Differential Effects of AZD-1208 and SMI-4a, Two Pim-1 Kinase Inhibitors on Primary HAM/TSP and ATL Cells

Special Article - Leukemia

Ann Carcinog. 2017; 2(1): 1008.

Differential Effects of AZD-1208 and SMI-4a, Two Pim-1 Kinase Inhibitors on Primary HAM/TSP and ATL Cells

Jean-Baptiste D1,2, Belrose G1,2, Meniane JC³, Lézin A1,2, Jeannin S4, Mesnard JM5, Olindo S4, Peloponese JM Jr5* and Césaire R1,2*

¹Laboratory of Virology, Martinique University Hospital, France

²EA 4537, Antilles University, France

³Service d’Hématologie Clinique, Martinique University Hospital, France

4Departments of Neurology, Radiology, Vascular Surgery, Martinique University Hospital, France

5IRIM (ex-CPBS)-UMR 9004, Research Institute in Infectiology of Montpellier, University Montpellier, France

*Corresponding author: Raymond Césaire, Laboratoire de Virologie, Centre Hospitalier Universitaire de Fort-de-France, BP 632, 97261 Fort-de-France, Martinique, France

Jean-Marie Péloponèse, Institut de Recherche en Infectiologie de Montpellier (ex CPBS) UMR9004 CNRS, 1919 Route de Mende, 34293 Montpellier Cedex 5, France

Received: February 09, 2017; Accepted: April 18, 2017; Published: April 25, 2017


Adult T-cell Leukemia-lymphoma (ATL), an aggressive neoplasm etiologically associated with HTLV-1, is a chemoresistant malignancy. Proviral integration site for Moloney murine leukemia virus-1 (Pim-1) is a critical enzyme that is involved in cell growth, differentiation, survival, apoptosis, senescence and drug resistance. Interaction of Pim-1 with different proteins and association with various signaling pathways make it one of the important antitumor targets. Aberrant elevation of Pim-1 kinase is associated with numerous types of cancer. In this study, we showed that Pim-1 kinase is highly expressed in ATL, as well as in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Numerous Pim-1 inhibitors are under preclinical studies or clinical trials, such as AZD1208. An increasing number of new Pim-1 inhibitors are still developing and undergoing preclinical investigations. Next, we compared the effect of two PIM-1 inhibitors, AZD-1208 and SMI-4a on HTLV-1 -derived cells lines and ex vivo cultured primary HAM/TSP and ATL leukemic cells. Our results show a differential effects between AZD on survival and proliferation of vs. HTLV-1 derived cells lines. Our results underscore the strong therapeutic potential of Pim kinase inhibition for the treatment of HTLV related pathogenesis such as HAM/TSP and ATL 3

Keywords: PIM-1; Adult T-cell leukemia; HAM/TSP


Adult T-cell Leukemia (ATL) is caused by Human T-Lymphotropic Virus-1 (HTLV-1), which is also the etiologic agent of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) [1,2]. The estimated lifetime risk of developing ATL in HTLV-1 carriers is 2–7%, and the disease occurs at least 20–30 years after the HTLV-1 infection [3]. ATL is classified as a peripheral T-lymphocytic malignancy of CD4+ T phenotype. The diversity in clinical features and evolution has led to its classification into 4 clinical subtypes: smoldering, chronic, acute, and lymphoma-type ATL [4]. Patients with acute or lymphoma forms have High-Risk ATL (HR-ATL). HR-ATL has a very poor prognosis because of multidrug resistance phenotype of ATL cells, rapid proliferation and large tumor burden, hypercalcemia, and/or infectious complications subsequent to reduced immunologic competence [5,6]. The combination of zidovudine and interferon-alpha with chemotherapy has slightly improved survival in HR-ATL [7,8], but prognosis still remains very poor with three years survival of less than 30% and high relapse rate [9]. In these aggressive forms, allogeneic hematopoietic stem-cell transplantation may improve survival rates [10] and to prevent relapse, Okamura et al. have suggested the possibility of a graft-versus-ATL and graft-versus-HTLV-1 [11].

