Multidrug resistance protein 4/ ATP binding cassette transporter 4: a new potential therapeutic target for acute myeloid leukemia

Less than a third of adults patients with acute myeloid leukemia (AML) are cured by current treatments, emphasizing the need for new approaches to therapy. We previously demonstrated that besides playing a role in drug-resistant leukemia cell lines, multidrug resistance protein 4 (MRP4/ABCC4) regulates leukemia cell proliferation and differentiation through the endogenous MRP4/ABCC4 substrate, cAMP. Here, we studied the role of MRP4/ABCC4 in tumor progression in a mouse xenograft model and in leukemic stem cells (LSCs) differentiation. We found a decrease in the mitotic index and an increase in the apoptotic index associated with the inhibition of tumor growth when mice were treated with rolipram (PDE4 inhibitor) and/or probenecid (MRPs inhibitor). Genetic silencing and pharmacologic inhibition of MRP4 reduced tumor growth. Furthermore, MRP4 knockdown induced cell cycle arrest and apoptosis in vivo. Interestingly, when LSC population was isolated, we observed that increased cAMP levels and MRP4/ABCC4 blockade resulted in LSCs differentiation. Taken together, our findings show that MRP4/ABCC4 has a relevant role in tumor growth and apoptosis and in the eradication of LSCs, providing the basis for a novel promising target in AML therapy.Fil: Copsel, Sabrina Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Bruzzone, Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: May, Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Beyrath, Julien. Radboud Universiteit Nijmegen; Países BajosFil: Wargon, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Cany, Jeannette. Radboud Universiteit Nijmegen; Países BajosFil: Russel, Frans G. M.. Radboud Universiteit Nijmegen; Países BajosFil: Shayo, Carina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Davio, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Immunotherapy of hepatocellular carcinoma: is there a place for regulatory T-lymphocyte depletion?

Item does not contain fulltextImmunotherapy represents a potential therapeutic option for patients with hepatocellular carcinoma (HCC), especially as secondary treatment to prevent recurrence. It has been shown that a patient's survival is directly correlated to the type and number of tumor-infiltrating immune cells, indicating that immune responses have a direct effect on the clinical course of the disease. We have assessed the potential of immunotherapy against HCC in preclinical models of low tumor burden. An antigen-specific strategy targeting alpha-fetoprotein, and consisting of immunization with a DNA-based synthetic vector (DNAmAFP/704), was tested on an autochthonous model of chemical hepatocarcinogenesis and led to an important (65%) reduction of the tumor burden. A nonspecific approach of CD25(+) T-cell depletion by injection of PC61 antibody was also tested on an orthotopic HCC model and led to a significant protection against tumor development. Antigen-specific immunotherapy and Treg depletion are promising strategies in physiologically relevant HCC preclinical models. Future clinical trials will demonstrate if a combination of Treg depletion with an antigen-specific immunotherapy will also translate into clinical responses in HCC patients.1 april 201

PD-L1 microSPECT/CT Imaging for Longitudinal Monitoring of PD-L1 Expression in Syngeneic and Humanized Mouse Models for Cancer

Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive responses in subgroups of patients with cancer. PD-L1 expression in tumors seems to be a prerequisite for treatment response. However, PD-L1 is heterogeneously expressed within tumor lesions and may change upon disease progression and treatment. Imaging of PD-L1 could aid in patient selection. Previously, we showed the feasibility to image PD-L1(+) tumors in immunodeficient mice. However, PD-L1 is also expressed on immune cell subsets. Therefore, the aim of this study was to assess the potential of PD-L1 micro single-photon emission tomography/computed tomography (microSPECT/CT) using radiolabeled PD-L1 antibodies to (i) measure PD-L1 expression in two immunocompetent tumor models (syngeneic mice and humanized mice harboring PD-L1 expressing immune cells) and (ii) monitor therapy-induced changes in tumor PD-L1 expression. We showed that radiolabeled PD-L1 antibodies accumulated preferentially in PD-L1(+) tumors, despite considerable uptake in certain normal lymphoid tissues (spleen and lymph nodes) and nonlymphoid tissues (duodenum and brown fat). PD-L1 microSPECT/CT imaging could also distinguish between high and low PD-L1-expressing tumors. The presence of PD-L1(+) immune cells did not compromise tumor uptake of the human PD-L1 antibodies in humanized mice, and we demonstrated that radiotherapy-induced upregulation of PD-L1 expression in murine tumors could be monitored with microSPECT/CT imaging. Together, these data demonstrate that PD-L1 microSPECT/CT is a sensitive technique to detect variations in tumor PD-L1 expression, and in the future, this technique may enable patient selection for PD-1/PD-L1-targeted therapy

