SMI of Bcl-2 TW-37 is active across a spectrum of B-cell tumors irrespective of their proliferative and differentiation status

Design, synthesis, purification, and chemical characterization of TW-37 N-[(2-tert-butyl-benzenesulfonyl)-phenyl]-2,3,4-trihydroxy-5-(2-isopropyl-benzyl)-benzamide is described in detail in ref [14]; in the inactive congener TW-37a, all three hydroxyl groups in the polyphenolic ring have been substituted with a methyl group, resulting in a 100-fold loss of binding.

The acute lymphoblastic leukemia (WSU-pre-B-ALL), diffuse large cell lymphoma cell line (WSU-DLCL₂), follicular small cleaved cell lymphoma (WSU-FSCCL) and Waldenstrom's macroglobulinemia (WSU-WM) cell lines were established in our laboratory at the Wayne State University School of Medicine [20‐23]. The WSU-pre-B-ALL cell line is CD10+, CD19+, CD20+, TdT+; the WSU-DLCL2 and WSU-FSCCL are both mature (SIg+), CD20+ cell lines. The WSU-WM cell line is IgM-secreting cell line. Fresh peripheral blood samples were obtained from patients with active chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) or marginal zone lymphoma (MZL) in leukemic phase under IRB-approved protocol and used to assess the TW-37 cytotoxic effect on primary lymphoma cells. The CLL/SLL cells expressed CD5, CD19, CD20 and faint monotypic SIg. The MZL cells were CD5-, CD19+ and CD20+. Mononuclear cells were separated by Ficoll-Hypaque density centrifugation (Lymphoprep™, Fresenius Kabi Norge AS, Oslo, Norway), washed twice with PBS and then cell pellet was resuspended in RPMI-1640 culture medium.

Cells from established lines (above) were plated in 24-well culture clusters (Costar, Cambridge, MA) at a density of 2 × 10⁵ viable cells/ml/well. Triplicate wells were treated with 0.0–750 nM TW-37. Plates were incubated at 37°C in a humidified incubator with 5% CO₂. All cultures were monitored throughout the experiment by cell count and viability every 24 hr for 72 hr using 0.4% trypan blue stain (Gibco BRL, Grand Island, NY) and a hemacytometer. Fresh primary lymphoma cells isolated from patients were processed similarly except cells were seeded at a density of 5 × 10⁵/ml/well. Statistical analysis was performed using the t test, two-tailed, with 95% confidence intervals between treated and untreated samples. P value < 0.05 were used to indicate statistical significance.

After exposure to various concentrations of TW-37 for 48 or 72 hr, cells were collected by centrifugation and resuspended into 25 μl of PBS. One microliter of AO/EB mix was added to each sample prior to analysis by fluorescent microscope. Using fluorescence microscope, cells seen in orange or light orange were counted as apoptotic whereas cells in green or light green were counted as viable [24]. Data analysis was done using "GraphPad Prism 4.03" software.

Bcl-2 family protein expression profile without TW-37 treatment among 4 WSU lymphoma cell lines was determined as baseline as previously described [25]. Cells were seeded and cultured in T-75 cell culture flasks and harvested at exponential growth phase. Cells were lysed by buffer containing 50 mM Tris-HCL, 1% NP-40, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na₃VO₄ and protease inhibitor and total protein quantification determined using Protein Assay (BioRad, Hercules, CA). For Western Blotting, 40 or 100 μg of total protein was separated by 12% or 15% SDS-PAGE gel electrophoresis then transferred to nitrocellulose membrane (BioRad, Hercules, CA). Membranes were blocked with 5% Fat Free Dry Milk and subjected to immunoblotting using antibodies against individual human Bcl-2 family proteins [Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit or Pro-Survival Bcl-2 Family Antibody Sampler Kit (Cell Signaling Technology, Beverly, MA)] at 4°C overnight with agitation. After 3 washings, of 15 min each, membranes were blotted with horseradish peroxidase HRP-conjugated secondary antibody at room temperature for 2 hr. Following 3 washings of each membrane, protein was detected by ECL Western blotting detect reagent (GE Healthcare, Piscataway, NJ). Fresh patient samples were analyzed by the same method. All membranes in each experiment were stripped, blocked and further immunoblotted with anti-β-actin (Santa Cruz, Santa Cruz, CA) antibody to confirm equal loading and as reference for quantification of Bcl-2 family protein expression level among each cell line and sample. Expression level of each Bcl-2 family protein was determined by scanning band density using "AlphaEaseFC" software and normalized to density of the β-actin band of same sample and the quantification of the Bcl-2 family protein inventory, relative to β-actin, was tabulated. Similar procedures were used for TW-37 or TW-37a-treated cells and to detect caspase 3, 8, 9 and PARP cleavage using appropriate antibodies (Cell Signaling Technology, Beverly, MA).

