1. OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells
by Ta-Ming Liu, Yonghua Ling, Jennifer A. Woyach, Kyle Beckwith, Yuh-Ying Yeh, Erin Hertlein, Xiaoli Zhang, Amy Lehman, Farrukh Awan, Jeffrey A. Jones, Leslie A. Andritsos, Kami Maddocks, Jessica MacMurray, Santosh B. Salunke, Ching-Shih Chen, Mitch A. Phelps, John C. Byrd, and Amy J. Johnson
Blood
Volume 125(2):
January 8, 2015
©2015 by American Society of Hematology

2. OSU-T315 induces preferential cytotoxicity in CLL cells
OSU-T315 induces preferential cytotoxicity in CLL cells. 1E7/mL primary CLL cells from patients; (A) healthy B cells (CD19+) and (B) T cells (CD3+) from leukopaks purified by the CD19+ or CD3+ enrichment kit, respectively, were incubated in complete RPMI wi...
OSU-T315 induces preferential cytotoxicity in CLL cells. 1E7/mL primary CLL cells from patients; (A) healthy B cells (CD19+) and (B) T cells (CD3+) from leukopaks purified by the CD19+ or CD3+ enrichment kit, respectively, were incubated in complete RPMI with 10% fetal bovine serum followed by increasing dose of OSU-T315 treatment. Cells viability was analyzed by flow cytometry at 24 hours. (C) The response toward OSU-T315 in CLL with cytogenetic abnormalities were analyzed by del(17p13.1) and IGVH status or (D) del(11q22.3) and del(13q14.3). (E) 1E6/mL cells in complete RPMI with 10% fetal bovine serum was incubated and treated with increasing concentration of OSU-T315. Cells viability on treatment in Mec-1 or OSU-CLL cells was examined at 24 hours by Annexin V/PI staining. (F) Three ibrutinib-resistant samples were treated with OSU-T315 for 24 hours in comparison with nonresistant CLL cells; 1 μM ibrutinib was used for treatment for 30 minutes and then washed out; 1 μM CAL-101 was used for treatment for 24 hours.
Ta-Ming Liu et al. Blood 2015;125:
©2015 by American Society of Hematology

3. The apoptotic machineries were induced on T315 treatment.
The apoptotic machineries were induced on T315 treatment. (A) CLL cells were incubated in α-IgM coated plates or (C) fibronectin-coated plates or 1 μg/mL VCAM-1 with vehicle or OSU-T315 for 16 hours. Cell lysate was analyzed by immunoblotting and normalized to GAPDH, and the ratio of densitometry to GAPDH was shown beneath the blots. (B) Mcl-1 expression level was quantified according to the results of immunoblotting in the separated conditions of plate-coated α-IgM, 0.5 μg/mL CD40L, 3.2 μM CpG-ODN, or (D) fibronectin-coated plates or 1 μg/mL VCAM-1 along with the treatment of vehicle or 4 μM OSU-T315 for 16 hours. (E) Caspase 3/7 activity was accessed by Caspase-Glo 3/7 Assay Systems (Promega) and normalized to vehicle after 16-hour treatment; 20 μM of Q-VD-OPh served as the negative control, whereas 500 nM flavopiridol served as the positive control. (F) Mec-1 or OSU-CLL cells pretreated with vehicle or 20 μM z-VAD-FMK prior to 4 μM OSU-T315 treatment were analyzed by Annexin V/PI staining after 24 hours.
Ta-Ming Liu et al. Blood 2015;125:
©2015 by American Society of Hematology

4. OSU-T315 targets intrinsic AKT and ERK signals cascades in CLL cells.
OSU-T315 targets intrinsic AKT and ERK signals cascades in CLL cells. (A) Lysate from Mec-1 and OSU-CLL cells treated with serial concentrations of OSU-T315 for 15 minutes are subjected to western blot analysis. (B) Lysate from primary CLL cells treated with OSU-T315 were subjected to analyze PDK1 (Ser241) and subsequent AKT (Thr308) activation. (C) In vitro kinase activity of class I PI3K is evaluated by the PI3 Kinase Activity/Inhibitor Assay Kit (Millipore) according to the instruction manual; 100 nM Wortmannin was applied as positive control. The biotinylated-PIP3 was set as 100%. The kinase reactions with vehicle or OSU-T315 were referenced to the biotinylated-PIP3 signal to have the relative percentage of inhibition. (D) RAS activity in 697 cells on treatments was measured by the Active Ras Detection Kit (Cell Signaling) according to the instruction manual. Guanosine triphosphate γS (positive control) and guanosine diphosphate (negative control) ensured the immunoprecipitation procedures worked properly, whereas insulin-like growth factor-1 served as a positive control to activate Ras. (E) Mec-1 and OSU-CLL cells pretreated with Okadaic acid (1 μM) were incubated with either OSU-T315 (4 μM) or OSU (5 μM), the PDK1 inhibitor, and the total lysate was subjected to immunoblotting to verify downstream signaling.
Ta-Ming Liu et al. Blood 2015;125:
©2015 by American Society of Hematology

