Ayad Al-Katib

Preclinical Studies of TW-37, a New Nonpeptidic Small-Molecule Inhibitor of Bcl-2, in Diffuse Large Cell Lymphoma Xenograft Model Reveal Drug Action on Both Bcl-2 and Mcl-1

Purpose: Overexpression of Bcl-2 protein has been observed in more than 80% of B-cell lymphomas, including diffuse large cell lymphoma (DLCL), the most common subtype of non-Hodgkins lymphoma. We have previously employed the natural product (−)-gossypol to test its therapeutic potential as a small-molecule inhibitor of Bcl-2 for the treatment of B-cell lymphomas. Experimental Design: Recently, we have used a structure-based strategy to design a new class of potent small-molecule inhibitor acting on Bcl-2. One such lead compound is the benzenesulfonyl derivative TW-37, which was designed to target the BH3-binding groove in Bcl-2 where proapoptotic Bcl-2 proteins, such as Bak, Bax, Bid, and Bim bind. Results: In our fluorescence polarization–based binding assays using recombinant Bcl-2, Bcl-XL, and Mcl-1 proteins, TW-37 binds to Bcl-2, Bcl-XL, and Mcl-1 with Ki values of 290, 1,110 and 260 nmol/L, respectively. Hence, TW-37 is a potent inhibitor of Bcl-2 and has >3-fold selectivity over Bcl-XL. In vitro, TW-37 showed significant antiproliferative effect in a de novo chemoresistant WSU-DLCL2 lymphoma cell line and primary cells obtained from a lymphoma patient with no effect on normal peripheral blood lymphocytes. Coimmunoprecipitation experiments showed that TW-37 disrupted heterodimer formation between Bax or truncated-Bid and antiapoptotic proteins in the order Mcl-1 > Bcl-2 >> Bcl-XL. As expected, TW-37 caused apoptotic death. Pre-exposure of lymphoma cells to TW-37 significantly enhanced the killing effect of cyclophosphamide-doxorubicin-vincristine-prednisone (CHOP) regimen. The maximum tolerated dose of TW-37 in severe combined immunodeficient (SCID) mice was 40 mg/kg for three i.v. injections when given alone and 20 mg/kg, ×3 when given in combination with CHOP. Using WSU-DLCL2-SCID mouse xenograft model, the addition of TW-37 to CHOP resulted in more complete tumor inhibition compared with either CHOP or TW-37 alone. Conclusions: We conclude that the administration of TW-37, as a potent Bcl-2 and Mcl-1 inhibitor, to standard chemotherapy may prove an effective strategy in the treatment of B-cell lymphoma.

Phase I study of bryostatin 1 in patients with relapsed non-Hodgkin's lymphoma and chronic lymphocytic leukemia.

PURPOSE To define, in a phase I study in relapsed non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia (CLL), the maximum-tolerated dose (MTD), major toxicities, and possible antitumor activity of bryostatin 1, a macrocyclic lactone. PATIENTS AND METHODS Bryostatin 1 was delivered by 72-hour continuous infusion every 2 weeks to patients with relapsed NHL or CLL, at doses that ranged from 12 microg/m2 to 180 microg/m2 per course. Correlative investigations included evaluations of total protein kinase C (PKC) in peripheral blood and lymphoid differentiation in patient tumor tissue. RESULTS Twenty-nine patients were treated, including three patients with CLL and 26 with NHL. Generalized myalgia was the dose-limiting toxicity (DLT) and occurred in two of three patients treated with bryostatin 1 at 180 microg/m2 per course. Myalgias were dose-related and cumulative, and often started in the thighs and calves, improved with activity, were somewhat responsive to analgesics, and often took weeks to resolve once taken off study. Six patients were treated at the MTD of 120 microg/m2 per course. Myalgia, headache, and fatigue were common. Hematologic toxicity was uncommon. Total cumulative doses of bryostatin 1 up to 1,134 microg/m2 have been administered without untoward toxicity. Eleven patients achieved stable disease for 2 to 19 months. An in vitro assay for total PKC evaluation in patient peripheral-blood samples demonstrated activation within the first 2 hours with subsequent downregulation by 24 hours, which was maintained throughout the duration of the 72-hour infusion. CONCLUSION This phase I study defined the MTD and recommended phase II dose of bryostatin 1, when administered over 72 hours every 2 weeks, to be 120 microg/m2 (40 microg/m2/d for 3 days). Generalized myalgia was the DLT. Future studies will define the precise activity of bryostatin 1 in subsets of patients with lymphoproliferative malignancies and its efficacy in combination with other agents.