Cells were seeded on white Luminometer 96-well plate (Fisher Scientific, Hanover Park, IL) at 2 × 10⁴ cells per 100 μl/well with various concentrations of TW-37 or 300 nM of TW-37a and cultured at 37°C, 5% CO₂. Caspase activity assay was performed after 4, 8, 2 and 24 hr of treatment using Caspase-Glo3/7 Assay and Caspase-Glo 9 Assay kit (Promega, Madison, WI). Assay procedure was done following manufacture's instruction using culture media without cells as blank control. One hundred μl of pre-mixed Caspase-Glo mixture was added to each assaying well with shake at 300 rpm for 30 seconds then incubated at room temperature protected from light for 1 to 3 hr. Luminescence was measured by Tecan Multifunction microplate reader at OD₄₅₀ nm versus OD₅₉₅ nm. Data was normalized by substituting substrate with blank control and analyzed by "GraphPad Prism 4.03" software. Statistical analysis was done using two-tailed t-test.

WSU-DLCL₂ and WSU-FSCCL cells were exposed to TW-37 or its trimethylated enantiomer (TW-37a) for 24 and 48 hr. 4 × 10⁶ cells were harvested from each condition and subsequently analyzed for DNA fragmentation using Apoptotic DNA Ladder Kit (Roche, Indianapolis, IN). DNA extraction procedure was done following manufacturer's instruction. DNA ladder was visualized by UV spectrometer after 1% agarose gel electrophoresis.

WSU-FSCCL cells were exposed to 1 or 2 μM TW-37 or TW-37-A for 24 hr then lysed in buffer containing 50 mM Tris·HCL, 1% CHAPS, 0.1% SDS, 150 mM NaCL, 1 mM EDTA, 1 mM PMSF, 1 mM Na₃VO₄ and protease inhibitor. 300 μg of total protein from each lysate was subjected for immunoprecipitation anti-Bim (Calbiochem, Darmstadt, Germany) in a total volume of 200 μl at 4°C with agitation. Supernatant was detected by Western blot with anti-Bim, anti-BclX_L (Calbiochem, Darmstadt, Germany) or anti-Mcl-1 (BD Biosciences, San Diego, CA) antibody and further detected with anti-Actin antibody (Santa Cruz, Santa Cruz, CA).

Four-week-old female ICR-SCID mice were obtained from Taconic Laboratory (Germantown, NY). The mice were adapted for several days and WSU-DLCL₂ xenografts were developed as described previously [26]. Each mouse received 10⁷ WSU-DLCL₂ cells (in serum-free RPMI-1640) subcutaneously (SC) in each flank area. When SC tumors developed to approximately 1500 mg, mice were euthanized, tumors dissected and mechanically dissociated into single-cell suspensions. Mononuclear cells were separated by Ficoll-Hypaque density centrifugation and washed twice with RPMI-1640 medium. These cells were subjected to phenotypic analysis for comparison with the established tumor cell line to insure the human origin and its stability. After formation of SC tumors, serial propagation was accomplished by excising the tumors, trimming extraneous materials, cutting the tumors into fragments of 20 to 30 mg that are transplanted SC using a 12 gauge trocar into the flanks of a new group of mice.

