CDC25 Inhibition in Acute Myeloid Leukemia–A Study of Patient Heterogeneity and the Effects of Different Inhibitors

Cell division cycle 25 (CDC25) protein phosphatases regulate cell cycle progression through the activation of cyclin-dependent kinases (CDKs), but they are also involved in chromatin modulation and transcriptional regulation. CDC25 inhibition is regarded as a possible therapeutic strategy for the treatment of human malignancies, including acute myeloid leukemia (AML). We investigated the in vitro effects of CDC25 inhibitors on primary human AML cells derived from 79 unselected patients in suspension cultures. Both the previously well-characterized CDC25 inhibitor NSC95397, as well as five other inhibitors (BN82002 and the novel small molecular compounds ALX1, ALX2, ALX3, and ALX4), only exhibited antiproliferative effects for a subset of patients when tested alone. These antiproliferative effects showed associations with differences in genetic abnormalities and/or AML cell differentiation. However, the responders to CDC25 inhibition could be identified by analysis of global gene expression profiles. The differentially expressed genes were associated with the cytoskeleton, microtubules, and cell signaling. The constitutive release of 28 soluble mediators showed a wide variation among patients and this variation was maintained in the presence of CDC25 inhibition. Finally, NSC95397 had no or only minimal effects on AML cell viability. In conclusion, CDC25 inhibition has antiproliferative effects on primary human AML cells for a subset of patients, and these patients can be identified by gene expression profiling.

. Single genes identified in the bioinformatical comparison of responders versus non-responders to CDC25 inhibitors. A total of 81 annotated genes were identified, and only 46 of them have a known function. A major subset of 18 genes encode proteins that are directly involved in cell cycle regulation (1 gene), the function of microtubuli/cytoskeleton (9 genes) and intracellular organelle formation/trafficking (4); 4 additional genes are involved in protein ubiquitination that is important for regulation of cell cycle progression/CDC25 modulation. These genes may thus represent a functional entity that is important for cell cycle progression/mitosis. The remaining genes are transcriptional regulators together with a DNA ligase (6 molecules), surface membrane molecules or surface membrane receptors (7 molecules), and molecules involved in intercellular communication (2 genes) or intracellular signaling (3 molecules). Thus, none of the molecules involved in regulation of the early steps of the cell cycle showed differential expression, but several genes important for mitosis/cell division differed between the two groups.

