempts, optimizations, clinical trials undertaken and the promising candidates at hand.

Abstract 939: The combination of liposomal Bcl-2 antisense oligonucleotide (BP1002) with decitabine is efficacious in venetoclax-resistant cells

Abstract Background: B-cell lymphoma 2 (Bcl-2) is an oncoprotein that promotes tumor cell survival and drug resistance. Elevated Bcl-2 expression in numerous human cancers has made it an ideal drug target. Venetoclax, the first FDA approved Bcl-2 inhibitor, is indicated for hematologic malignancies. Accompanying its promising clinical results, however, is the emergence of venetoclax resistance. A recent study found that AML patients who had relapsed from frontline venetoclax-based treatment were refractory to salvage therapy and had a median survival of less than 3 months. Thus, novel treatment approaches for such patients are urgently needed. BP1002, a non-toxic neutral dioleoylphosphatidylcholine liposome incorporating a nuclease-resistant P-ethoxy antisense oligonucleotide (oligo), was developed to target Bcl-2 mRNA. The safety and efficacy of BP1002 is being investigated in patients with advanced lymphoid malignancies. Venetoclax in combination with decitabine is an approved treatment for elderly AML patients. Here we determine the effects of BP1002 plus decitabine in venetoclax-resistant cells. Methods: Cell lines: Venetoclax-resistant hematologic cell lines, MV-4-11- R, SU-DHL-2-R, SU-DHL-6-R, were established from the parental cells by exposing the cells to increasing doses of venetoclax for 10-12 weeks.BP1002: A Bcl-2 antisense oligo, which targets the translation initiation site of human Bcl-2 mRNA, was incorporated in BP1002. Combination study: Parental and venetoclax-resistant cells were treated with decitabine for 2 days followed by the addition of BP1002 for another 4 days of incubation. Resazurin-based cell viability assays and Western blots were conducted to determine the inhibitory effects of BP1002 in parental and resistant cells. Results: The IC50 values of venetoclax in resistant cells were 3 to 40 times higher than those in parental cells. MV-4-11-R, SU-DHL-6-R, SU-DHL-2-R cells are resistant up to 0.1 μM, 0.8 μM, and 7 μM venetoclax, respectively. Compared to parental cells, venetoclax-resistant cells displayed 2- to 3-fold elevated levels of Bcl-2 protein and other anti-apoptotic proteins such as Mcl-1. BP1002 in combination with decitabine decreased the viability of parental cells. BP1002 reduced Bcl-2 levels in venetoclax-resistant cells. Also, the BP1002 plus decitabine combination was more potent than the venetoclax plus decitabine combination in reducing the viability of the resistant cells, by about 50-70%. Conclusions: Our data indicate that, despite expressing higher levels of anti-apoptotic proteins, venetoclax-resistant cells are sensitive to the BP1002 plus decitabine combination. These promising preclinical results suggest that the BP1002 plus decitabine combination is a potential treatment for patients who have relapsed from frontline venetoclax-based therapies. Citation Format: Maria Gagliardi, Ana Tari Ashizawa. The combination of liposomal Bcl-2 antisense oligonucleotide (BP1002) with decitabine is efficacious in venetoclax-resistant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 939.

Abstract CT240: A phase I clinical trial to study the safety, pharmacokinetics, and efficacy of BP1002 (L-Bcl-2) antisense oligonucleotide in patients with advanced lymphoid malignancies

