DT2216 and DT2216 negative compound (DT2216 NC) were synthesized in our lab according to the protocol as described previously . ABT263 (Cat. No. S1001), ABT199 (Cat. No. S8048), S63845 (Cat. No. S8383), MG132 (Cat. No. S2619), doxorubicin (Cat. No. S1208), etoposide (Cat. No. S1225), and vincristine (Cat. No. S1241) were purchased from Selleckchem (Houston, TX, USA). A-1155463 (Cat. No. HY-19725) was purchased from MedChem Express (Monmouth Junction, NJ, USA).
The TCL cell lines MyLa, MJ, MAC2A, L82, FEPD, SMZ1, and DL40 were cultured in RPMI medium supplemented with 10 or 20% FBS (Cat. No. 97068-085, VWR, Atlanta, GA, USA), and 1% penicillin-streptomycin (Cat. No. 15140122, Thermo Fisher Scientific) in a humidified incubator at 37 °C and 5% CO2. Specifically, MyLa cells were cultured in RPMI medium supplemented with 10% FBS and 100 units/mL IL-2 (PHC0021, Cat. No. 12430054, Thermo Fisher Scientific, Waltham, MA, USA). MJ and DL40 cells were cultured in RPMI medium supplemented with 20% FBS. MAC2A, L82, and SMZ1 cells were cultured in RPMI medium supplemented with 10% FBS. Human 786-O renal cell adenocarcinoma cells (Cat. No. CRL-1932) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in DMEM (Cat. No. 12430054, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and 1% penicillin-streptomycin.
Cell and platelet viability assay
Cells from different TCL cell lines were seeded into 96-well plates at 1 × 105/well and treated for 72 h or indicated time points with various agents. Platelets were isolated from human platelet-rich plasma (PRP, Cat. No. SER-PRP-SDS, Zenbio, Research Triangle Park, NC, USA) . Briefly, PRP was transferred into a 50-mL tube containing 5 mL acid citrate buffer (Cat. No. sc-214744, Santa Cruz Biotechnology, Dallas, TX, USA). To prevent clotting, prostaglandin E1 (PGE1, Cat. No. sc-201223A, Santa Cruz Biotechnology) and apyrase (Cat. No. A6237, Sigma-Aldrich) were added to final concentrations of 1 μM and 0.2 units/mL, respectively. After gently mixing the solution, platelets were pelleted by centrifugation at 1200×g for 10 min without a break. Pelleted platelets were gently washed in 2 mL HEPES Tyrode’s buffer (Cat. No. PY-921WB, Boston BioProducts, Ashland, MA, USA) containing 1 μM PGE1 and 0.2 units/mL apyrase. After washing, pellets were suspended in 10 mL HEPES Tyrode’s buffer containing 1 μM PGE1, 0.2 units/mL apyrase, and 10% FBS. Platelet number was counted using the HEMAVET 950FS hematology analyzer (Drew Scientific, Miami Lakes, FL, USA). For viability assays, platelet number was adjusted to 2 × 108/mL in HEPES Tyrode’s buffer containing 1 μM PGE1, 0.2 units/mL apyrase and 10% FBS. Each treatment was performed in 2 mL platelet suspension in 15 mL polypropylene tubes. The tubes were placed on a rotating platform at room temperature, and the viability of platelets was measured after treatment for indicated time points. For measuring the viability, platelets were transferred to a 96-well plate (200 uL/well).
Cell and platelet viabilities were measured by the tetrazolium-based MTS assay according to the manufacturer’s instructions. Briefly, MTS reagent (2 mg/mL stock, Cat. No. G1111, Promega Madison, WI, USA) was freshly supplemented with phenazine methosulfate (PMS, 0.92 mg/mL stock, Cat. No. P9625, Sigma-Aldrich, St. Louis, MO, USA) at a 20:1 ratio, and 20 μL of this mixture was added to each control and treatment well. The cells and platelets were incubated for 4 h at 37 °C and 5% CO2, and then, the absorbance was recorded at 490 nm using Biotek’s Synergy Neo2 multimode plate reader (Biotek). The half maximal effective concentration (EC50) values of individual agents were calculated with the GraphPad Prism 7 software (GraphPad Software, La Jolla, CA, USA). The combination index (CI), EC25, EC50, and EC75 values were calculated using the Compusyn software (http://www.combosyn.com).
