A Subset of Patients with Acute Myeloid Leukemia Has Leukemia Cells Characterized by Chemokine Responsiveness and Altered Expression of Transcriptional as well as Angiogenic Regulators

Acute myeloid leukemia (AML) is an aggressive and heterogeneous bone marrow malignancy, the only curative treatment being intensive chemotherapy eventually in combination with allogeneic stem cell transplantation. Both the AML and their neighboring stromal cells show constitutive chemokine release, but chemokines seem to function as regulators of AML cell proliferation only for a subset of patients. Chemokine targeting is therefore considered not only for immunosuppression in allotransplanted patients but also as a possible antileukemic strategy in combination with intensive chemotherapy or as part of disease-stabilizing treatment at least for the subset of patients with chemokine-responsive AML cells. In this study, we characterized more in detail the leukemia cell phenotype of the chemokine-responsive patients. We investigated primary AML cells derived from 79 unselected patients. Standardized in vitro suspension cultures were used to investigate AML cell proliferation, and global gene expression profiles were compared for chemokine responders and non-responders identified through the proliferation assays. CCL28-induced growth modulation was used as marker of chemokine responsiveness, and 38 patients were then classified as chemokine-responsive. The effects of exogenous CCL28 (growth inhibition/enhancement/no effect) thus differed among patients and was also dependent on the presence of exogenous hematopoietic growth factors as well as constitutive AML cell cytokine release. The effect of CCR1 inhibition in the presence of chemokine-secreting mesenchymal stem cells also differed among patients. Chemokine-responsive AML cells showed altered expression of genes important for (i) epigenetic transcriptional regulation, particularly lysine acetylation; (ii) helicase activity, especially DExD/H RNA helicases; and (iii) angioregulatory proteins important for integrin binding. Thus, chemokine responsiveness is part of a complex AML cell phenotype with regard to extracellular communication and transcriptional regulation. Chemokine targeting in chemokine-responsive patients may thereby alter AML cell trafficking and increase their susceptibility toward antileukemic treatment, e.g., conventional chemotherapy or targeting of other phenotypic characteristics of the chemokine-responsive cells.


EMP2
Epithelial membrane protein 2. This protein regulates cell membrane composition and has been associated with endocytosis, cell signaling, cell proliferation, cell migration, cell adhesion and cell death. It is a negative regulator of caveolin-1, a scaffolding protein which is the main component of the caveolae plasma membrane invaginations. Through activation of PTK2 it positively regulates vascular endothelial growth factor A, and it also modulates the function of specific integrin isomers in the plasma membrane. Up-regulation of this gene has been linked to cancer progression in multiple different tissues.
Surface membrane FBLN5 Fibulin 5. This is a secreted, extracellular matrix protein containing an Arg-Gly-Asp (RGD) motif and calcium-binding EGF-like domains. It promotes adhesion of endothelial cells through interaction of integrins and the RGD motif. Its expression is induced in injured vessels, notably in intimal vascular smooth muscle cells and endothelial cells. Therefore, the protein may play a role in vascular development and remodeling.

ICAM3
Intercellular adhesion molecule 3. The protein belongs to the intercellular adhesion molecule (ICAM) family; all ICAM proteins are type I transmembrane glycoproteins that bind to the LFA-1 integrin protein. LFA-1 is expressed by all leucocytes as well as endothelial cells (12).

Adhesion IMPAD1
Inositol monophosphatase domain containing 1. This protein is a member of the inositol monophosphatase family, is localized to the Golgi apparatus and catalyzes the hydrolysis of phosphoadenosine phosphate to AMP. Possibly involved in carcinogenesis (13).
Golgi ITGB1BP1 Integrin beta 1 binding protein 1. The encoded protein binds to the beta1 integrin cytoplasmic domain. The longer isoform form is a phosphoprotein and the extent of its phosphorylation is regulated by the cell-matrix interaction. Integrin beta 1 is important in angiogenesis (12,14,15).

ITGB6
Integrin, beta 6. This integrin forms a dimer with an alpha v chain and this heterodimer can bind to ligands like fibronectin and transforming growth factor beta 1. This integrin seems important in angioregulation (16).

Integrin JAM3
Junctional adhesion molecule 3. The protein only forms weak homotypic interactions between cells as a member of the junctional adhesion molecule protein family and acting as a receptor for another family member. It also exist in a soluble form that is important in angiogenesis (including tumor angiogenesis), and modulates VR-cadherin-mediated cell-cell contact (17)(18)(19).