Abstract Background: Bcl-2 protein is essential for cancer cell survival. Inhibition of Bcl-2 expression may lead to cancer cell death. A nuclease-resistant, hydrophobic Bcl-2 antisense oligonucleotide (oligo) incorporated in a neutral dioleoylphosphatidylcholine liposome (L-Bcl-2, also known as BP1002) is utilized to inhibit Bcl-2 expression. Preclinical studies showed that L-Bcl-2 inhibited Bcl-2 protein expression and in vivo tumor growth. Bcl-2 expression is frequently altered in human cancers, including lymphomas. We postulate that BP1002 will have anti-tumor activity against lymphomas. This first-in-human BP1002 study is proposed as a dose-escalation monotherapy study in patients with advanced lymphoid malignancies. Methods: Subjects: BP1002 will be given to adult subjects diagnosed with refractory/relapsed chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, non-Hodgkin lymphoma (B-cell and T-cell) and Waldenströms macroglobulinemia. Study Design: The study will utilize a single-arm, sequential, dose-escalation design (3+3) to identify dose-limiting toxicities (DLTs), recommended Phase II dose (RP2D), pharmacokinetics (PK), pharmacodynamics, and efficacy of BP1002. BP1002 will be administered to subjects intravenously twice weekly per 28-day cycle (8 doses). There are 2 planned dose levels, 20 and 40 mg/m2. Successive cohorts of eligible patients will be treated with BP1002. A higher dose level cannot be opened until a minimum of three patients have completed an entire therapy cycle at the preceding dose level without having DLTs. Any DLT observed will trigger an expansion of a cohort from 3 to 6 patients. A second DLT at any dose level will identify the maximum dose. The dose below that will be the maximum tolerated dose (MTD). The RP2D will be determined using the MTD data and all information regarding safety, biological activity, PK and pharmacodynamics. Primary objectives: To determine • Safety and tolerability of escalating doses of BP1002 • RP2D of BP1002 • PK of BP1002 • BP1002 activity on Bcl-2 expression Secondary objectives: To obtain evidence of tumor response (overall response rate = complete remission + partial remission + minor response), and estimates for other response parameters (time to progression, progression-free survival, event-free survival) Exploratory objectives: To correlate treatment response with biological activity, cytogenetic and molecular characteristics Safety assessments will include identification of DLTs, serious adverse events, treatment-emergent adverse events and treatment-emergent laboratory abnormalities Response assessments will be determined per the 2018 International Workshop Group on CLL (iwCLL) response criteria, the International Working Group consensus response evaluation criteria in lymphoma (RECIL 2017), or the Eighth International Workshop for Waldenströms Macroglobulinemia response criteria. Citation Format: Ana Tari Ashizawa, Locke Bryan, William Wierda. A phase I clinical trial to study the safety, pharmacokinetics, and efficacy of BP1002 (L-Bcl-2) antisense oligonucleotide in patients with advanced lymphoid malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT240.

Oligonucleotide delivery with cell surface binding and cell penetrating Peptide amphiphile nanospheres.

A drug delivery system designed specifically for oligonucleotide therapeutics can ameliorate the problems associated with the in vivo delivery of these molecules. The internalization of free oligonucleotides is challenging, and cytotoxicity is the main obstacle for current transfection vehicles. To develop nontoxic delivery vehicles for efficient transfection of oligonucleotides, we designed a self-assembling peptide amphiphile (PA) nanosphere delivery system decorated with cell penetrating peptides (CPPs) containing multiple arginine residues (R4 and R8), and a cell surface binding peptide (KRSR), and report the efficiency of this system in delivering G-3129, a Bcl-2 antisense oligonucleotide (AON). PA/AON (peptide amphiphile/antisense oligonucleotide) complexes were characterized with regards to their size and secondary structure, and their cellular internalization efficiencies were evaluated. The effect of the number of arginine residues on the cellular internalization was investigated by both flow cytometry and confocal imaging, and the results revealed that uptake efficiency improved as the number of arginines in the sequence increased. The combined effect of cell penetration and surface binding property on the cellular internalization and its uptake mechanism was also evaluated by mixing R8-PA and KRSR-PA. R8 and R8/KRSR decorated PAs were found to drastically increase the internalization of AONs compared to nonbioactive PA control. Overall, the KRSR-decorated self-assembled PA nanospheres were demonstrated to be noncytotoxic delivery vectors with high transfection rates and may serve as a promising delivery system for AONs.

