Deregulated expression of Kruppel-like factors in acute myeloid leukemia

doi:10.1016/j.leukres.2011.03.010

Leukemia Research

Volume 35, Issue 7, July 2011, Pages 909-913

https://doi.org/10.1016/j.leukres.2011.03.010 Get rights and content

Abstract

The known participation of Kruppel-like transcription factors (KLF) in cellular differentiation prompted us to investigate their expression in acute myeloid leukemia (AML) blast cells that are typically blocked in their differentiation. We determined the expression patterns of KLFs with a putative role in myeloid differentiation in a large cohort of primary AML patient samples, CD34⁺ progenitor cells and granulocytes from healthy donors. We found that KLF2, KLF3, KLF5 and KLF6 are significantly lower expressed in AML blast and CD34⁺ progenitor cells as compared to normal granulocytes. Moreover, we found markedly increased KLF levels in acute promyelocytic leukemia patients who received oral ATRA. Accordingly, we observed a strong induction of KLF5/6 upon ATRA-treatment in NB4 and HT93 APL but not in ATRA-resistant NB4-R cells. Lastly, knocking down KLF5 or KLF6 in NB4 cells significantly attenuated neutrophil differentiation. In conclusion, we found a significant repression of KLF transcription factors in primary AML samples as compared to mature neutrophils and further show that KLF5 and KLF6 are functionally involved in neutrophil differentiation of APL cells.

Introduction

The Krüppel-like factor (KLFs) family consists of 17 members (KLF 1-17) with highly conserved DNA-binding domains that bind to GC-rich elements in promoters of a variety of genes. In contrast, the KLF amino-terminal termini are highly variable [1] and depending on the cellular context, e.g., expression of co-activators or -repressors, these proteins either repress or activate their target genes [2]. In general, the KLF family participates in regulating a broad range of important cellular functions including proliferation, development, differentiation and signal transduction.

As to the role of KLFs in hematopoiesis, several links have already been established. KLF2, for example, induces cell growth arrest in lymphoid Jurkat cells by transcriptional activation of the cell cycle inhibitor p21^WAF1/CIP1 [3]. Similar to KLF1, KLF2 is a positive regulator of beta-like globin and it is required for the maturation of erythroid cells [4]. Furthermore, Klf3^−/− mice display a myeloproliferative disorder and KLF3 is highly expressed in erythroid cells [5]. KLF5 regulates genes involved in cell cycle regulation, apoptosis, migration and differentiation [6]. Some KLF5 target genes, such as p15^INK4b and p27^KIP1, are involved in myeloid differentiation pointing to a putative role of KLF5 in this process. Moreover, Klf6^−/− mice are embryonically lethal and show aberrant hematopoiesis [7]. In general, the current literature suggests that KLFs are important downstream signaling targets during leukocyte maturation [8].

Since the function of KLFs in neutrophil differentiation is not well studied, we analyzed the expression patterns of selected KLFs in primary normal and leukemic myeloid cells as well as in neutrophil differentiation models. We found that KLFs are generally inhibited in primary acute myeloid leukemia (AML) patient samples compared to mature neutrophils from healthy donors. Moreover, KLF5/6 expression is induced during neutrophil differentiation of acute promyelocytic leukemia (APL) cells and inhibiting these KLFs attenuated their differentiation. Our data point to an important role for KLF5/6 in neutrophil differentiation of APL cells.

Section snippets

Cell lines, culture conditions and primary cells

The human acute promyelocytic leukemic (APL) cells NB4 and HT93 as well as ATRA-resistant NB4-R2 cells were a kind gift of B.E. Torbett. All cells were maintained in RPMI-1640 cell culture medium (Sigma–Aldrich, Buchs, Switzerland) and supplemented with 10% foetal bovine serum (FBS). Cells were cultured in a humidified atmosphere containing 5% CO₂ at 37 °C.

For differentiation experiments, cells were seeded at a density of 0.2 × 10⁶ cells/ml and treated with 1 μM all-trans retinoic acid (ATRA).

KLF2/3/5/6 expression is inhibited in primary AML patient samples but can be restored in APL upon ATRA therapy

Since certain KLFs are involved in hematopoietic differentiation, we were wondering whether their expression is downregulated in acute myeloid leukemic cells that are blocked in their differentiation. To this end we analyzed KLF2, 3, 5 and 6 mRNA expression in primary AML patient samples, CD34⁺ progenitor cells and granulocytes form healthy donors using Taqman Low Density Arrays. Since we are particularly interested in neutrophil differentiation of AML cells, we excluded KLF1 and KLF4 from our

Discussion

We found significantly elevated KLF2/3/5/6 levels in granulocytes from healthy donors compared to CD34⁺ progenitor or primary AML blast cells. Moreover, their expression was increased in APL patients as well as in APL cell line models upon ATRA-therapy. Generally, our data indicate that increased expression of KLF2/3/5/6 is associated with a more mature myeloid phenotype, whereas low expression is associated with immature myeloid cells and support earlier publications showing a role for

Conflict of interest

All authors have no conflict of interest to declare.

Acknowledgements

Gustav Arvidsson is gratefully acknowledged for excellent technical support and the Molecular Biology Group of Prof. R. Jaggi for their support in analyzing the RQ-PCR data. This work was supported by grants from the Swiss National Science Foundation 3100–067213 (to MFF and AT), the Marlies-Schwegler Foundation, the Ursula-Hecht-Foundation for Leukemia Research, and the Bernese Foundation of Cancer Research (to MFF), the Werner and Hedy Berger-Janser Foundation of Cancer Research (to MFF and

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¹: These two authors equally contributed to this study.

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Deregulated expression of Kruppel-like factors in acute myeloid leukemia

Abstract

Introduction

Section snippets

Cell lines, culture conditions and primary cells

KLF2/3/5/6 expression is inhibited in primary AML patient samples but can be restored in APL upon ATRA therapy

Discussion

Conflict of interest

Acknowledgements

Blood

Int J Biochem Cell Biol

Blood

Blood

Leuk Res

J Biol Chem

J Biol Chem

FEBS Lett

Blood Cells Mol Dis

J Biol Chem

J Biol Chem

Sp1- and Kruppel-like transcription factors

Genome Biol