Cell apoptosis assays
Cell apoptosis assay was done as described previously . Briefly, cells were treated with vehicle or 10 μM Q-VD-OPh (QVD, Cat. No. S7311, Selleckchem, Houston, TX, USA) for 4 h prior to the addition of DT2216 for 24 h. Cells were harvested in polystyrene round-bottom tubes (Cat. No. 352058, Falcon, Corning, NY, USA). The cells were stained with Alexa Fluor 647-Annexin V (1:50, Cat. No. 640912, BioLegend, San Diego, CA, USA) and propidium iodide (PI, 10 μg/mL, Cat. No. 421301, BioLegend, San Diego, CA, USA) at room temperature for 30 min. Apoptotic cells were analyzed using flow cytometry (LSR II, BD Biosciences, San Jose, CA, USA).
Proteins in cell lysates and tissue homogenates of the primary tumors from MyLa cell-engrafted mice or the spleens from DFTL-28776 cell-engrafted PDX mice were extracted using the RIPA buffer (Cat. No. BP-115DG, Boston BioProducts, Ashland, MA, USA) supplemented with 1% protease and phosphatase inhibitor cocktail (Cat. No. PPC1010, Sigma-Aldrich, St. Louis, MO, USA). Samples were lysed on ice for 30 min and then kept at − 80 °C freezer overnight. After centrifugation at 15,000×g at 4 °C for 15 min, the supernatant was collected and the protein concentration was measured using the Pierce BCA protein assay kit (Cat. No. 23225, Thermo Fisher Scientific). An equal amount of proteins (30–60 μg/lane) was loaded to a precast gel (Mini-PROTEAN TGX, Cat. No. 456-1094, Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes (Invitrolon, Cat. No. LC2002, Life Technologies, Carlsbad, CA, USA) by electrophoresis. The membranes were blocked with 1X TBS-Tween (TBST, Cat. No. J77500, Affymetrix, Santan Clara, CA, USA) containing 5% non-fat dry milk (Cat. No. sc-2324, Santa Cruz Biotechnology, Dallas, TX, USA) and subsequently probed with primary antibodies at a predetermined optimal concentration overnight at 4 °C. The primary antibodies including Bcl-xL (Cat. No. 2762), Mcl-1 (Cat. No. 5453), Bcl-2 (Cat. No. 4223), Bax (Cat. No. 2772), Bak (Cat. No. 12105), Bim (Cat. No. 2933), Noxa (Cat. No. 14766), VHL (Cat. No. 68547), Caspase-3 (Cat. No. 9662), cleaved Caspase-3 (Cat. No. 9661), PARP (Cat. No. 9532), β-actin (Cat. No. 4970), and the secondary horse radish peroxidase (HRP)-linked antibody (Cat. No. 7074) were purchased from Cell Signaling Technology (Danvers, MA, USA). After washing with TBST for 3 times (10 min each time), the membranes were incubated with the secondary antibody for 2 h at room temperature. Following sufficient washing with TBST, the membranes were incubated with chemiluminescent HRP substrate (Cat. No. WBKLS0500, MilliporeSigma, Billerica, MA, USA). The blotting membranes were recorded using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). The immunoblots were quantified using the ImageJ version 1.52a software.
Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted using an RNeasy Mini Kit (Cat. No. 74106, Qiagen, Gaithersburg, MD, USA). RNA (1 μg) was reverse-transcribed with the High-Capacity cDNA Reverse Transcription Kit (Cat. No. 4368813, Thermo Fisher Scientific). Real-time PCR was performed using specific TaqMan probes (Cat. No. 4351370, BCL2L1 id: Hs00236329_m1; BCL2 id: Hs00608023_m1 and MCL1 id: Hs01050896_m1; GAPDH id: Hs02758991_g1) and the TaqMan Fast Advanced Master Mix (Cat. No. 4444965, Thermo Fisher Scientific). All reactions were run in triplicate on an ABI QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Data were normalized to GAPDH and calculated using the 2– ∆∆CT method .
MyLa TCL xenograft mouse model and treatment
The animal work described in this study was approved by and done in accord with the Institutional Animal Care and Use Committee of the University of Florida. Female NOD-scid IL2Rgnull (NOD/SCID) mice were purchased from The Jackson Laboratory (Stock No. 005557; The Jackson Laboratory, Bar Harbor, ME, USA) at 5 weeks of age. They received food and water ad libitum and were allowed to acclimatize for 1 week before being used for experiments at the age of 6 weeks. One million (1 × 106) MyLa cells were suspended in 100 μL of regular RPMI 1640 Medium (Cat. No. 22400089, Thermo Fisher Scientific, Waltham, MA, USA): Matrigel (Cat. No. 356231, Corning, New York, USA) 1:1 mixture and s.c. implanted in the right flank of NOD/SCID mice. Tumor growth was monitored daily and tumors were measured twice a week using Vernier caliper or digital calipers. Tumor volume was determined using the formula [(L × W2) × 0.5], where L is the length/long dimension in millimeter (mm) and W is the width/short dimension in millimeter. The treatment started once the average tumor volume reached 100 mm3. The animals were randomly assigned to treatment groups (n = 8 mice per group at the start of treatment) such that each group had a nearly equal starting average tumor volume. Mice were weighed twice a week, and the treatments were given according to average mouse weight within each group before initiation of treatment. They were treated with vehicle (10% ethanol, 30% PEG 400, and 60% PHOSAL 50 PG, once a day [qd] by gavage [p.o.]; 50% PHOSAL 50 PG, 45% MIGLYOL® 810 N, and 5% polysorbate 80, every 4 days [q4d] via intraperitoneal injection [i.p.]), ABT263 (formulated in 10% ethanol, 30% polyethylene glycol 400 (PEG 400), and 60% PHOSAL 50 PG; 50 mg/kg, qd/p.o.), or DT2216 (formulated in 50% PHOSAL 50 PG, 45% MIGLYOL® 810 N, and 5% polysorbate 80; 15 mg/kg, q4d/i.p.). PEG 400 (Cat. No. HR2-603) was from Hampton Research (Aliso Viejo, CA, USA). Ethanol (Cat. No. BP2818500) was from Fisher Scientific (Pittsburgh, PA, USA). PHOSAL 50 PG (Cat. No. 368315-3130003/020) was from American Lecithin Company (Oxford, CT, USA). MIGLYOL® 810 N was from IOI Oleochemical (Hamburg, Germany). Polysorbate 80 (Cat. No. P0138) was from Spectrum Chemical (New Brunswick, NJ, USA). Blood was collected 1 day after the first dose of the drugs for complete blood cell count (CBC) analysis. The mice were euthanized when the maximum tumor size reached the humane endpoint according to institutional policy concerning tumor endpoints in rodents. In addition, to prevent excessive pain or distress, the mice were euthanized if the tumors became ulcerated or the mice showed any signs of ill health. Mice were euthanized by CO2 suffocation followed by cervical dislocation, and various tissues including tumors were harvested for further analyses.