Dacarbazine with or without oblimersen (a Bcl-2 antisense oligonucleotide) in chemotherapy-naive patients with advanced melanoma and low–normal serum lactate dehydrogenase

In a previous large randomized, open-label study, retrospective subset analysis revealed that the addition of the Bcl-2 antisense oligonucleotide oblimersen to dacarbazine (Dac) significantly improved overall survival, progression-free survival, and the response rate in chemotherapy-naive patients with advanced melanoma and normal baseline serum lactate dehydrogenase (LDH) levels. To confirm and expand on this observation, we conducted a prospective double-blind, placebo-controlled study to determine whether oblimersen augmented the efficacy of Dac in advanced melanoma patients with low-normal baseline LDH levels. A total of 314 chemotherapy-naive patients were randomly assigned to receive Dac (1000 mg/m(2)) preceded by a 5-day continuous intravenous infusion of either oblimersen sodium (7 mg/kg/day) or placebo every 21 days for less than eight cycles. Co-primary efficacy endpoints were overall survival and progression-free survival. Response and progression of the disease were assessed by independent blinded review of computed tomography scan images. No difference in overall nor progression-free survival was observed between the Dac-oblimersen and Dac-placebo groups. Although the overall (17.2 vs. 12.1%) and durable (10.8 vs. 7.6%) response rates numerically favored Dac-oblimersen over Dac-placebo, they did not differ significantly (P=0.19 and 0.32, respectively). The incidence of hematologic adverse events, particularly thrombocytopenia and neutropenia, was higher in the Dac-oblimersen group than in the Dac-placebo group. Withdrawals from the study because of treatment-related adverse events were low (i.e. <2.5%) in both groups. The addition of oblimersen to Dac did not significantly improve overall survival nor progression-free survival in patients with advanced melanoma and low-normal levels of LDH at baseline.

The Price of Hope: Personalized Medicine in 2014

In 1994, shortly after taking my first faculty position, I was invited to give Medical Grand Rounds at my new institution. The title of my talk was “Novel Therapeutic Applications of Cancer Biology.” Today, the title would be “Personalized Medicine in Oncology.” My Kodachrome slides covered numerous biologically rational molecular targets and their proposed treatments, including RAS (farnesyl transferase inhibitors), MYC (retinoic acid—my own research interest at the time), P53 (gene therapy), BCL2 (antisense oligonucleotides), and growth factor receptors (receptor antagonists), only the last of which has proven to have any clinical utility. At that time, the notion of targeting cancer cells based on their molecular derangements was being evaluated mainly in preclinical studies and early-phase clinical trials. Much has changed in the past 20 years. Today, most people don’t know what a Kodachrome slide is, and, more importantly, several molecularly targeted anticancer agents have demonstrated dramatic clinical activity in molecularly defined patient populations. The “poster child” for targeted therapy is imatinib, which has kept many patients with chronic myelogenous leukemia (CML) in remission for well over a decade. But most cancers are not as molecularly homogeneous as chronic-phase CML. The results achieved with targeted agents in solid tumors have been far more modest, because of widespread tumor cell heterogeneity and energetic exploitation of alternative growth and survival pathways leading to the relatively rapid emergence of resistance. Nonetheless, we have learned that defining patient subsets based on predictive biomarkers allows us to target specific treatments to those who will benefit most, while avoiding undue toxicity to those who will not benefit. In thoracic oncology, we are finally starting to understand how to use epidermal growth factor receptor (EGFR) inhibitors most effectively, thanks to the commitment of thousands of patients and many investigators who participated in relevant clinical trials over the past decade. Being enticed by the marvels of molecular technology and the promise of personalized medicine is easy, particularly when confronted by the limitations of current therapy. I recently received an e-mail from the husband of a woman with advanced non–small cell lung cancer (NSCLC) harboring wild-type EGFR, ALK, and ROS1. The plaintive tone was all too familiar. He had done his online research and, to his credit, was turning to the science of molecular tumor profiling rather than to laetrile. For only $40,000, the e-mail said, he could ensure that his wife would get “the best and most appropriate care.” For him, this was the price of hope. But separating the hope from the hype is important, as is clarifying the line between research and clinical reality. A rapidly growing number of commercial vendors offer molecular tumor profiling services that purportedly guide personalized therapy. However, the number of predictive biomarkers and targeted therapies proven to benefit patients with solid tumors remains relatively small, a fact that is not clearly evident from current news stories or most oncology journals. The promise of personalized therapy is not yet commensurate with the hype it has generated. But our patients only hear the hype, from both the lay press and direct-to-consumer advertising. The FDA is to be applauded for the recent warning regarding the need to validate the benefits of direct-to-consumer testing. As academic oncologists, we could also present a more balanced reality when interacting with the press. The impact of this hype extends beyond our patients. Many of the calls I receive from community oncologists reflect a growing confusion about the prevailing standard Gregory P. Kalemkerian, MD