FJX1
Four jointed box 1 Important for embryonic development in Drosophila, unknown function in humans. MUC4 MUCIN 4 Encoding an integral membrane glycoprotein found on the cell surface. SASS6 SAS-6 centriolar assembly protein SAS6 is necessary for centrosome duplication and functions during procentriole formation; SAS6 functions to ensure that each centriole seeds the formation of a single procentriole per cell cycle. ACOT1 Acyl-CoA thioesterase 1 Cell metabolism. LOC643176 ABCA5 ATP-binding cassette, subfamily A, member 5 A membrane-associated protein that is a member of the superfamily of ATP-binding cassette transporters (ABC1 subfamily). These proteins transport various molecules across extra-and intracellular membranes. LOC642169 DOK7 Docking protein 7 The encoded protein can induce autophosphorylation of receptor kinase; the protein may also be implicated in breast cancer development [1,2]. ANO7 ANOCTAMIN 7 The protein is a member of the anoctamin family; these proteins are proposed to have eight transmembrane domains with both termini facing the cytoplasm and a C-terminal domain of unknown function. GPBP1L1 GC-rich promoter binding protein 1-like 1 KIR2DL5A Killer cell immunoglobulinlike receptor, two domains, long cytoplasmic tail, 5 Killer cell immunoglobulin-like receptors (KIRs) are transmembrane glycoproteins classified by the number of extracellular immunoglobulin domains (2D or 3D) and by whether they have a long (L) or short (S) cytoplasmic domain. KIR proteins with the long cytoplasmic domain transduce inhibitory signals upon ligand binding. LOC728667 DNAI2 Dynein, axonemal, intermediate chain 2 Belongs to the dynein intermediate chain family and part of the dynein complex of respiratory cilia and sperm flagella (see also FLJ20397). Cytoplasmic dynein is a large minus-end-directed microtubule motor complex, involved in many different cellular processes including intracellular trafficking, organelle positioning, and microtubule organization. Furthermore, dynein plays essential roles during cell division where it is implicated in multiple processes including centrosome separation, chromosome movements, spindle organization, spindle positioning, and mitotic checkpoint silencing [3]. COL3A1 Collagen, type III, alpha 1 This gene encodes the extracellular fibrillar pro-alpha1 chains of type III collagen. FAM134C Family with sequence similarity 134, member C A nuclear respiratory factor-1 (NRF-1) transcriptional target expressed in neuroblastoma cells, regulates cellular outgrowth. An orphan G protein-coupled receptor, its ligand prosaposin (also known as sulphated glycoprotein-1) promotes its endocytosis, induces phosphorylation of ERK, stimulates (35)S-GTPγS binding, inhibits cAMP production and protects against oxidative stress [5]. Dynein, axonemal, assembly factor 5 The protein is essential for the preassembly or stability of axonemal dynein arms (see also DNAI2 above). Cytoplasmic dynein is a large minus-end-directed microtubule motor complex, involved in many different cellular processes including intracellular trafficking, organelle positioning, and microtubule organization. Furthermore, dynein plays essential roles during cell division where it is implicated in multiple processes including centrosome separation, chromosome movements, spindle organization, spindle positioning, and mitotic checkpoint silencing [3]. LOC653346 WDR23 DDB1 and CUL4 associated factor 11 This gene encodes a WD repeat-containing protein that interacts with the COP9 signalosome, a macromolecular complex that interacts with cullin-RING E3 ligases and regulates their activity by hydrolysing cullin-Nedd8 conjugates. Cullin-Ring E3 ubiquitin ligases are important for actin cytoskeletal organization [8]. RAB4A RAB4A, member RAS oncogene family Belongs to the Ras superfamily of small GTPases, it is associated with early endosomes and is involved in their sorting/recycling and recycling of receptors from endosomes to the plasma membrane. MAP2 Microtubule-associated protein 2 Belongs to the microtubule/cytoskeletal-associated protein family, possibly involved in microtubule assembly. This gene encodes a member of the autophagin protein family that is important for autophagy, the protein also being designated as a member of the C-54 family of cysteine proteases. LOC729260 MACROD2 MACRO domain containing 2 The protein can mediate chemoresistance in breast cancer cells [9]. ITIH3 Inter-alpha-trypsin inhibitor heavy chain 3 This gene encodes the heavy chain subunit of the pre-alpha-trypsin inhibitor complex; this complex may stabilize the extracellular matrix through its ability to bind hyaluronic acid. ZGRF1 ( Fasciculation and elongation protein zeta 1 CLASP2 and NEK1 proteins are present in a centrosomal complex and participate in cell cycle and cell division mechanisms, and they colocalize with FEZ1 in a perinuclear region and interact with endogenous gamma-tubulin [10]. BOLA2 BolA family member 2 The protein belongs to the family of BolA-like proteins probably involved in cell proliferation or cell-cycle regulation. LOC100130703 LOC643302 GPR173 G protein-coupled receptor 173 This gene encodes a member of the G-protein coupled receptor 1 family.

MAP2K3
Mitogen-activated protein kinase 3 The protein belongs to the MAP kinase family. It phosphorylates and thus activates MAPK14/p38-MAPK.

MDFI MyoD family inhibitor
This protein is a transcription factor. THAP8 THAP domain containing 8 Unknown DMD Dystrophin Dystrophin is a large cytoskeletal protein, it is part of the dystrophin-glycoprotein complex (DGC) that bridges the inner cytoskeleton (Factin) and the extra-cellular matrix, it seems to interact with microtubules and have a role in cell division/cell cycle regulation, and it is expressed in hematopoietic cells [11,12]. HSPC157 Long intergenic nonprotein coding RNA 33 Unknown CCKAR Cholecystokinin A receptor This gene encodes a G-protein coupled receptor. CAB39L Calcium binding protein 39like CABL39L can activate the LKB1 tumour suppressor [13].

DMRTA1
Doublesex and mab-3 related transcription factor like family A1 The protein is a Pax6 activated transcription factor [14].

TRIM34 tripartite motif containing 34
The protein is a member of the tripartite motif (TRIM) family, the TRIM motif including three zinc-binding domains, a RING, B-box type 1 and B-box type 2 domain, and a coiled-coil region. Unknown functions. AKAP2 A kinase (PRKA) anchor protein 2 The encoded protein binds to the regulatory subunit of protein kinase A and is found associated with the actin cytoskeleton.