Equilibrative nucleoside transporter-2 (hENT2) protein expression correlates with ex vivo sensitivity to fludarabine in chronic lymphocytic leukemia (CLL) cells

Abstract Nucleoside derivatives are widely employed in the treatment of hematological malignancies. In particular, fludarabine is considered the treatment of choice for most patients with chronic lymphocytic leukemia (CLL). Anticancer therapy using nucleoside-derived analogs is dependent on drug uptake and metabolic activation. Although some enzymes have been shown to be suitable biomarkers of drug metabolism and may determine response to therapy, the role of transporters in determining nucleoside-derived drug bioavailability is less known. The uptake of these drugs into cells is mediated by one or more of the different nucleoside transporters that have recently been cloned. These proteins belong to two non-related gene families, SLC28 and SLC29, encoding CNT (Concentrative Nucleoside Transporters) and ENT (Equilibrative Nucleoside Transporters) proteins, respectively. In primary CLL cells we have described a high variability in ENT1, ENT2, CNT2 and CNT3 mRNA expression, whereas no CNT1 mRNA is detected. We have also shown that, in CLL cells, the uptake of fludarabine relies mainly ENT-type carriers and that ex vivo sensitivity significantly correlates with this ENT mediated uptake. In order to elucidate the role of ENT1 and ENT2 in the uptake and cytotoxicity of fludarabine, we have analyzed ENT1 and ENT2 expression by real time RT-PCR and Western Blot in cells from 19 CLL patients. ENT1 and ENT2 mRNA expression showed a high variability. ENT2 mRNA levels showed a range of variability of nearly 4-fold, whereas ENT1 variability was of 19-fold. Polyclonal antibodies against ENT1 and ENT2 consistently identified a single band of 50–55 kDa in all samples. Semiquantitative analysis of ENT1 and ENT2 expression was achieved by calculating the densitometry ratios versus α-tubulin, in a range of protein concentrations in which densitometric signal has been previously shown to be linear. Contrary to the mRNA expression, Western Blot analysis of ENT1 showed much less variability among patients (three-fold) than ENT2 (11-fold). The amounts of ENT1 protein did not significantly correlate with ENT1 mRNA expression, transporter function, or ex vivo fludarabine cytotoxicity. In contrast, although ENT2 protein does not bear any significant relationship with ENT2 mRNA, its expression correlates with transport activity. Moreover, ex-vivo sensitivity to fludarabine, at a pharmacological concentration (7.5 μM), directly correlated with the amount of ENT2 protein. Similar correlations were observed when the drug was tested at 3 and 15 μM. In summary, this study strongly suggests that fludarabine action on CLL cells is determined by the equilibrative nucleoside transporter ENT2. This is the first time ENT2 has been implicated in the therapeutic response to a nucleoside analog and might be useful to predict fluradabine response in CLL patients.

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Identification of TIGAR in the equilibrative nucleoside transporter 2-mediated response to fludarabine in chronic lymphocytic leukemia cells

Haematologica ◽

10.3324/haematol.13186 ◽

2008 ◽

Vol 93 (12) ◽

pp. 1843-1851 ◽

Cited By ~ 14

Author(s):

M. Lopez-Guerra ◽

L. Trigueros-Motos ◽

M. Molina-Arcas ◽

N. Villamor ◽

F. J. Casado ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Nucleoside Transporter ◽

Equilibrative Nucleoside Transporter

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Cytotoxic Effect of R-Etodolac (SDX-101) in Combination with Purine Analogues or Monoclonal Antibodies on Ex-Vivo B-Cell Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v106.11.2122.2122 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2122-2122

Author(s):

Pawel Robak ◽

Anna Linke ◽

Barbara Cebula ◽

Lorenzo M. Leoni ◽

Tadeusz Robak ◽

...

Keyword(s):