The maximum tolerated dose (MTD) for TW-37 is defined as the dose that will lead to no deaths of any of the animals and no more than 10% loss of body weight during treatment, followed by weight gain. To test the efficacy of TW-37 in vivo, small fragments of WSU-DLCL₂ xenograft were implanted SC bilaterally into naïve SCID mice as previously described. Mice were checked three times per week for tumor development. Once transplanted WSU-DLCL₂ fragments developed into palpable tumors (60–100 mg), groups of five animals were removed randomly and assigned to receive TW-37 or diluent (as control). Mice were observed for measurement of SC tumors, changes in weight and side effects of the drug. SC tumors were measured three times per week.

The end-points for assessing anti-tumor activity were according to standard procedures used in our laboratory and are as follows: Tumor weight (mg) = (A × B²)/2, where A and B are the tumor length and width (in mm), respectively; Tumor growth inhibition (T/C) is calculated by using the median tumor weight in the treated group (T) when the median tumor weight in the control group (C) reached approximately 900 mg. Tumor growth delay (T-C) is the difference between the median time (in days) required for the treatment group tumors (T) to reach 900 mg and the median time (in days) for the control group tumors (C) to reach the same weight and tumor cell kill (log₁₀) total = (T-C)-/(3.32)(Td).

All studies involving mice were performed under Animal Investigation Committee (AIC)-approved protocols. Tumor weights in SCID mice were plotted against time on a semi-log sheet with the growth pattern resembling an S-shape. Tumor doubling (Td) is the time (in days) required in order for the tumor to double its weight during the exponential growth phase.

The structure of TW-37 is given in Figure 1. The cell lines selected span the spectrum of the B-cell lineage. In addition, fresh peripheral blood samples of patients with CLL or leukemic phase of NHL were obtained under IRB-approved protocol. In each case, cells were exposed to TW-37 and TW-37a over 72 hr, and cell viability was determined. In general, exposure to TW-37 resulted in a dose-dependant inhibition of cell proliferation. The TW-37 concentration resulting in 50% growth inhibition (IC₅₀) of the established cell lines (Fig. 1A) were as follows: WSU-pre-B-ALL 180 nM (A.1); WSU-DLCL₂ 300 nM (A.2); WSU-FSCCL 165 nM (A.3); WSU-WM 320 nM (A.4). We have similarly tested growth inhibitory effect of TW-37 on 8 patient samples (pt) obtained from 7 patients. Patients 1–6 have a diagnosis of CLL/SLL whereas patient-7 has a diagnosis of marginal zone lymphoma (MZL). Two samples were obtained from case #6; one before therapy (pt.6a), and the second (pt.6) while the patient was on therapy with Rituximab and prednisone. None of the other patients were under active therapy at the time of obtaining blood samples except pt.2 who was receiving pulse dose chlorambucil and prednisone. There was no significant increase in cell numbers of control cultures after 72 hr; however, TW37-treated cultures showed progressive decrease in cell numbers, which was dose dependent (Fig. 1B). This was true of all patient samples although the effect was less profound in cells from pt.2 and pt.6 who were under treatment with chemotherapy for CLL/SLL. The inactive congener TW-37a had no effect (data not shown). Moreover, TW-37 had no effect on normal PBL (Fig. 1C).

Since TW-37 targets proteins in the apoptotic pathway; we investigated its ability to induce apoptotic cell death in lymphoid cell lines and patients samples:

TW-37 induced significant apoptosis in the cell lines and fresh patient samples (Fig. 2). This effect was specific since there was significant difference between TW-37 and TW-37a used under the same conditions. The highest proportion of cells in apoptosis was observed in WSU-FSCCL indicating higher sensitivity to TW-37 whereas the lowest was in WSU-WM (Fig. 2A). Similarly, TW-37 induced apoptosis on each of the three patient samples examined (Fig. 2B) with lower values in pt.2 that also showed less growth inhibition (Fig. 1B). Interestingly, the Bax-to-Mcl-1 ratio positively correlated with induction of apoptosis in the cell lines and in the 2 fresh cases studied (R² = 0.9682 and 0.9653 after 48 and 72 h of exposure to TW-37, respectively, Figure 2C).