Antitumor effects of mutant endostatin are enhanced by Bcl-2 antisense oligonucleotides in UM-UC-3 bladder cancer cell line.

Endostatin is a potent inhibitor of tumor angiogenesis. In the preliminary studies, we developed a mutant endostatin containing Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) sequences. In this study, we compared the antitumor effects of mutant endostatin and Bcl-2 antisense oligonucleotides both in combination and individually. The artificially synthesized Bcl-2 ASODN (antisense oligonucleotides) included a translation-initiation site and was transfected into the bladder cancer cells by Lipofectamine. Cell growth was investigated by the tumor cell growth chart, MTT assay, caspase-3 activity detection assay, AO/EB fluorescein stain, and the annexin V-FITC apoptosis detection assay. In the in vivo study, UM-UC-3 bladder cancer cells were subcutaneously implanted into nude mice and the growth of tumor was examined. The ultrastructure of the tumor tissues in the treated and control groups were observed. The cell growth chart showed that the cell population of the treated combination group decreased by 52.04% compared to the control group. The inhibition rate of the treated combination group was (79.66 ± 6.79)%, whereas those of the individual ASODN and ES groups were (53.39 ± 3.22)% and (50.22 ± 5.46)% respectively. In the caspase-3 activity detection using AO/EB fluorescein stain and annexin V-FITC apoptosis detection assay, the co-inhibitory effect was higher than the individual inhibitory effects (P < 0.05). There were significant differences in the inhibition of the solid tumor growth in the in vivo study. Our findings indicated that Bcl-2 antisense oligonucleotides enhance the antitumor effects of mutant endostatin both in vitro and in vivo. We noted the synergistic effects of Bcl-2 antisense oligonucleotides combined with mutant endostatin.

Oblimersen in combination with temozolomide and albumin-bound paclitaxel in patients with advanced melanoma: a phase I trial

The combination of oblimersen, a bcl-2 antisense oligonucleotide, and dacarbazine lead to superior progression-free survival in advanced melanoma patients. Albumin-bound paclitaxel (nab-paclitaxel) has single-agent activity in melanoma. In a phase I trial, chemotherapy-naïve patients with metastatic melanoma and normal LDH levels were enrolled on 3 cohorts. The treatment regimen consisted of 56-day cycles of oblimersen (7 mg/kg/day continuous IV infusion on day 1-7 and 22-28 in cohort 1 and 2; 900 mg fixed dose, twice weekly in weeks 1-2, 4-5 for cohort 3), temozolomide (75 mg/m(2), days 1-42), and nab-paclitaxel (175 mg/m(2) in cohort 1 and 3, 260 mg/m(2) in cohort 2 on day 7 and 28). Apoptosis markers were tested in pre- and post-treatment specimens of a subset of patients. Six grade 3 events (neutropenia, renal insufficiency, hyponatremia, elevated creatinine, allergic reaction, and neuropathy) and 2 grade 4 events (neutropenia and thrombocytopenia) were seen in 32 patients. The objective response rate was 40.6% (2 complete responses and 11 partial responses) and 11 patients had stable disease, for a disease control rate of 75%. The combination of oblimersen, temozolomide, and nab-paclitaxel was well tolerated and demonstrated encouraging activity in patients with advanced melanoma.