Preclinical studies of Apogossypolone: a new nonpeptidic pan small-molecule inhibitor of Bcl-2, Bcl-XL and Mcl-1 proteins in Follicular Small Cleaved Cell Lymphoma model

Elevated expression of anti-apoptotic Bcl-2 family proteins have been linked to a poor survival rate of patients with Follicular Lymphoma (FL). This prompted us to evaluate a very potent non-peptidic Small-Molecule Inhibitor (SMI) targeting Bcl-2 family proteins, Apogossypolone (ApoG2) using follicular small cleaved cell lymphoma cell line (WSU-FSCCL) and cell isolated from lymphoma patients. ApoG2 inhibited the growth of WSU-FSCCL significantly with a 50% growth inhibition of cells (IC50) of 109 nM and decreased cell number of fresh lymphoma cells. ApoG2 activated caspases-9, -3, and -8, and the cleavage of Poly (ADP-ribose) polymerase (PARP) and Apoptosis Inducing Factor (AIF). In the WSU-FSCCL-SCID xenograft model, ApoG2 showed a significant anti-lymphoma effect, with %ILS of 84% in the intravenous and 63% in intraperitoneal treated mice. These studies suggest that ApoG2 can be an effective therapeutic agent against FL.

Chromosomal analyses of 52 cases of follicular lymphoma with t(14;18), including blastic/blastoid variant.

We have identified 52 patients of follicular lymphoma (FL) with t(14;18)(q32;q21). Histologically, the lymphomas were placed into six groups according to their cellular composition and growth pattern. Chromosome analysis revealed that all cases but one had additional secondary chromosomal abnormalities. The most frequent numerical aberrations were gains of chromosomes 7 (38%), X (36%), 5 (15%), 12 (15%), 18/der(18)t(14;18) (25%), and 21 (15%). Structural abnormalities of chromosome 1 were seen in 19 tumors (36%) affecting both arms with breakpoints clustered at 1p36. Other structural abnormalities included partial deletions of 6q, 10q, and 13q. Breakpoint at 8q24 was seen in four cases. The chromosome aberrations were correlated with the morphological subtypes of follicular lymphoma. Gain of chromosome 7 appeared to be associated with follicular large cell lymphoma. The incidence of trisomy 5 and 12, and 13q- was higher in follicular lymphoma with aggressive histological features than in low-grade lymphoma. In addition, complexity of the karyotype and high degree of polyploidy increased with the grade. The most valuable cytogenetic markers in the t(14;18) lymphomas are those involving 8q24 which was found exclusively in the blastic/blastoid variant FL. Therefore, chromosome analysis in relation to histologic pattern of follicular lymphoma can provide additional information in predicting tumor evolution and transformation to a higher-grade malignancy.

Bax:Bcl-2 ratio modulation by bryostatin 1 and novel antitubulin agents is important for susceptibility to drug induced apoptosis in the human early pre-B acute lymphoblastic leukemia cell line, Reh

The ratio of Bax to Bcl-2 protein can determine whether cells will die via apoptosis or be protected from it. Reh was found to express a high basal level of Bcl-2 but was lacking of Bax protein expression. Treatment with bryostatin 1 induced a down-regulation in Bcl-2 protein that was not accompanied by an obvious Bax protein induction or apoptosis. These results suggest that a decreased level of Bcl-2 alone in this cell line is not sufficient for apoptosis induction. In an effort to identify the mechanism whereby apoptosis could be induced in this ALL model, we treated Reh cells with three microtubule inhibitors: dolastatin 10, auristatin PE and vincristine, in the presence and absence of bryostatin 1. When used alone, only dolastatin 10 induced apoptosis that was detected morphologically, and by flow cytometry. Western blots revealed that dolastatin 10-induced apoptosis was accompanied by the induction of Bax protein and the reduction in Bcl-2 protein. Auristatin PE and vincristine induced both Bax and Bcl-2 protein, leaving the Bax:Bcl-2 ratio constant. Reh cells pretreated for 24 h with bryostatin 1 followed by dolastatin 10, auristatin PE or vincristine showed significant apoptosis which was accompanied by Bcl-2 protein down regulation and Bax protein up regulation. We conclude that: (1) expression of bax is necessary for apoptosis-induction in this model; (2) a decrease in Bcl-2 level alone is not sufficient and might not be necessary for apoptosis-induction; and (3) the ratio of Bax:Bcl-2 plays a critical role in susceptibility to apoptosis in Reh cells. The results from this study should prove useful in guiding the clinical application of these novel agents in the treatment of acute lymphoblastic leukemia.