Exposure of WSU-FSCCL cells to TW-37 induced activation of caspase 9 and caspase 3 activity and PARP cleavage (Fig. 3A.1). Using luminescent assay, Caspase activation was evident within 24 hr (Fig. 3A.2) and became more pronounced with longer incubation. Caspase 3 and 9 activation was evident as early as 4 hr after exposure to TW-37, which was again specific to TW-37. There was no activation of caspase 8. TW-37 also induced caspase 3 and 9 activation on WSU-DLCL₂ cells (Fig. 3B.1). To confirm induction of apoptosis, there was clear evidence of DNA fragmentation of extracts from both WSU-FSCCL and WSU-DLCL₂ cells (Fig. 3A.3 and 3B.2).

To determine if certain Bcl-2 family protein expression profiles are associated with increased susceptibility to TW-37, we determined the expression of major proteins in this family in all 4 cell lines and 5 of the fresh cases using Western Blotting analysis (Fig. 4). In all cases, fresh and cell lines, cells expressed at least 2 of the 3 anti-apoptotic proteins examined (Bcl-2, Bcl-X_L, and Mcl-1). Bcl-2 was over-expressed in all fresh cases, and cell lines except the WSU-WM (expressed low levels), Bcl-X_L was expressed in all patient cells and cell lines (except WSU-ALL cell line) and Mcl-1 was low only in WSU-ALL, WSU-DLCL2 and pt4. There was variation in the expression of the pro-apoptotic proteins examined. In every case there was at least 3 pro-apoptotic proteins expressed.

In general Western Blot analysis conducted on all 4 cell lines exposed to different concentrations of TW-37 at various time points showed no major changes in Bcl-2 family protein levels (Fig. 5A–D). There was apparent increase of Mcl-1 in WSU-pre-B-ALL cell line at 24 and 48 hr (Fig. 5A) but similar finding was not seen in other cell lines (Fig. 5B–D). Similarly, Bcl-X_L was more abundantly expressed in WSU-DLCL₂ after exposure to TW-37 for 72 hr (Fig. 5B) but the finding did not extend to other cell lines. The failure of drug treatment to induce consistent change in the steady-state level of Bcl-2 family proteins implies that baseline (i.e., not drug treated) quantitation of these proteins closely approximates the quantitation in drug-treated cells, at least over the 48 to 72 hr interval.

Protein lysates of TW-37-treated WSU-FSCCL cells were immunoprecipitated with antibody to Bim BH3-only proapoptotic protein. Immunoprecipitates were separated by SDS-PAGE and electroblotted to a membrane. Subsequent immunoblotting with Mcl-1 and Bcl-X_L revealed a decrease in Bim-Mcl-1 and Bim-Bcl-X_L complexes in the WSU-FSCCL-treated cells compared with untreated (control) cell lysates (Fig. 6). The blocking of Bim-Mcl-1 heterodimerization is evident at 1 μM TW-37 and increased at 2 μM; the blocking of Bim-Bcl-X_L heterodimerization is evident only at the highest drug concentration. This finding confirms the ability of TW-37 to block Bim-Mcl-1 and Bim-Bcl-X_L heterodimerization. Using similar technique, previously we have shown that TW-37 blocks Bid-Bcl-2 and Bid-Mcl-1 but not Bid-Bcl-X_L in WSU-DLCL₂ cell lysate [27].

The MTD of TW-37 in SCID mice was determined to be 120 mg/kg when given alone as intravenous (iv) injections (40 mg/kg daily × 3 doses). Animals at this dose experienced weight loss of < 5% and had scruffy fur, however with full recovery 48–72 hours after completion of treatment.

Antitumor activity of TW-37 at its MTD against WSU-DLCL₂-bearing SCID mice as measured by tumor growth inhibition (T/C), tumor growth delay (T-C) and log₁₀kill was 28%, 10 days and 1.5, respectively (Table 1). A T/C value of 42% or less is considered significant anti-tumor activity by NCI, the drug evaluation branch of the division of cancer treatment. Therefore, TW-37 is considered active against WSU-DLCL₂ tumor and resulted in significant growth delay (p ≤ 0.01) compared with control (Fig. 7).