DFTL-28776 T cell prolymphocytic leukemia (T-PLL) PDX model and treatment
Four-week-old female NOD/SCID mice from The Jackson Laboratory were allowed to acclimatize for 1 week before being used for experiments at the age of 5 weeks. DFTL-28776 cells were purchased from PRoXe (https://www.proxe.org/) and expanded once in NOD/SCID mice according to the instructions from PRoXe. They were injected into NOD/SCID mice via the tail veins at 1 × 106 cells/mouse. Mice were weighed twice a week and DFTL-28776 cell engraftment in blood was monitored weekly by measuring human CD45+CD2+ cell engraftment in blood as described below. For the initial efficacy and survival study, DFTL-28776 cells were freshly harvested from the spleen of a host mouse with 60% of blood engraftment of DFTL-28776 cells and were immediately engrafted into NOD/SCID mice for further study. Mice with substantial engraftment of DFTL-28776 cells in blood (> 1.0%) were randomly assigned to different treatment groups (n = 5 mice for VEH and n = 4 mice for other groups at the start of treatment). They received treatment with vehicle, ABT199 (50 mg/kg, qd/p.o.), DT2216 (15 mg/kg, q4d/i.p.), or DT2216 plus ABT199. The mice were euthanized when they became moribund according to institutional policy concerning tumor endpoints in rodents by CO2 suffocation followed by cervical dislocation. The spleens were harvested from the mice for immunoblotting analyses of Bcl-xL, Bcl-2, and Mcl-1 expression. To assess additive vs. synergistic activity of DT2216 + ABT199 in DFTL-28776 TCL PDX mice, the Bliss independence model  was adapted to survival analysis as previously described . A second study was performed to further characterize the effects of ABT199 and/or DT2216 on DFTL-28776 T-PLL PDX, in which previously frozen DFTL-28776 cells were used for the xenografts. Mice with a detectable engraftment of DFTL-28776 cells in blood (≥ 0.1%) were randomly assigned to different treatment groups (n = 6 mice per group); the average blood engraftment of DFTL-28776 cells per group was around 0.1%. They received treatment with vehicle, ABT263 (50 mg/kg, qd/p.o.), ABT199 (50 mg/kg, qd/p.o.), DT2216 (15 mg/kg, q4d/i.p.), or DT2216 plus ABT199. Vehicle-treated mice were euthanized 27 days after the initiation of the treatment due to their deteriorating condition, while the other groups were euthanized 7 days later. The spleen, liver, bone marrow, and blood were harvested. Age-matched female NOD/SCID control (CTL) mice (n = 5) without engraftment or any other treatments were included for comparative purposes. The weight of the spleens and livers from each mouse was measured and recorded. DFTL-28776 cell engraftment in the spleen, bone marrow, and blood were analyzed as described below.
Analysis of DFTL-28776 cell engraftment in the spleen, bone marrow, and blood
Spleens were disaggregrated into RPMI 1640 medium supplemented with 2% FBS by grinding with plunger of a 3-mL sterile syringe (Cat. No. 309657, BD Biosciences, San Jose, CA, USA). The suspension was filtered through 70-μm cell strainers (Cat. No. 22-363-548, Thermo Fisher Scientific, Waltham, MA, USA). Bone marrow cells from the femora and tibiae were flushed into RPMI 1640 supplemented with 2% FBS using a 3-mL sterile syringe. Approximately 50 uL of blood was collected from each mouse via submandibular plexus route in an EDTA tube (Cat. No. 077051, RAM Scientific, Inc., Nashville, TN, USA). After red blood cells had been lysed with 1X BD Pharm Lyse™ lysing solution (Cat. No. 555899, BD Biosciences, San Jose, CA, USA), about 200,000 splenocytes, bone marrow cells, and white blood cells were stained with the mouse anti-human CD45 monoclonal antibody conjugated to BD Horizon™ V450 (Cat. No. 560367; BD Biosciences, San Jose, CA, USA) and mouse anti-human CD2 monoclonal antibody conjugated to APC (Cat. No. 300214; BioLegend, San Diego, CA, USA). Human CD45+CD2+ cells were analyzed on an Aurora flow cytometer (Cytek Aurora, Fremont, CA, USA).
