Clinical Trials For Hematological Malignancies Research Articles

Anti-Leukemia Activity of MS-275 Histone Deacetylase Inhibitor Implicates 4-1BBL/4-1BB Immunomodulatory Functions

Histone deacetylase inhibitors (HDACi) have demonstrated promising therapeutic potential in clinical trials for hematological malignancies. HDACi, such as SAHA/Vorinostat, Trichostatin A, and MS-275 were found to induce apoptosis of leukemic blasts through activation of the death receptor pathway and transcriptional induction of the Tumor Necrosis Factor (TNF)-related pro-apoptotic family members, TRAIL and FasL. The impact of HDACi on TNF-related costimulatory molecules such as 4-1BB ligand (4-1BBL/TNFSF9) is however not known. Following exposure to SAHA/Vorinostat, Trichostatin A, and MS-275, transcript levels were determined by real time PCR in Jurkat, Raji and U937 cells. Treatment with HDACi up-regulated TNFSF9 gene expression in the three leukemia cell lines, yet to different extend and with distinct kinetics, which did not require de novo protein synthesis and was not associated with DNAse I hypersensitive chromatin remodeling. Transcriptional activity of TNFSF9 promoter-luciferase constructs was induced up to 12 fold by HDACi, and implication of Sp1/Sp3 transcription factors binding to functional GC-box elements was evidenced by reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays. Functionality of modulated target genes was assessed in allogeneic mixed leukocyte reaction experiments. MS-275- and to a lesser extent Trichostatin A- and SAHA-treated Raji cells significantly up regulated T lymphocytes proliferation which was reduced by about 50% by a 4-1BB blocking recombinant protein, while MS-275- but neither Trichostatin A- nor SAHA-treated cells up-regulated IFNγ secretion by T lymphocytes. Our results identify 4-1BBL/4-1BB as a downstream target of HDACi, especially of MS-275 anti-leukemia action in vitro. Thus, HDACi such as MS-275 displaying dual TNF-dependent proapoptotic and costimulatory activities might be favored for inclusion in HDACi-based anti-cancer therapeutic strategies.

A unique RNA-directed nucleoside analog is cytotoxic to breast cancer cells and depletes cyclin E levels

In contrast to deoxyribose or arabinose containing nucleoside analogs that are currently established for cancer therapeutics, 8-chloro-adenosine (8-Cl-Ado) possesses a ribose sugar. This unique nucleoside analog is RNA-directed and is in a phase I clinical trial for hematological malignancies. RNA-directed therapies are effective for the treatment of many malignancies as their activities are primarily aimed at short-lived transcripts, which are typically encoded by genes that promote the growth and survival of tumor cells such as cyclin E in breast cancer. Based on this, we hypothesized that 8-Cl-Ado, a transcription inhibitor, will be effective for the treatment of breast cancer cells. The metabolism of 8-Cl-Ado and the effect on ATP in the breast cancer cell lines MCF-7 and BT-474 were measured using HPLC analysis. In these cells, 8-Cl-Ado was effectively taken up, converted to its cytotoxic metabolite, 8-Cl-ATP, and depleted the endogenous ATP levels. This in turn led to an inhibition of RNA synthesis. The RNA synthesis inhibition was associated with a depletion of cyclin E expression, which is indicative of a diminished tumorigenic phenotype. The final outcome of 8-Cl-Ado treatment of the breast cancer cells was growth inhibition due to an induction of apoptosis and a loss of clonogenic survival. These results indicate that 8-Cl-Ado, which is currently in clinic for hematological malignancies, may be an effective agent for the treatment of breast cancer.

LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors

Multiple clinical trials are investigating the use of the DNA methylation inhibitors azacitidine and decitabine for the treatment of solid tumors. Clinical trials in hematological malignancies have shown that optimal activity does not occur at their maximum tolerated doses but selection of an optimal biological dose and schedule for use in solid tumor patients is hampered by the difficulty of obtaining tumor tissue to measure their activity. Here we investigate the feasibility of using plasma DNA to measure the demethylating activity of the DNA methylation inhibitors in patients with solid tumors. We compared four methods to measure LINE-1 and MAGE-A1 promoter methylation in T24 and HCT116 cancer cells treated with decitabine treatment and selected Pyrosequencing for its greater reproducibility and higher signal to noise ratio. We then obtained DNA from plasma, peripheral blood mononuclear cells, buccal mucosa cells and saliva from ten patients with metastatic solid tumors at two different time points, without any intervening treatment. DNA methylation measurements were not significantly different between time point 1 and time point 2 in patient samples. We conclude that measurement of LINE-1 methylation in DNA extracted from the plasma of patients with advanced solid tumors, using Pyrosequencing, is feasible and has low within patient variability. Ongoing studies will determine whether changes in LINE-1 methylation in plasma DNA occur as a result of treatment with DNA methylation inhibitors and parallel changes in tumor tissue DNA.