Treatment strategies for nodal and gastrointestinal follicular lymphoma: Current status and future development

In recent years, therapies for follicular lymphoma (FL) have steadily improved. A series of phase III trials comparing the effect of rituximab with chemotherapy vs chemotherapy alone in treating FL have indicated significant improvements in progression-free survival (PFS) and overall survival. Recent studies have found that prolonged response durations and PFS were obtained with maintenance therapy using rituximab or interferon after completion of first line therapy. For patients with relapsed or refractory FL, phase II studies have assessed the effectiveness of combination therapies using a Toll-like receptor-9 agonist (1018ISS), oblimersen sodium (a Bcl-2 antisense oligonucleotide), bendamustine, and rituximab, as well as veltuzumab, a new humanized anti-CD20 antibody, and epratuzumab. In addition, the effectiveness of yttrium-90 ibritumomab tiuxetan and iodine-131 tositumomab as radioimmunotherapies has been reported. Furthermore, three phase III studies on an idiotype vaccine are near completion. Unfortunately, these vaccines, which appeared highly effective in phase I and II trials, do not appear to result in prolonged PFS. This report will summarize the current knowledge on therapies for treatment of FL, and will conclude with a brief discussion of feasible future options for effective treatments. Lastly, we added descriptions of the management of gastrointestinal FL, which is considered to be controversial because it is rare.

The Amount of Spontaneous Apoptosis Is An Independent Strong Disease Progression Indicator in B-Cell Chronic Lymphocytic Leukemia (B-CLL)

Abstract 1252▪▪This icon denotes an abstract that is clinically relevant.Poster Board I-274Bcl-2 levels has emerged as the most important protein in predicting survival between 11 proteins in CLL cells that are implicated in the control of apoptosis, proliferation and differentiation (Faderl, 2002). In fact, malignant cells are arrested in the G0/early G1 phase of the cell cycle, and inhibition of spontaneous apoptosis with upregulation of the anti-apoptotic protein bcl-2 may define clinical prognosis. The today availability both of bcl-2 antisense oligonucleotides and of novel pro-apoptotic BH3 peptidomimetic prompted us to evaluate the real impact of apoptosis pathways on B-CLL prognosis. The primary aims of our study were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon bax/bcl-2 ratio, 2) whether bax/bcl-2 ratio and ZAP-70 show additive prognostic impact and finally 3) whether bax/bcl-2 is an independent prognostic factor. Therefore we investigated 356 patients (pts), median age 65 years (range 37-89), 164 males and 192 females. With regard to modified Rai stages, 115 pts had a low stage, 229 an intermediate stage and 12 a high stage. Bax/bcl-2 ratio was determined by flow cytometry, dividing mean fluorescence intensity (MFI) of bax by MFI of bcl-2 on CD19+CD5+ B-CLL cells. We obtained the bax/bcl-2 ratio and the threshold was set at the median value >1.45 (range 0.27-13.6). ZAP-70 was quantified by multicolor flow cytometry and the cut-off was fixed at >20%. Two hundred-three pts were bax/bcl-2 ratio positive (203/356; 57%). Higher bax/bcl-2 ratio was significantly associated with low Rai stage (80/115; P=0.003), lymphocyte doubling time >12 months (183/299; P=0.0003), beta-2 microglobulin (B-2M) <2.2 mg/dl (129/200; P=0.001) and soluble CD23 (sCD23) <70 U/ml (141/228; P=0.005). Moreover, there were significant correlations between higher bax/bcl-2 ratio and IgVH gene mutated status (216 cases, 96/154; P=0.015) or low risk (normal or 13q-) FISH cytogenetics (255 cases, 109/187; P=0.011). Noteworthy, a very strict association was found between higher bax/bcl-2 ratio and lower ZAP-70 (147/220; P<0.00001), suggesting that low ZAP-70 expression is characterized by high apoptosis levels. With regard to clinical outcome, significant shorter PFS and OS were observed in pts with lower bax/bcl-2 ratio (10% vs 60% at 14 years; P<0.00001 and 51% vs 74% at 16 years; P=0.005, respectively) as well as in ZAP-70+ pts (5% vs 57% at 12 years; P<0.00001 and 30% vs 85% at 16 years, respectively). To further explore the prognostic impact of bax/bcl-2 ratio, we investigated its expression associated with ZAP-70 protein. As a matter of fact, higher bax/bcl-2 ratio plus ZAP-70 <20% identified the pts subset with the longest PFS (70% vs 2% at 12 years; P <0.00001, Figure) and OS (92% vs 33%; P<0.00001). The discordant pts presented an intermediate outcome (Figure). In multivariate analysis of PFS, in which cytogenetics, IgVH status, ZAP-70, CD38, bax/bcl-2, sCD23 entered, bax/bcl-2 (P=0.02), cytogenetics (P=0.02) and ZAP-70 (P=0.04), resulted to be independent prognostic factors. In conclusion, our apoptotic index (bax/bcl-2 ratio), performed by flow cytometry, was very useful to identify pts at different progression rate and since the ZAP-70 negative subset represents a large and heterogeneous B-CLL population with a variable progression, other biological factors, such as the amount of apoptosis, have to be added in order both to identify early and treat timely progressive pts. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Docetaxel plus oblimersen sodium (Bcl-2 antisense oligonucleotide): an EORTC multicenter, randomized phase II study in patients with castration-resistant prostate cancer