New therapeutic agents are needed to treat and to improve ATL outcome. Some well-known molecular hallmarks of ATL cells are essential to consider for innovative treatment strategies [12]. The HTLV-1 proviral genome is characterized by the pX region between env and the 3’ Long Terminal Repeat (LTR). The pX-encoded Tax protein activates viral transcription, but is also considered as an oncoprotein [4,13]. Tax has been extensively studied, as a key player at the initial phase of the multistep process of HTLV-1 leukemogenesis. Tax deregulates many cellular signaling pathways related with cell cycle and apoptosis. Tax is pro-mitotic and propels CD4+ T-cell into proliferation [4,13]. At the same time, Tax is the immune-dominant target recognized by the CTL response [14]. Interestingly, tax gene is frequently inactivated in 4 ATL cells [4,13,15]. The HTLV-1 Basic leucine Zipper factor (HBZ), encoded by the pX minus strand is also suspected of down-regulating Tax transcription and contributing to immune escape [16]. HBZ remains the only gene that is consistently expressed in all ATL cases [15,17,18], and is able to induce T-cell lymphoma in transgenic mice [19]. HBZ modulates several cell signaling pathways involved in cell growth and differentiation [9]. HBZ mRNA promotes CD4+ T-lymphocyte proliferation. Evidences are accumulating about the critical role of HBZ in the maintenance of HTLV-1-induced transformation [9]. Proviral integration site for Moloney murine leukemia virus-1 (Pim-1) kinase is observed to interact with numerous proteins participating in various signaling pathways [20-23]. The Pim-1 gene was originally identified as a proviral integration site for Moloney murine leukemia virus-1. Pim- 1 is a proto-oncogene that encodes a serine/threonine kinase, which has a crucial role in oncogenesis [20-24]. This proto-oncogene was originally found in hematopoietic cells as a member of the Pim family (Pim-1, Pim-2 and Pim-3). Transcription of Pim-1 can be activated by several interleukins, such as interleukin-2 (IL-2), IL-3 and IL- 6. It has been shown that the Pim-1 kinase has an essential role in cytokine-induced signal transduction by controlling transcription factors [20,22,24,25] Upregulation of Pim-1 is correlated with cell proliferation induced by mitogens or cytokines, while downregulation of Pim-1 is correlated with growth retention due to the absence of cytokines [20,22,24,25]. Additionally, deficiency of Pim-1 kinase leads to failure in cell survival and growth [20,22,24,25]. Recent studies have shown that Pim-1 is required in drug resistance and has important roles in prostate cancer [26]. Inhibition of Pim kinase activity provides a novel therapeutic approach to the treatment of cancer. In this study, we evaluated the effect of two second generation PIM-1 inhibitor, AZD-1208 and SMI-4a, on HTLV-1 derived cell lines and on primary cells isolated from Asymptomatic Carrier (AC), HAM/TSP and ATL patients. Our finding shows that AZD-1208 preferentially reduces cell proliferation from HAM/TSP patients while SMI-4a 5 inhibits the growth of ATL cells during short-term culture. These results underscore the therapeutic potential of Pim kinase inhibition for the treatment of HTLV related diseases such as HAM/TSP and ATL.

Patients, Materials and Methods


Blood samples from HTLV-1 infected patients and non-infected donors were obtained from the CHU of Fort-de-France in Martinique. Patients suffering from ATL or HAM/TSP were recruited according to World Health Organization (WHO) criteria. AC had no neurologic or hematological symptoms. According to the French Bioethics laws, the collection of samples from ATL, HAM/TSP and AC has been declared to the French Ministry of Research. Because the protocol is non-interventional (e.g. blood samples collected for routine health care with no additional samplings or specific procedures for subjects), no informed consent was required, as stated by the French Public Health code and the study was conducted anonymously. Clinical collection of samples for research purpose is stored at the Center of Biological Resources of Martinique (CeRBiM).

Cell culture and PIM-1 inhibitors treatment

HTLV-I negative Jurkat, CEM and HTLV-I-positive MT-2, HUT102, C81-66 human T-cell lines, were propagated in RPMI 1640 with 10% fetal calf serum (FCS). PBMC were isolated from EDTAanticoagulated blood samples on Ficoll-density gradients, and washed in phosphate-buffered saline (PBS). CD8+ cells were removed using anti-CD8 paramagnetic microbeads, following the manufacturer’s instructions (Miltenyi Biotec, Paris, France). CD8+-cell–depleted PBMC were then placed in culture wells (round-bottomed 24-well plate) at 106/mL in 1 mL RPMI 1640 medium, supplemented with 10% fetal calf serum, glutamine (2 mmol/L), penicillin (100 IU/mL), and streptomycin (100 μg/mL) (Eurobio, Paris, France). AZD-1208,a benzylidene-1,3-thiazolidine-2,4-dione, and SMI-4a, (5Z)-5-[[3- 7(Trifluoromethyl)phenyl]methylene]-2,4-thiazolidinedione,(Z)- 5-(3-Trifluoromethylbenzylidene) thiazolidine-2,4-dione (Sigma Aldrich) were diluted in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO). AZD-1208 and SMI-4a were diluted into the medium at the indicated concentration. Cells and culture supernatants were harvested after different times of incubation at 37°C in 5% CO2, from day 0 (D0) up to D5, depending on the analysis performed.