Flow Cytometry ◽

Chronic Lymphocytic Leukemia ◽

Protein Expression ◽

Cytotoxic Effect ◽

Caspase 3 ◽

Ex Vivo ◽

Fold Increase ◽

Lymphocytic Leukemia ◽

Apoptotic Protein ◽

Cell Chronic Lymphocytic Leukemia

Abstract Background: SDX-101 (R-etodolac) is the R isomer of the non-steroidal anti-inflammatory drug Etodolac. SDX-101 decreased the in vitro survival of B-cell chronic lymphocytic leukemia (B-CLL) cells and showed synergistic activity with chlorambucil. In multiple myeloma cells, SDX-101 showed cytotoxic activity and displayed a synergistic cytotoxic effect with dexamethasone. Currently, SDX-101 is being tested in phase II clinical trial for treatment of refractory B-CLL in combination with chlorambucil. Aims: To evaluate the ex-vivo cytotoxic effect of SDX-101 in combination with agents proven to be effective first line treatment in B-CLL: fludarabine (FA), cladribine (2-chlordeoxyadenosine, 2-CdA), anti-CD-52 (alemtuzumab, ALT) or anti-CD-20 (rituximab, RTX). Methods: Tumor cells were obtained from 20 untreated patients with B-CLL. Cytotoxicity of SDX-101 and other study drugs was assessed using a propidium iodide-based flow cytometry assay. Apoptosis was assessed measuring activity of caspase-3 and DNA-content (sub-G1 fraction) by flow cytometry, and by measuring protein expression of p53 protein family members (p53 and p73), and BCl-2 family members (pro-apoptotic Bax, Bak and Bid; anti-apoptotic Bcl-2 and Mcl-1). The percentage of apoptotic cells and the expression of apoptosis-regulating proteins were measured at 0 h and 24 h. IC50 values were defined as the concentration of agents that achieved 50% decrease in cell viability. The combination index (CI) was used to estimate sub-additive (CI value 1.2), additive (0.8–1.2) or synergistic (<0.8) interactions. Results: Dose-ranging experiments indicated that the SDX-101 IC50 in CLL cells was 800 μM, and that 400 μM of SDX-101 reduced CLL viability by 11% on average after 24 hrs. An SDX-101 dose of 400 μM was selected for combination studies with FA (used at 3.5 μM, yielding a viability reduction of 9.9%), 2-CdA (0.175 μM, 10.1%), RTX (20μg/ml, 6.1%), and ALT (20μg/ml, 8.4%). The combination of SDX-101+FA provided a drop of 20.9% in cell viability (p<0.03, vs. single drugs alone). The SDX-101+2-CdA combination reduced viability by 27.8% (p<0.006 vs. single drugs), SDX-101+RTX reduced viability by 22.3% (p<0.01 vs. single drugs), and that of SDX-101+ALT combination reduced it by 18.7% (n.s.). The CIs for those combinations were 0.89, 1.17, 0.95, and 1.25, respectively. Apoptosis analysis by sub-G1 fraction and caspase-3 activity confirmed the viability results for each tested combination. Analysis of apoptosis-regulating proteins showed, that SDX-101 combined with RTX or 2-CdA distinctly up-regulated Bax (p<0.02, vs. single drugs), whereas other combinations did not significantly increase its expression. The expression of p73 protein showed 2–4 fold increase after treatment with SDX-101 alone, and all combinations further up-regulated p73, especially that with RTX (up to 6-fold increase in p73 expression). Interestingly, although SDX-101 (400 μM at 24 hr) increased protein expression of Mcl-1, SDX-101 combination with RTX, 2-CdA or FA caused a 2-3-fold decrease of its expression. Conclusion: SDX-101 in combination with low doses of either 2-CdA, FA or RTX exerts an additive cytotoxic effect on ex-vivo B-CLL cells, indicating a possible clinical benefit of such a combination treatment. Up-regulation of pro-apoptotic protein expression (p73 and Bax), and down-regulation of anti-apoptotic protein (Mcl-1) seem to play a mechanistic role in these additive cytotoxic effects.

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Quantitative analysis of nucleoside transporter and metabolism gene expression in chronic lymphocytic leukemia (CLL): identification of fludarabine-sensitive and -insensitive populations

Blood ◽

10.1182/blood-2004-03-1046 ◽

2005 ◽

Vol 105 (2) ◽

pp. 767-774 ◽

Cited By ~ 52

Author(s):

John R. Mackey ◽

Carlos M. Galmarini ◽

Kathryn A. Graham ◽

Anil A. Joy ◽

Alain Delmer ◽

...