Decitabine enhances targeting of AML cells by CD34(+) progenitor-derived NK cells in NOD/SCID/IL2Rg(null) mice

Combining natural killer (NK) cell adoptive transfer with hypomethylating agents (HMAs) is an attractive therapeutic approach for patients with acute myeloid leukemia (AML). However, data regarding the impact of HMAs on NK cell functionality are mostly derived from in vitro studies with high nonclinical relevant drug concentrations. In the present study, we report a comparative study of azacitidine (AZA) and decitabine (DAC) in combination with allogeneic NK cells generated from CD34(+) hematopoietic stem and progenitor cells (HSPC-NK cells) in in vitro and in vivo AML models. In vitro, low-dose HMAs did not impair viability of HSPC-NK cells. Furthermore, low-dose DAC preserved HSPC-NK killing, proliferation, and interferon gamma production capacity, whereas AZA diminished their proliferation and reactivity. Importantly, we showed HMAs and HSPC-NK cells could potently work together to target AML cell lines and patient AML blasts. In vivo, both agents exerted a significant delay in AML progression in NOD/SCID/IL2Rg(null) mice, but the persistence of adoptively transferred HSPC-NK cells was not affected. Infused NK cells showed sustained expression of most activating receptors, upregulated NKp44 expression, and remarkable killer cell immunoglobulin-like receptor acquisition. Most importantly, only DAC potentiated HSPC-NK cell anti-leukemic activity in vivo. Besides upregulation of NKG2D- and DNAM-1-activating ligands on AML cells, DAC enhanced messenger RNA expression of inflammatory cytokines, perforin, and TRAIL by HSPC-NK cells. In addition, treatment resulted in increased numbers of HSPC-NK cells in the bone marrow compartment, suggesting that DAC could positively modulate NK cell activity, trafficking, and tumor targeting. These data provide a rationale to explore combination therapy of adoptive HSPC-NK cells and DAC in patients with AML

DNA/Amphiphilic Block Copolymer Nanospheres Promote Low-dose DNA Vaccination

Intramuscular (i.m.) DNA vaccination induces strong cellular immune responses in the mouse, but only at DNA doses that cannot be achieved in humans. Because antigen expression is weak after naked DNA injection, we screened five nonionic block copolymers of poly(ethyleneoxide)-poly(propyleneoxide) (PEO-PPO) for their ability to enhance DNA vaccination using a β-galactosidase (βGal) encoding plasmid, pCMV-βGal, as immunogen. At a high DNA dose, formulation with the tetrafunctional block copolymers 304 (molecular weight [MW] 1,650) and 704 (MW 5,500) and the triblock copolymer Lutrol (MW 8,600) increased βGal-specific interferon-γ enzyme-linked immunosorbent spot (ELISPOT) responses 2–2.5-fold. More importantly, 704 allowed significant reductions in the dose of antigen-encoding plasmid. A single injection of 2 µg pCMV-βGal with 704 gave humoral and ELISPOT responses equivalent to those obtained with 100 µg naked DNA and conferred protection in tumor vaccination models. However, 704 had no adjuvant properties for βGal protein, and immune responses were only elicited by low doses of pCMV-βGal formulated with 704 if noncoding carrier DNA was added to maintain total DNA dose at 20 µg. Overall, these results show that formulation with 704 and carrier DNA can reduce the dose of antigen-encoding plasmid by at least 50-fold

Homing receptor expression profile of UCB-NK cells.