DFTL-28776 PDX cell purification and analyses
When DFTL-28776 cell engraftment in blood reached approximately 60%, DFTL-28776 PDX mice were euthanized by CO2 suffocation followed by cervical dislocation. The spleens were harvested from the mice for the preparation of single splenocyte suspension. DFTL-28776 cells were isolated using the EasySep Mouse/Human Chimera Isolation Kit (Cat. No. 19849; StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer’s instructions. Cell viability of isolated DFTL-28776 cells after treatment with different agents was measured by tetrazolium-based MTS assay as described above with a slight modification. Briefly, DFTL-28776 cells were seeded into 96-well plates at 2 × 105/well and treated for 24 h with various agents, the absorbance was read after adding the MTS reagents and incubation with cells overnight. All other assays for DFTL-28776 cells were done in a way similar to the assays for other TCL cell lines described above.
Hematoxylin & eosin staining and anti-hCD45 immunostaining of the spleens and livers of DFTL-28776 PDX mice
The formalin-fixed tissues of mice from different groups were embedded in paraffin, sectioned into 5-μM slices, and mounted onto glass slides. Hematoxylin & eosin (HE) staining was done by the Molecular Pathology Core of the University of Florida. A set of slides was used for immunostaining of human CD45 (Cat. No. M0701, DAKO, Agilent Technologies, Inc., Santa Clara, CA, USA). Slides were immersed twice (5 min each time) in xylene solution followed by serial washes with 100, 95, and 75% ethanol, respectively (2 min each time). Then, the slides were dipped in water twice and then incubated in trilogy solution (Cat. No. 920P-10, Cell Marque Corporation, Rocklin, CA, USA) at 95 °C for 25 min. After this incubation, the slides were washed under running water for 1 min and then washed with 1X TBST for 5 min. After incubation in 1.5% horse serum (Cat. No. ZE0122, Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min, the slides were washed with TBST again and then sequentially incubated with avidin and biotin blocking solutions in a commercial kit (Cat. No. ZE0919, Vector Laboratories, Inc., Burlingame, CA, USA) following the manufacturer’s instructions. The slides were washed with TBST and incubated with mouse monoclonal anti-human CD45 primary antibody (1:50) overnight followed by three washes with TBST. Slides were incubated with anti-mouse secondary antibody (1:200) for 30 min, washed again with TBST, followed by incubation with reagents in the Vectastain Alkaline Phosphatase Standard AK-5100 kit (Cat. No. ZE0628, Vector Laboratories, Inc., Burlingame, CA, USA). Finally, the slides were washed with TBST and mounted with a coverslip using the mounting medium with DAPI (Cat. No. ZD0421C, Vector Laboratories, Inc., Burlingame, CA, USA). The slides were visualized under a fluorescent microscope (Olympus BX43 with Olympus DP80 camera) at a × 200 magnification to obtain images.
Complete blood cell counts
Approximately 50 μL of blood was collected from each mouse in EDTA tubes via the submandibular plexus. The blood was immediately used for CBC analysis using the HEMAVET 950FS hematology analyzer (Drew Scientific, Miami Lakes, FL, USA). The data were expressed as the number of different blood cells or platelets per microliter of blood.
Most of the graphs presented in this manuscript were made and statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA). For analysis of means of three or more groups, analysis of variance (ANOVA) tests were performed. In the event that ANOVA justified post hoc comparisons between group means, the comparisons were conducted using Tukey’s multiple comparison test. Two-sided unpaired Student’s t test was used for comparisons between the means of two groups. Kaplan-Meier test was used for the survival rate analysis, and the data were statistically analyzed using log-rank (Mantel-Cox) test. The Bliss independence model was adapted to assess additive vs. synergistic activity of DT2216 + ABT199 in the DFTL-28776 TCL PDX mice as we previously described . p < 0.05 was considered statistically significant.