Background: This randomized, phase II study assessed the activity of oblimersen sodium, a Bcl-2 antisense oligonucleotide, administered before docetaxel (Taxotere) to patients with castration-resistant prostate cancer.Patients and methods: Chemotherapy-naive patients with prostate-specific antigen (PSA) progression and testosterone ≤0.5 ng/ml received docetaxel 75 mg/m2 on day 1 or oblimersen 7 mg/kg/day continuous i.v. infusion on days 1–7 with docetaxel 75 mg/m2 on day 5 every 3 weeks for ≤12 cycles. Primary end points were confirmed PSA response (Bubley criteria) and major toxic events.Results: Confirmed PSA response was observed in 46% and 37% of 57 and 54 patients treated with docetaxel and docetaxel–oblimersen, respectively. Partial response (RECIST) was achieved in 18% and 24%, respectively. Oblimersen added to docetaxel was associated with an increase in the incidence of grade ≥3 fatigue, mucositis, and thrombocytopenia. Major toxic events were reported in 22.8% and 40.7% of patients with docetaxel and docetaxel–oblimersen, respectively.Conclusions: The primary end points of the study were not met: a rate of confirmed PSA response >30% and a major toxic event rate <45% were not observed with docetaxel–oblimersen.

SiRNA-Lipoplex-Mediated Bcl-2 and Bcl-xL Gene Silencing Induces Apoptosis in MCF-7 Human Breast Carcinoma Cells

Bcl-2 family genes play a central role in cell apoptosis and cell proliferation, and are implicated in the pathol- ogy of many malignancies. We explored different ways of introducing siRNA duplexes into cells, comparing naked with lipoplex- and polyplex-based formulations in order to decrease the level of the Bcl-2 and Bcl-xL proteins. Our results show that siRNA binds efficiently to all cationic liposomes used. Upon binding, siRNA reduces the zeta potential of the particles, although in most cases they remain positively charged. 70% of MCF-7 cells took up fluorescently-labeled siRNA after 24 h. All siRNA sequences caused growth inhibition of cells, with variable efficiency in a dose-dependent manner. Significant decreases in Bcl-2 and Bcl-xL proteins were caused by two siRNA sequences. Both caused signifi- cant growth inhibition in concentrations as low as 100 nM. These two siRNAs caused the greatest increase in caspase-7 activity and DNA fragmentation level. Addition of CaCl 2 as a transfection enhancer resulted in marked increase of growth inhibition and Bcl-2 gene suppression by siRNA. Our lipoplexes containing siRNA showed equal or superior efficacy in comparison with commercial siRNA transfection kits. Efficiency of cell growth inhibition per RNA molecule using siRNA was found to be twenty fold higher than by a well-established Bcl-2 antisense oligonucleotide (ODN) molecule af- ter optimization of ODN delivery to cells in culture. This study indicates the potential for efficient delivery of siRNA for treatment of various malignancies.

