Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.


Supplementary Figure 4: Synergistic effect of pan-PIM inhibitor(s) with Ponatinib in a subset of T-ALL cell lines. (A)
H-SB2 cells were incubated with AZD1208 (1 µM) and Ponatinib (10 nM) for 18 h and cell lysates were immunoblotted with specified antibodies. (B and C) H-SB2 and KOPT-K1 cells were treated with AZD1208 either alone or in combination with Ponatinib for 72 h and percentage viable cells was determined by XTT assay. The percent growth inhibition of drugs alone and in combination was determined and the combination index (CI) for Ponatinib 5 nM/10 nM and indicated concentrations of AZD1208 combination was determined by Combosyn, Inc. The combination index value of <, = and > 1 indicate synergism, additive effect and antagonism, respectively. (D) DU.528 and CUTLL1 cell lines were treated with DMSO, AZD1208 (1µM), either alone or in combination with Ponatinib (10 nM) for 72 h. Percent viability was determined by XTT assay. The growth of DMSO control cells is considered 100% and percentage cell growth for individual treatment is reported relative to the DMSO control cells.
(E) DU.528 cells treated similar to D, and percentage apoptosis was determined by Guava Nexin assay followed by flow cytometry. (F and G) SUP-T1 cells were treated with DMSO, AZD1208 (1 µM), LGB-321 (1 µM) either alone or in combination with Ponatinib (500 nM) for 72 h. Percent relative viability was determined by XTT assay. Percentage apoptosis was determined by Guava Nexin assay followed by flow cytometry. XTT and Apoptosis data shown are the average +/− S.D. of three independent experiments.

Supplementary Figure 5: Effect of pan-PIM inhibitor(s) in combination with Dasatinib in T-ALL cell lines.
(A) KOPT-K1 cell lines were treated with indicated concentrations of LGB-321 either alone or in combination with Dasatinib for 72 h and percentage viable cells was determined by XTT assay. (B) SUP-T1 cells were treated with indicated concentrations of AZD1208 or NVPLGB-321 either alone or in combination with Dasatinib for 72 h and percentage viable cells was determined by XTT assay. The growth of DMSO control cells is considered 100% and percentage cell growth for individual treatment is reported relative to the DMSO control cells. XTT data shown is the average +/− S.D. of three independent experiments.

Cell viability (XTT) assay
For drug cytotoxicity experiments, T-ALL cells were seeded into 96-well plates at a density of 20,000 cells per well, and pa-PIM kinase inhibitors (AZD1208 & LGB-321), Ponatinib or combinations were added at a range of doses for 72 h, using DMSO as control. Cell viability was measured using XTT cell proliferation assay (Trevigen Cat # 4891-025-K) following manufacturer's protocol. Briefly, XTT reagent was added to cell culture (1:2 dilution) and incubated for 4 h at 37°C and 5% CO 2 . The absorbance of the colored formazan product was measured at 450 nm.

Development of SUP-T1 persister cells
Persister cells were developed as described previously (24) by repeatedly exposing SUP-T1 cells to a 1 µM of Gamma Secretase Inhibitor, Compound E (EMD4 Biosciences) over the period of 7 weeks, replenishing the inhibitor every 3-4 days. Persister cells were characterized as reported previously.

Real-Time PCR
Total RNA was isolated using RNAeasy kit and 1 ug RNA was used to synthesize cDNA (Bio-Rad, Cat # 1708890) following the manufacturers protocol. The quantification of real-time PCR products was performed using SsoAdvanced ™ Universal SYBR ® Green Supermix (Bio-RAD, Cat#1725271) on a CFX96 Real-Time System. Samples were assayed in triplicate and the data were normalized to 18S mRNA levels. Primer sequences are provided in TableS4.

Cell cycle analysis
Cell cycle progression was analyzed by Guava Easy Cyte flow cytometer. After drug treatment cells were centrifuged, washed with PBS, and fixed in 70% ethanol solution overnight at −20°C. Fixed cells were centrifuged and pellet was washed with PBS before stained using a propidium iodide-RNase A solution (PBS containing 50 ug/ml Propidium Iodide, 0.1 mg/ml RNase A, and 0.05% Triton X-100) for 30-40 min at 37°C in the dark.

Assessment of apoptosis by annexin V staining
The percentage of apoptotic cells were detected by using Guava Nexin reagent (Millipore #4500-0450) following the manufacturer's instructions using the Guava Easy Cyte flow cytometer. Briefly, cells were mixed with Guava Nexin reagent (1:2 dilution) and incubated for 20 minutes at room temperature in the dark. In the Guava Nexin assay, Annexin V-PE positive stained cells are in the bottom right quadrant of the display and represents early apoptotic cells. While the cells with 7-AAD staining appear in the top right quadrant and represents the late apoptotic and dead cell populations.

CFSE staining
T-ALL cells were stained using cell Trace CFDA SE dye, (Vybrant CFDA SE cell tracer kit, Invitrogen, Cat # V12883) diluted in PBS to give a final staining concentration of 5 μM. The staining reagent containing the cells were mixed well until homogeneity and incubated at room temperature for 10 min. The solution was then centrifuged at 800 rpm for 5 min and the supernatant was discarded. The cells were washed by resuspending the pellet in 1-2 ml of fresh RPMI Medium, centrifuging at 800 rpm for 5 min and discarding the supernatant. After washing, the cells were treated with DMSO or AZD1208 for 48 hours and the percentage CFSE positive cells quantitated by flow cytometry.

Generation of the luciferase-expressing, H-SB2 cells
T-ALL cells line, HSB2 was electroporated with plasmid (Pluc-ff-Zeo), using Human CD34+ cell Nucleofector kit and Nucleofector II Device (Amaxa scientific, Allendale, NJ). Positive cells were selected by gradually increasing concentrations of Zeocin. Luciferase activity of selected cells were tested by Dual-Glo luciferase kit (Promega, Madison, WI).