SOD1
Superoxide dismutase 1, soluble. The protein a cytoplasmic protein that is one of two isozymes responsible for destroying free superoxide radicals. The protein is a regulator of intracellular protein phosphorylation; inhibition of the enzyme has antiangiogenic effects (24,25).

THBS4
Thrombospondin 4 is a thrombospondin family member and mediates cell-to-cell and cell-to-matrix interactions. This gene is activated during the stromal response to invasive cancer, and it is also important in the regulation of angiogenesis (26,27).

Angiogenesis THY1
Thy-1 cell surface antigen. This cell surface glycoprotein is involved in cell adhesion and cell communication, and it is used as a marker for hematopoietic stem cells. This gene may function as a tumor suppressor, and may also be important in angioregulation (28)(29)(30).

Cell membrane TNN
Tenascin N. This is a protein-encoding gene. Extracellular matrix proteins of the tenascin family resemble each other in their domain structure, and also share functions in modulating cell adhesion and cellular responses to growth factors (31). These molecules may also be involved in angiogenesis (32)(33)(34)(35)(36).
VWF von Willebrand factor. This is a glycoprotein involved in hemostasis. The encoded preproprotein is processed into large multimeric complexes that function in platelet adhesion and transport of various proteins in the blood. This molecule has several other functions, including modulation of angiogenesis (37,38).

Coagulation Angiogenesis
Supplementary Table 2 The GO term Histone acetylation; differential gene expression when comparing AML cell with and without CCL28-induced growth modulation. The table is based on information from the GENE database and additional references given in the reference list.

ACTL6A
Actin-like 6A. The protein is a family member of actin-related proteins (ARPs); it is a 53 kDa subunit protein of the BAF (BRG1/brm-associated factor) complex thought to facilitate transcriptional activation of specific genes by antagonizing chromatin-mediated transcriptional repression.
Chromatin modulation BAT3 BCL2-associated athanogene 6. This gene encodes a nuclear protein implicated in the control of apoptosis. In addition, the protein forms a complex with E1A binding protein p300 and is required for p53 acetylation in response to DNA damage. General transcription factor IIIC, polypeptide 4, 90kDa.

HCFC1
Host cell factor C1. This gene is a member of the host cell factor family; the protein is involved in control of the cell cycle and transcriptional regulation.

Transcription Cell cycle regulation ING3
Inhibitor of growth family, member 3. This is a tumor suppressor protein that interacts with TP53, inhibit cell growth, and induce apoptosis. It contains a PHDfinger, a common motif in proteins involved in chromatin remodeling. Initiation of transcription by RNA polymerase II requires the activities of more than 70 polypeptides coordinated by the basal transcription factor TFIID. TFIID is composed of the TATA-binding protein (TBP) and a group of evolutionarily conserved proteins known as TBP-associated factors or TAFs. TAFs may participate in basal transcription, serve as coactivators, function in promoter recognition or modify general transcription factors (GTFs) to facilitate complex assembly and transcription initiation. This gene encodes the largest subunit of TFIID, but it also binds to other transcriptional regulators, possesses acetyltransferase activity and can act as an ubiquitin-activating/conjugating enzyme.
Transcription TAF1L TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 210kDa-like. This locus is intronless, and the product has been shown to function interchangeably with the TAF1 product.  Table 3 The GO term Helicase Activity; differential gene expression when comparing AML cell with and without CCL28-induced growth modulation. The table is based on information from the GENE database.

ASCC3
Activating signal cointegrator 1 complex subunit 3. This protein belongs to a family of helicases that are involved in the ATP-dependent unwinding of nucleic acid duplexes. The encoded protein is the largest subunit of the activating signal cointegrator 1 complex that is involved in DNA repair and resistance to alkylation damage. DEAD (Asp-Glu-Ala-Asp) box helicase 3, X-linked. The protein is a member of the DEAD-box protein family. This protein has a high level of RNAindependent ATPase activity, and unlike most DEAD-box helicases, the ATPase activity is thought to be stimulated by both RNA and DNA. It is thought to play roles in both the nucleus and cytoplasm. Nuclear roles include transcriptional regulation, mRNP assembly, pre-mRNA splicing, and mRNA export. In the cytoplasm, this protein is thought to be involved in translation, cellular signaling, and viral replication. Misregulation of this gene has been implicated in tumorigenesis.