Phase III randomised study of dexamethasone with or without oblimersen sodium for patients with advanced multiple myeloma

Upregulation of the Bcl-2 antiapoptotic protein is reported to be associated with aggressive clinical course in multiple myeloma. Oblimersen sodium is a bcl-2 antisense oligonucleotide complementary to the first six codons of the open-reading frame of bcl-2 mRNA that can decrease transcription of Bcl-2 protein and increase myeloma cell susceptibility to cytotoxic agents. In this phase III randomised trial, we investigated in patients with relapsed/refractory myeloma whether addition of oblimersen to dexamethasone improved clinical outcomes vs. dexamethasone alone. Two hundred and twenty-four patients were randomised to receive either oblimersen/dexamethasone (N = 110) or dexamethasone alone (N = 114). The primary endpoint was time to tumor progression (TTP). Final results of this study demonstrated no significant differences between the two groups in TTP or objective response rate. The oblimersen/dexamethasone regimen was generally well tolerated with fatigue, fever and nausea, the most common adverse events reported.

Phase I/II Study of G3139 (Bcl-2 Antisense Oligonucleotide) in Combination with Doxorubicin and Docetaxel in Breast Cancer

Preclinical data showed enhancement of breast cancer cell death when G3139 was combined with anthracyclines and taxanes. We evaluated the efficacy and safety of a Bcl-2 antisense oligonucleotide, G3139, in combination with doxorubicin (A) and docetaxel (T) in patients with locally advanced breast cancer (LABC). Following a brief phase I to determine the phase II dose, patients with locally advanced breast cancer received G3139 administered by continuous i.v. infusion for 5 to 7 days with bolus A (50 mg/m2) and T (75 mg/m2) administered on either day 3 or 6 of therapy with G3139. Cycles were repeated every 21 days x 6 in the neoadjuvant setting. Serial plasma samples were obtained for pharmacokinetic analysis. Tissue samples were obtained before and after therapy for pharmacodynamic analysis of Bcl-2 expression. Thirty patients (median age, 49 years; range, 24-71 years) received 160 cycles. During the phase I portion of the trial, the dose of G3139 was escalated from 3 to 7 mg/kg/d (i.v. for 5 days) in combination with AT. During the phase II portion of the trial, several doses and schedules of G3139 were evaluated. There were no pathologic complete responses. Pharmacodynamic studies showed limited Bcl-2 down-regulation in the primary tumors. G3139 in combination with doxorubicin and docetaxel is well tolerated. No pathologic complete response was seen and pharmacodynamic studies showed very little down-regulation of Bcl-2 in primary tumors, perhaps related to issues with insufficient drug delivery to the intact tumor.

Novel Agents in Chronic Lymphocytic Leukemia: Efficacy and Tolerability of New Therapies

Alkylating agents and purine analogues have been the mainstays of therapy for chronic lymphocytic leukemia (CLL) for decades. The past decade witnessed the general clinical use of monoclonal antibodies such as rituximab and alemtuzumab, both as single agents and in combination regimens with cytotoxic drugs, for previously untreated and relapsed CLL. First-line chemoimmunotherapy regimens combining rituximab and purine analogues have greatly improved initial response rates and progression-free survival. Despite these advances in first-line therapy, patients with CLL invariably experience relapse and often acquire high-risk chromosomal abnormalities, such as del(11q22) and del(17p13), which result in resistance to current therapies. Patients who are refractory to fludarabine-based therapy have a median survival of < 1 year. Therefore, new agents with novel mechanisms of action are needed for the treatment of patients with relapsed CLL, particularly for patients with high-risk genetic features. Recent clinical studies have examined the tolerability and efficacy of several novel agents in relapsed CLL: (1) the alkylator bendamustine, (2) the cyclin-dependent kinase inhibitor flavopiridol, (3) the immunomodulating drug lenalidomide, (4) the bcl-2 antisense oligonucleotide oblimersen, and (5) the Bcl-2 smallmolecule inhibitor obatoclax. While these agents have demonstrated exciting clinical activity against genetically high-risk CLL, they have also induced toxicities that have not been commonly observed with previous CLL therapies. The most notable toxicities have been tumor lysis syndrome and tumor flare, which are potentially serious or even fatal complications of these new therapies. Thus, further studies are needed to define these agents' biologic mechanism(s) of action, clinical activity, and safety.