Keyword(s):

Gene Expression ◽

Chronic Lymphocytic Leukemia ◽

Disease Progression ◽

Hierarchical Cluster ◽

Complete Response ◽

Lymphocytic Leukemia ◽

Nucleoside Transport ◽

Nucleoside Transporter ◽

Equilibrative Nucleoside Transporter ◽

Risk Of Disease

AbstractResistance to fludarabine is observed in the clinic, and molecular predictive assays for benefit from chemotherapy are required. Our objective was to determine if expression of nucleoside transport and metabolism genes was associated with response to fludarabine therapy in patients with chronic lymphocytic leukemia (CLL). CLL cells from 56 patients were collected prior to treatment with fludarabine. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) was performed on sample RNA to determine the relative levels of mRNA of 3 nucleoside transporters that mediate fludarabine uptake (human equilibrative nucleoside transporter 1 [hENT1], human equilibrative nucleoside transporter 2 [hENT2], and human concentrative nucleoside transporter 3 [hCNT3]), deoxycytidine kinase (dCK), and 3 5′-nucleotidases (ecto-5′nucleotidase [CD73], deoxynucleotidase-1 [dNT-1], and cytoplasmic high-Km 5-nucleotidase [CN-II]). Two-dimensional hierarchical cluster analysis of gene expression identified 2 distinct populations of CLL. Cluster 2 patients experienced a 3.4-fold higher risk of disease progression than cluster 1 patients (P = .0058, log-rank analysis). Furthermore, independent analysis of the individual genes of interest revealed statistically significant differences for risk of disease progression (adjusted hazard ratios [HRs]) with underexpression of dNT-1 (HR = 0.45; P = .042), CD73 (HR = 0.40; P = .022), and dCK (HR = 0.0.48; P = .035), and overexpression of hCNT3 (HR = 4.7; P = .0007) genes. Subjects with elevated hCNT3 expression experienced a lower complete response rate to fludarabine therapy (11% vs 69%; P = .002). No hCNT3-mediated plasma membrane nucleoside transport was detected in CLL samples expressing hCNT3 message, and hCNT3 protein was localized to the cytoplasm with immunohistochemical and confocal microscopy.

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Targeting CD38 is lethal to Breg-like chronic lymphocytic leukemia cells and Tregs, but restores CD8+ T-cell responses

Blood Advances ◽

10.1182/bloodadvances.2019001091 ◽

2020 ◽

Vol 4 (10) ◽

pp. 2143-2157 ◽

Cited By ~ 1

Author(s):

Alak Manna ◽

Timothy Kellett ◽

Sonikpreet Aulakh ◽

Laura J. Lewis-Tuffin ◽

Navnita Dutta ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Mononuclear Cells ◽

Transforming Growth Factor ◽

Ex Vivo ◽

Xenograft Model ◽

Lymphocytic Leukemia ◽

Dependent Manner

Abstract Patients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+κ/λ+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]–like CLL cells) produce high amounts of IL-10 and transforming growth factor β (TGF-β) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-β-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-γ and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL–patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.

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Immunological and genetic kinetics from diagnosis to clinical progression in chronic lymphocytic leukemia

Biomarker Research ◽

10.1186/s40364-021-00290-z ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Isabel Jiménez ◽

Bárbara Tazón-Vega ◽

Pau Abrisqueta ◽

Juan C. Nieto ◽

Sabela Bobillo ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Ex Vivo ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Clinical Progression ◽

Molecular Changes ◽

Main Driver ◽

Altered Gene

Abstract Background Mechanisms driving the progression of chronic lymphocytic leukemia (CLL) from its early stages are not fully understood. The acquisition of molecular changes at the time of progression has been observed in a small fraction of patients, suggesting that CLL progression is not mainly driven by dynamic clonal evolution. In order to shed light on mechanisms that lead to CLL progression, we investigated longitudinal changes in both the genetic and immunological scenarios. Methods We performed genetic and immunological longitudinal analysis using paired primary samples from untreated CLL patients that underwent clinical progression (sampling at diagnosis and progression) and from patients with stable disease (sampling at diagnosis and at long-term asymptomatic follow-up). Results Molecular analysis showed limited and non-recurrent molecular changes at progression, indicating that clonal evolution is not the main driver of clinical progression. Our analysis of the immune kinetics found an increasingly dysfunctional CD8+ T cell compartment in progressing patients that was not observed in those patients that remained asymptomatic. Specifically, terminally exhausted effector CD8+ T cells (T-betdim/−EomeshiPD1hi) accumulated, while the the co-expression of inhibitory receptors (PD1, CD244 and CD160) increased, along with an altered gene expression profile in T cells only in those patients that progressed. In addition, malignant cells from patients at clinical progression showed enhanced capacity to induce exhaustion-related markers in CD8+ T cells ex vivo mainly through a mechanism dependent on soluble factors including IL-10. Conclusions Altogether, we demonstrate that the interaction with the immune microenvironment plays a key role in clinical progression in CLL, thereby providing a rationale for the use of early immunotherapeutic intervention.

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