MLN4924, a Novel Small Molecule Inhibitor of Nedd8-Activating Enzyme, Demonstrates Potent Anti-Tumor Activity in Diffuse Large B-Cell Lymphoma

MLN4924 is a first-in-class, small molecule inhibitor of the Nedd8 Activating Enzyme (NAE) in Phase I clinical trials in hematological malignancies. Inhibition of NAE by MLN4924 leads to decreased neddylation and inhibition of cullin-dependent ubiquitin ligase (CDL) activity. CDLs are enzyme complexes which control the ubiquitination and degradation of proteins with important roles in cell cycle progression and cell survival. CDL-mediated degradation of pIkBa regulates NF-kB signaling by freeing cytoplasmic NF-kB transcription factors to translocate to the nucleus promoting cell proliferation and survival. In tumors dependent on the NF-kB pathway for growth and survival, we hypothesized that MLN4924 inhibition of CDL activity would prevent pIkBa degradation and inhibit NF-kB signaling.We utilized models of ABC-like Diffuse Large B-cell Lymphoma (ABC-like DLBCL, OCI-Ly10 and OCI-Ly3 cells) dependent on NF-kB signaling for survival and Germinal Center B-cell like DLBCL (GCB-like DLBCL, OCI-Ly19 and OCI-Ly7 cells) that are not dependent on NF-kB signaling for survival. In vitro, we show that NAE inhibition by MLN4924 in ABC-like DLBCL produces marked stabilization of pIkBa, inhibits p65 nuclear translocation and NF-KB gene transcription demonstrating an inhibition of NF-kB signaling. The inhibition of NF-KB signaling in Ly10 cells results in a G1 phenotype and an acute induction of apoptosis. In contrast, in GCB-like DLBCL we observed an elevation of multiple substrates of the CDLs, an accumulation of cells with increased DNA content (>4N) followed by a DNA damage response and induction of cell death. This mechanism of action in GCB-like DLBCL cells is observed in other tumor cell lines that are not dependent on NF-kB signaling for survival.In vivo administration of MLN4924 to mice bearing xenograft tumors of OCI-Ly10 and OCI-Ly19 resulted in a pharmacodynamic response of NAE pathway inhibition. In both models, a single dose of MLN4924 resulted in time and dose-dependent inhibition of total neddylated cullin levels and stabilization of CDL substrates including the CDL3Keap1 substrate, Nrf-2. Notably, in the OCI-Ly10 model, a single dose of MLN4924 resulted in a marked elevation of pIkBa levels, indicative of NF-kB pathway inhibition, and induction of apoptosis. In both OCI-Ly10 and OCI-Ly19 xenograft models, inhibition of the NAE pathway following repeated daily and intermittent dosing of MLN4924 translated into significant tumor growth inhibition. In the OCI-Ly10 model tumor regressions were observed showing this model to be particularly sensitive to MLN4924 treatment, reflecting the addiction of these tumors to NF-kB signaling. Additionally we demonstrate an inhibition of the NAE pathway and NF-KB signaling in a primary human tumor DLBCL xenograft model (PHTX-22L) resulting in tumor regressions following MLN4924 treatment.In summary, in tumors dependent on NF-kB signaling for growth and survival, MLN4924 inhibition of CDL activity provides a novel mechanism for targeted NF-kB pathway modulation and therapeutic intervention. In addition, these data demonstrate that MLN4924 is a novel agent that has broad activity in pre-clinical models of lymphoma.

Activity of ARRY-614, an Inhibitor of p38 Map Kinase and Angiogenic Targets, in Hematologic Malignancies.

Multiple Myeloma (MM) is a malignancy characterized by the clonal expansion of plasma cells within the bone marrow microenvironment. Within this compartment, the proliferative capacity of MM cells is enhanced by pro-inflammatory cytokines, and there is growing recognition that bone marrow stromal cells play an important role in the support of MM growth and chemo-resistance. We have identified a series of compounds, of which ARRY-614 is representative, that are potent inhibitors (EC50<10 nM) of cytokine synthesis through the inhibition of p38 MAPK. This compound is also a potent inhibitor of Abl tyrosine kinases and Tie2/Tek receptor tyrosine kinase. ARRY-614 is active against all of these targets on both the isolated enzymes and in cells. To confirm these activities, ARRY-614 was evaluated in relevant in vitro and in vivo models. ARRY-614 inhibited p38α in ex vivo stimulated human whole blood (EC50=2 nM) and the release of IL-6 and TNFα from SEA- or LPS-challenged mice (ED50<10 mg/kg). Further, ARRY-614 administered as a single agent was efficacious at inhibiting bFGF-driven angiogenesis in an in vivo matrigel invasion assay as well as inhibiting tumor growth in subcutaneous K562 (BCR-Abl dependent) and RPMI 8226 (multiple myeloma) tumor xenografts in mice, at doses ranging from 30 to 100 mg/kg qd, PO. In regulated safety studies, this compound was well-tolerated at doses up to 100 mg/kg qd and 30 mg/kg qd in rats and cynomolgus, respectively. Together, these data support the advancement of this agent into clinical trials for hematologic malignancies.