An MDM2 antagonist (MI-319) restores p53 functions and increases the life span of orally treated follicular lymphoma bearing animals

BackgroundMI-319 is a synthetic small molecule designed to target the MDM2-P53 interaction. It is closely related to MDM2 antagonists MI-219 and Nutlin-3 in terms of the expected working mechanisms. The purpose of this study was to evaluate anti-lymphoma activity of MI-319 in WSU-FSCCL, a B-cell follicular lymphoma line. For comparison purpose, MI-319, MI-219 and Nutlin-3 were assessed side by side against FSCCL and three other B-cell hematological tumor cell lines in growth inhibition and gene expression profiling experiments.ResultsMI-319 was shown to bind to MDM2 protein with an affinity slightly higher than that of MI-219 and Nutlin-3. Nevertheless, cell growth inhibition and gene expression profiling experiments revealed that the three compounds have quite similar potency against the tumor cell lines tested in this study. In vitro, MI-319 exhibited the strongest anti-proliferation activity against FSCCL and four patient cells, which all have wild-type p53. Data obtained from Western blotting, cell cycle and apoptosis analysis experiments indicated that FSCCL exhibited strong cell cycle arrest and significant apoptotic cell death; cells with mutant p53 did not show significant apoptotic cell death with drug concentrations up to 10 μM, but displayed weaker and differential cell cycle responses. In our systemic mouse model for FSCCL, MI-319 was tolerated well by the animals, displayed effectiveness against FSCCL-lymphoma cells in blood, brain and bone marrow, and achieved significant therapeutic impact (p < 0.0001) by conferring the treatment group a > 28% (%ILS, 14.4 days) increase in median survival days.ConclusionOverall, MI-319 probably has an anti-lymphoma potency equal to that of MI-219 and Nutlin-3. It is a potent agent against FSCCL in vitro and in vivo and holds the promises to be developed further for the treatment of follicular lymphoma that retains wild-type p53.

Bryostatin 1 induces apoptosis and augments inhibitory effects of vincristine in human diffuse large cell lymphoma

Bryostatin 1 (Bryo1), a macrocyclic lactone and a protein kinase C activator, is isolated from the marine bryozoan Bugula neritina. In this study we describe its effect, alone or after sequential use with vincristine (VCR), on the human diffuse large cell lymphoma cell line WSU-DLCL2. Our results show that both Bryo1 and VCR induced apoptosis as demonstrated by morphological examination, DNA flow cytometry (FCM), and DNA fragmentation on agarose gel electrophoresis. Cells pretreated for 24 h with Bryo1 and then exposed to VCR showed an increase in apoptosis compared to cells that were exposed to Bryo1 or VCR alone. We also studied the effects of Bryo1, VCR and their combination on cell growth, bcl-2 and p53 expression, and inhibition of cell proliferation as measured by [3H]-thymidine incorporation. Cell analysis showed significant growth inhibition of WSU-DLCL2 cells by the Bryo1/VCR combination as compared to either agent alone. Immunocytochemistry (ICC) revealed that relative bcl-2 oncoprotein expression was decreased in cells treated with Bryo1, or VCR separately and was abolished by combining both drugs. When examined by ICC, WSU-DLCL2 cells were initially negative for the p53 protein. However, upon treatment with the above agents, the relative expression of p53 was moderate on Bryo1-or VCR-treated cells and strong on cells treated with the Bryo1/VCR combination. Cell proliferation as measured by [3H]-thymidine incorporation revealed significant inhibition of tumor growth by exposure to the agents when compared to the control. In contrast, Bryo1, VCR and their combination did not show any inhibition of normal bone marrow growth. These findings taken together, suggest that the exposure of WSU-DLCL2 cells to Bryo1 prior to treatment with VCR enhances apoptosis, a phenomenon which might be exploited for future therapies.