Flow-cytometry analysis of apoptosis

Cells were washed in PBS, resuspended in annexin V-binding buffer, and incubated for 15 minutes at room temperature with fluorescein–isothiocyanate (FITC)-labeled annexin V (annexin) and propidium iodide (PI) reagents (BD Biosciences, San Jose, CA). 100,000 events in dual-labeled samples were analysed using flow cytometer (FACSCalibur, BD Biosciences). Percentages of viable and apoptotic cells were determined using Cellquest software (Becton-Dickinson Immunocytometry Systems, San Jose, CA) after appropriate compensations.

RNA isolation and qRT-PCR analysis

Cells were collected and cryopreserved as dry pellets until used. Nucleic acid was extracted using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Courtabeouf, France). To obtain first-strand cDNA, total RNA isolated from each sample was subjected to reverse transcription by Superscript II reverse transcriptase (Invitrogen, Cergy Pontoise, France) in the presence of oligo-dT 12-18 primer (Invitrogen). Realtime PCR was run in triplicate using Light Cycler 480 SYBR Green I Master Mix on Light Cycler 480 thermocycler (Roche Applied Science, Meylan, France). Relative mRNA quantification was performed using Cp (crossing point) 8 determined by the 2nd derivative peak of each amplification curve and normalized to housekeeping genes Hypoxanthine-guanine Phosphoribosyltransferase-1 (HPRT1) (forward primer: 5’ TGACACTGGCAAAACAATGCA-3’, reverse primer 5’-GGTCCTTTTCACCAGCAAGCT-3’). qRTPCR. Primer for PIM-1 were purchased from Biorad compagny.

CSFE Proliferation Assays

Cells were stained before culture with 5 μM of 5,6-Carboxyfluorescein diacetate Succinimidyl Ester (CFSE), according to the manufacturer’s instructions (Invitrogen). During 5 days of culture, cells were harvested every 24h, washed twice with PBS, and incubated with Peridinin chlorophyll labeled anti-CD3 and allophycocyanin-labeled anti-CD4. The CFSE fluorescence intensity was measured using FACSCanto II and ModFit LT 4.0 software (Verity Software House, Topsham, ME, USA). Proliferation was evaluated through the proliferation index (i.e. average number of cells that each original cell became), and the non-proliferative fraction (i.e. percent of cells that did not proliferate).

CCK8-Cell proliferation Assay

Cell proliferation was determined using the Cell Counting Kit- 8 (CCK-8) (Dojindo) according to manufacturer’s protocol. Briefly, 104/well cells were plated in 96-well plates. 10 μl WST-8 solutions (Dojindo) were added daily to each well and incubated for 4 hours. The cell viability in each well was determined by reading the OD at 450 nm.

Western blot analysis 9 Whole-cell lysates were prepared using RIPA buffer [10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.1% SDS and 1 mM DTT], separated by electrophoresis on SDS-PAGE gels, and transferred onto PVDF membranes (Millipore). Incubations with primary antibodies to detect PIM-1 (Santa Cruz Biotech) and Actine (Sigma-Aldrich) were followed by incubations with the appropriate secondary antibodies conjugated with Horseradish Peroxidase (HRP) (GE Healthcare) and by detection using enhanced luminescence (Roche).

Statistical analysis

(1) Pearson’s correlation for two-dimensional hierarchical clustering analysis; (2) two-tailed pared Student’s t test or 2-way ANOVA for in vitro cell lines and primary cells experiments, including qRT-PCR, cell growth assay. Data are presented as mean � SD. Differences were considered significant at *P<0.05, **P<0.01, and ***P<0.001. 10.


Pim-1L is overexpressed in cells isolated from HAM/TSP and ATL patients

The pim family genes were first identified as proviral integration sites for Moloney murine leukemia virus, but have later been shown to be involved in development of human lymphoid malignancies as well as solid tumors [24]. Aberrant elevation of Pim-1 kinase is associated with numerous types of cancer [24]. We first analyzed expression of PIM-1 in HTLV-1 related cell lines and in primary PBMCs from HTLV-1 infected patients (Figure 1). qRT-PCR analysis reveal that HTLV-1 related cell lines HUT102 and C81-66 expressed significantly (p< 0,0001) more messenger for PIM-1 than unrelated T-cells lines (Figure 1A). Next, we followed by quantitative RTPCR, the expression of pim-1 during culture of CD8+-cell–depleted PBMCs from HTLV-1 carriers without malignancy (AC), HAM/ TSP patients and ATL patients with acute subtype (Figure 1B). In cells derived from AC, we measured low level of pim-1. In contrast in HAM/TSP and in ATL leukemic cells, PIM-1-mRNA expression was spontaneously detectable (Figure 1B). Then, we analyzed PIM1 protein level by western Blot and confirmed that CD8+-depleted PBMCs from HAM/TSP and ATL express high level of PIM-1L in comparison to PBMCs from AC (p<0,0001) and no significant difference in PIM1-L protein level was observed between cells from HAM/TSP and ATL patients (Figure 1C and 1D).