Aplidin synergizes with cytosine arabinoside: functional relevance of mitochondria in Aplidin-induced cytotoxicity

Aplidin (plitidepsin) is a novel marine-derived antitumor agent presently undergoing phase II clinical trials in hematological malignancies and solid tumors. Lack of bone marrow toxicity has encouraged further development of this drug for treatment of leukemia and lymphoma. Multiple signaling pathways have been shown to be involved in Aplidin-induced apoptosis and cell cycle arrest in G1 and G2 phase. However, the exact mechanism(s) of Aplidin action remains to be elucidated. Here we demonstrate that mitochondria-associated or -localized processes are the potential cellular targets of Aplidin. Whole genome gene-expression profiling (GEP) revealed that fatty acid metabolism, sterol biosynthesis and energy metabolism, including the tricarboxylic acid cycle and ATP synthesis are affected by Aplidin treatment. Moreover, mutant MOLT-4, human leukemia cells lacking functional mitochondria, were found to be resistant to Aplidin. Cytosine arabinoside (araC), which also generates oxidative stress but does not affect the ATP pool, showed synergism with Aplidin in our leukemia and lymphoma models in vitro and in vivo. These studies provide new insights into the mechanism of action of Aplidin. The efficacy of the combination of Aplidin and araC is currently being evaluated in clinical phase I/II program for the treatment of patients with relapsed leukemia and high-grade lymphoma.

Personalized immunotherapy for the treatment of non‐Hodgkin's lymphoma: a promising approach

The efficacy of immunotherapeutic strategies for the treatment of lymphoid malignancies has been demonstrated in recent years. In patients with B-cell lymphomas, particularly indolent lymphoma, the use of passive immunotherapy, such as the anti-CD20 monoclonal antibody rituximab, has made an impressive impact on patient outcome. Personalized immunotherapy, a method that triggers the immune system to mount a response against tumor cells, has shown promising results in early clinical trials in hematologic malignancies. This therapeutic modality appears safe, with the most common adverse events being transient, local reactions at the site of injection. Furthermore, personalized immunotherapy has the potential to generate immunologic memory, which could provide prolonged remission. Currently, 3 large phase III studies are evaluating the efficacy and safety of personalized immunotherapy in patients with follicular lymphoma. It is hoped that the results of these studies will lead to the incorporation of this promising approach into the standard treatment of patients with lymphoma.

Mechanisms and Activity of PPARγ-Active Triterpenoids CDDO and CDDO-Me in Leukemias.

First-line therapy of acute myeloid leukemia (AML) consists of combinations of cytarabine and an anthracycline. While initial complete remissions are frequent, most patients succumb to resistant disease underlining the need for novel, more effective agents. The most striking progress in AML therapy was achieved by targeting the nuclear receptor RARα with ATRA. Research in our laboratory has demonstrated that the novel synthetic triterpenoid CDDO (2-cyano-3,12- dioxooleana-1,9-dien-28-oic acid) and its more active C28 methyl ester derivative CDDO-Me inhibit growth and induce apoptosis in a variety of cancers including AML, CLL and blast crisis CML. CDDO and to a much higher degree CDDO-Me are potent activators of the nuclear transcription factor Peroxisome Proliferator-Activated Receptor gamma (PPARγ). In a mammalian two-hybrid assay, the CDDO and CDDO-Me induced activation of PPARγ was associated with a marked increase in multiple coactivator recruitment (SRC-1, SRC-2, SRC-3, TRAP220/DRIP205, CARM-1 and PGC-1) that is qualitatively different from that induced by other PPARγ ligands. CDDO induced a higher degree of myelo-monocytic differentiation in DRIP205-overexpressing HL-60 cells suggesting that high cellular levels of DRIP205 co-activator modulate differentiation response to PPARγ ligation. CDDO induced p21 mRNA and protein in leukemic cells and transactivation of the p21 promoter in a p53-independent fashion. We have recently identified the PPARγ-independent depletion of mitochondrial glutathione (GSHm) as a novel mechanism of action resulting in redox disbalance and mitochondrial damage as mechanisms of pro-apoptotic effects of CDDO and CDDO-Me. Gene expression studies using cDNA arrays demonstrate that CDDO induces genes involved in the antioxidant response (AR) including phase II detoxifying enzymes (glutamate cysteine ligase, GSH transferase, etc.) and antioxidant enzymes (heme oxygenase 1, thioredoxin reductase). Cotreatment with the GSH precursor, n-acetyl cystein prevented apoptosis and loss of viability induced by CDDOs, whereas alkylation of intracellular thiols by diethylmaleate decreased the accumulation of a biotinylated derivative of CDDO, TP-301, in U937 leukemic cells suggesting that intracellular reduced thiols are functional targets of CDDO and its derivatives. The in vivo studies using liposomal CDDO-Me in a conditional leukemia model demonstrated significant reduction of leukemia burden as measured by bioluminescence imaging and prolongation of survival. Based on the ample pre-clinical evidence of anti-leukemia effects and on the favorable PK/toxicity profile of the parental compound, CDDO will enter Phase I clinical trials in hematologic malignancies in 3Q 2005 and CDDO-Me in 1Q 2006.