Selective inhibitors of nuclear export for the treatment of non-Hodgkin’s lymphomas

The nuclear export protein chromosome maintenance region 1, found to be elevated in non-Hodgkin’s lymphomas, controls localization of critical tumor suppressor proteins. Nuclear localization of tumor suppressor proteins is necessary for their cell surveillance function. However, their nuclear exclusion by chromosome maintenance region 1 renders them ineffective making this nuclear transporter an attractive therapeutic target. We have identified selective inhibitors of nuclear export that lock tumor suppressor proteins in the cell nucleus leading to apoptosis of lymphoid but not normal cells. Our inhibitors induce tumor suppressor protein nuclear retention-dependent growth inhibition and apoptosis in a panel of non-Hodgkin’s lymphoma cell lines. Western blot of nuclear protein fraction and confocal microscopy analysis demonstrated retention of major tumor suppressor proteins in the cell nucleus. Co-immunoprecipitation studies showed disruption of the tumor suppressor protein-chromosome maintenance region 1 interaction. Small inhibitor RNA knockdown of two major tumor suppressor proteins, p53 in wild-type protein-53 and protein 73 in mutant-protein-53, abrogated inhibitor activity. Oral administration of related inhibitor at 75 and 150 mg/kg resulted in 65 and 70% tumor reduction, respectively and subcutaneous injections of inhibitor (25 and 75 mg/kg) resulted in 70 and 74% suppression of non-Hodgkin’s lymphoma tumor growth with no toxicity; residual tumors showed activation of the protein 73 pathway. Our study verifies chromosome maintenance region 1 as a therapeutic target in non-Hodgkin’s lymphoma, indicating that this nuclear export protein warrants further clinical investigations.

The bcl-2 and p53 oncoproteins can be modulated by bryostatin 1 and dolastatins in human diffuse large cell lymphoma

The effects of dolastatin 10 (Dol10) and dolastatin 15 (Dol15) alone, and after treatment with bryostatin 1 (Bryo1), on human diffuse large cell lymphoma cell line (WSU-DLCL2) were studied. At a concentration of 1.0 ng/ml Dol10 and Dol15 showed significant growth inhibition (p < 0.05). This inhibition was intensified when the cells were pretreated for 24 h with 200 nM Bryo1. Bryo1, Dol10 and Dol15 induced apoptosis which was seen on morphological examination, by flow cytometry and DNA fragmentation on agarose gel electrophoresis. Cells pretreated with Bryo1 and then exposed to Dol10 showed an increase in apoptosis compared with cells that were treated with the Dol10, Dol15 alone. Immunocytochemistry revealed that WSU-DLCL2 cells express the bcl-2 oncoprotein constitutively. bcl-2 expression was decreased when cells were treated with Bryo1, Dol10 or Dol15 and abolished with the Bryo1/Dol10 combination. WSU-DLCL2 cells were negative for p53 protein expression, upon treatment with Bryo1 or Dol10, the expression of p53 was weak and moderate with the Bryo1/Dol10 combination. The inverse correlation between bcl-2 and p53 oncoprotein expression seems to be related to induction of apoptosis in this lymphoma cell line.

Phase II study of bryostatin 1 in patients with relapsed multiple myeloma.

Bryostatin 1, a macrocyclic lactoneisolated from the marine bryozoan Bugula neritina,is a protein kinase C (PKC) modulator which has shown bothpreclinical and clinical activity inlymphoid malignancies. We conducted aphase II trial of bryostatin 1 administeredat a dose of 120 μg/m2 by 72-hcontinuous infusion every 2 weeks inpatients with relapsed multiple myeloma. Treatment was well tolerated with myalgiasconstituting the primaray toxicity. Therewere no responses in nine evaluablepatients. The preclinical anti-lymphoidactivity is strong enough to supportfurther exploration of bryostatin 1 indifferent schedules and in combinationtherapy for multiple myeloma.