<p>(A–B) The expression level of homing receptors was analysed by flow cytometry on UCB-NK cells at the end of the culture process. (A) Dot plots gated on CD56⁺ cells from one representative donor; (B) Summary of 6 different donors analysed. (C) The capacity of UCB-NK cells to respond <i>in vitro</i> to gradients (10 to 250 ng/ml) of the chemokines CCL4 (CCR5 ligand), CCL20 (CCR6 ligand), CXCL10, CXCL11 (both CXCR3 ligand) and CXCL12 (CXCR4 ligand) was evaluated in transwell migration assays as described in materials and methods. Mean ± SEM of three independent experiments are shown, each performed with different UCB-NK cell donors. Migration towards specific chemokines was compared to non-specific migration (0 ng/ml) using a one way-ANOVA followed by Dunnett’s multiple comparison post-hoc test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

SPECT-CT imaging provides good sensitivity and specificity to track ¹¹¹In-labeled UCB-NK cells <b><i>in vivo</i></b><b>.</b>

<p>To address the feasibility of UCB-NK cell tracking <i>in vivo</i>, adult NSG mice were injected <i>i.v.</i> either with increasing doses of ¹¹¹In-NK cells (1, 5 or 12×10⁶¹¹¹In-NK cells per mouse equivalent to 1, 5 and 12 MBq respectively, n = 5 in total), with a lysate obtained from ¹¹¹In-NK cells (¹¹¹In-lysate, n = 2) or with ¹¹¹In-oxinate (n = 3). (A) Representative whole body SPECT scans acquired 1 h and 24 h after injection of ¹¹¹In-NK cells with major visualization of lungs (Lu), liver (Li), spleen (Sp, arrowhead) and bone marrow (BM, arrow). (B) Representative whole body SPECT scans acquired 24 h after injection of ¹¹¹In-NK cell lysate or ¹¹¹In-oxinate; kidneys (Ki), heart (He). (C) Quantitative biodistribution analysis performed 24 h after injection. For comparison, the relative ¹¹¹In-content of each tissue of interest was normalized to blood. Data are shown as mean ± SD. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Organ-distribution of UCB-NK cells following adoptive transfer.

<p>(A) For optimization of NK cell labelling procedure, increasing number of UCB-NK cells were incubated with 2MBq ¹¹¹In. Graphs show mean ± SD of three experiments performed each with a different UCB donor. Good labelling efficiency and cell recovery were achieved using >4×10⁶ cells per 2 MBq. Subsequently (panels B-D), 0.4MBq ¹¹¹In was added per 10⁶ UCB-NK cells to label. (B) The capacity of ¹¹¹In-NK cells to migrate in response to the chemokine CXCL12 was evaluated <i>in vitro</i> and compared to that of unlabeled UCB-NK cells. The same experimental procedure was employed to assess the transwell migration of unlabeled and ¹¹¹In-NK cells as described in materials and methods, except that the proportion of ¹¹¹In-labeled cells present in bottom chambers were quantified using a shielded 3-inch-well-type gamma counter (Wizard; Pharmacia LKB). (C) Biodistribution analysis of ¹¹¹In-NK cells 24 h after <i>i.v.</i> infusion, expressed as a percentage of injected activity per gram of tissue of interest or per organ. Combined results from two experiments expressed as mean ± SD (n = 6) are shown. *Extrapolated values according to physiological parameters. (D) Representative 2D-reconstruction analysis of SPECT-CT illustrating the accumulation of ¹¹¹In-NK cells (orange) in bones (grey).</p