Hu1D10 induces apoptosis concurrent with activation of the AKT survival pathway in human chronic lymphocytic leukemia cells

AbstractThe 1D10 antigen is the target for Hu1D10 (apolizumab), a humanized HLA-DR β-chain–specific antibody that is currently in clinical trials for hematologic malignancies. We demonstrate that Hu1D10 induces caspase-independent apoptosis following secondary cross-linking in primary chronic lymphocytic leukemia (CLL) cells. Generation of reactive oxygen species (ROS) and signal transduction, as evidenced by phosphorylation of Syk and AKT, were noted. The source of the Hu1D10-induced ROS was examined using the Raji lymphoblastic cell line with engineered defects in the mitochondrial respiratory chain. Hu1D10 treatment of clones with deficient mitochondrial respiration produced ROS suggesting a cytoplasmic source. Administration of ROS scavengers to primary CLL cells prior to Hu1D10 treatment diminished AKT activation. Treatment with Hu1D10 and the phosphatidylinositol 3-kinase inhibitor LY294002 demonstrated in vitro synergy with enhanced apoptosis. In conjunction with an ongoing clinical trial, blood samples were collected following intravenous infusion of Hu1D10 and analyzed for phosphorylation of AKT. Two of 3 patient samples showed a sustained increase in AKT phosphorylation following Hu1D10 administration. These data suggest that Hu1D10 ligation in CLL cells induces death and survival signals for which combination therapies may be designed to greatly enhance efficiency of both Hu1D10 and other class II antibodies in development.

Histone deacetylases as therapeutic targets in hematologic malignancies.

During the past 5 years, it has become increasingly apparent that deregulated transcriptional control is a root cause of hematologic malignancy. Chromosomal translocations yield novel fusion transcription factors that in turn either activate genes critical for cell growth or repress genes important for normal cellular differentiation. Many of the fusion proteins of myeloid leukemia are aberrant transcriptional repressors and share the property of recruiting histone deacetylases (HDACs) to target genes. HDACs, by acting on chromatin and on transcription factors themselves, can modulate gene regulation. HDACs also play major roles in the function of well-characterized tumor suppressors such as p53 and Rb. Thus, HDACs are a compelling therapeutic target for cancer therapy. Several classes of HDAC inhibitors induce differentiation and cell death in myeloid and lymphoid model systems. Some of these are now in clinical trials for hematologic malignancies. The nature of HDAC function, the classes of inhibitors available, and recent experimental and clinical data will be reviewed.

Campath-1H monoclonal antibody therapy

Monoclonal antibodies are receiving ever-increasing utilization in the treatment of hematologic malignancies. Campath-1 antibodies are directed against the surface antigen CD52 that is expressed on virtually all lymphocytes and monocytes. Murine forms, Campath-1G and Campath-1M, have been utilized extensively in allogeneic bone marrow transplants in order to purge the allograft of lymphocytes. The humanized form, Campath-1H, is currently the focus of many clinical trials in hematologic malignancies and autoimmune diseases. The genetically engineered Campath-1H has been utilized in the treatment of lymphomas and lymphoid leukemias with impressive results. T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphomas appear to be particularly good targets for this agent. Campath-1H may be administered intravenously or subcutaneously. Infectious complications are the most significant side effect associated with its usage, with fevers, chills, nausea, and vomiting most common. Antibiotic prophylaxis has made the infectious morbidity associated with Campath-1H more manageable. The efficacy demonstrated in clinical trials and manageable toxicities make Campath-1H an appealing agent in the treatment of hematologic malignancies.