5. OSU-T315 impairs AKT translocation to lipid raft subdomains in plasma membrane.
OSU-T315 impairs AKT translocation to lipid raft subdomains in plasma membrane. (A) Mec-1 cells were retro-virally transduced with pBABE-Myr-flag-AKT vector (Addgene). The total lysates were subjected to western blot to verify the myristoylated AKT expression. (B) Mec-1 expressing Myr-flag-AKT was treated with increasing concentration of OSU-T315. Total lysate was analyzed by immunoblotting. (C) Lipid raft from Mec-1 cells expressing Myr-flag-AKT was purified after vehicle or OSU-T315 (4 μM) treatment by the ultracentrifugation approach. The raft (R) and nonraft (NR) fractions were analyzed for raft-associated molecules, and Flotillin-1 serves as a lipid raft marker. (D) The 3 independent studies were quantified for AKT content in raft compartment. (E) Mec-1 cells with Myr-flag-AKT were subject to immunofluorescence staining with antibodies of α-AKT (Alexa 594) and α- Cholera toxin subunit B (CT-B) (Alexa 488). (F) The colocalization index was measured and analyzed by confocal microscope double blindly. (G) 697 cells were treated with plate-bounded α-IgM, 4 μM OSU-T315, or in combination for 1 hour, and the lipid raft fractions were extracted for analysis. Cholera toxin subunit B (CT-B) is the marker for lipid raft compartment.
Ta-Ming Liu et al. Blood 2015;125:
©2015 by American Society of Hematology

6. OSU-T315 inhibits BCR-, CD40L-, and CpG-induced survival signal.
OSU-T315 inhibits BCR-, CD40L-, and CpG-induced survival signal. (A) CLL cells were treated with either plate-bounded α-IgM, 0.5 μg/ml CD40L, 3.2 μM CpG-ODN, or (C) plate-bound fibronectin or 1 μg/ml VCAM-1 combined with vehicle or 4 μM OSU-T315 for 15 minutes. The total lysate was subjected to immunoblotting. (B,D) Data from individual patients were quantified for p-AKT level and normalized to GAPDH. (E-G) Cell viability was examined after 24 hours in the treated conditions of plate-bounded α-IgM, 0.5 μg/ml CD40L, or 3.2 μM CpG-ODN combined with vehicle or different doses of OSU-T315.
Ta-Ming Liu et al. Blood 2015;125:
©2015 by American Society of Hematology

7. Prolonged survival of TCL1 leukemia-engrafted mice by OSU-T315.
Prolonged survival of TCL1 leukemia-engrafted mice by OSU-T315. (A) C57BL/6 mice engrafted with TCL1 leukemia cells were treated orally with vehicle or 50 mg/kg OSU-T315 daily after appearance of 10% leukemia cells in the peripheral blood. WBCs were monitored by blood smear slides weekly until euthanization was required. Data represent WBC counts after 4-week treatment. (B) Overall survival was analyzed after treatment starts (n = 6 per group). (C) C57BL/6 mice engrafted with TCL1 leukemia cells were treated by intraperitoneal injection with vehicle or 25 mg/kg OSU-T315 once daily for 2 weeks after the appearance of 5% CD19+, CD5+ leukemia cells among CD45+ peripheral blood mononuclear cells. OSU-T315 was formulated in phosphate-buffered saline containing 10% Cremophor EL (Sigma). After a 2-week daily scheme, leukemic mice were treated every other day with vehicle or 25 mg/kg OSU-T315 to prevent weight loss. The percent of leukemic cells was monitored weekly by flow cytometry until euthanization was required. Overall survival was analyzed (n = 9 for each group). (D) Data represent the pharmacokinetics of OSU-T315 in plasma after intravenous, intraperitoneal, or oral dosing (n = 6 per group).
Ta-Ming Liu et al. Blood 2015;125:
©2015 by American Society of Hematology