Pretransplant systemic metabolic profiles in allogeneic hematopoietic stem cell transplant recipients - identification of patient subsets with increased transplant-related mortality.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is used in the treatment of high-risk acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS); however, the treatment has high risk of severe transplantation-related mortality (TRM). In this study, we examined pretransplantation serum samples derived from 92 consecutive allotransplant recipients with AML or MDS. Using nontargeted metabolomics, we identified 1274 metabolites including 968 of known identity (named biochemicals). We further investigated metabolites that differed significantly when comparing patients with and without early extensive fluid retention, pretransplantation inflammation (both being associated with increased risk of acute graft-versus-host disease [GVHD]/nonrelapse mortality) and development of systemic steroid-requiring acute GVHD (aGVHD). All three factors are associated with TRM and were also associated with significantly altered amino acid metabolism, although there was only a minor overlap between these three factors with regard to significantly altered individual metabolites. Furthermore, steroid-requiring aGVHD was especially associated with altered taurine/hypotaurine, tryptophan, biotin, and phenylacetate metabolism together with altered malate-aspartate shuttle and urea cycle regulation. In contrast, pretransplantation inflammation was associated with a weaker modulation of many different metabolic pathways, whereas extensive fluid retention was associated with a weaker modulation of taurine/hypotaurine metabolism. An unsupervised hierarchical cluster analysis based on the 13 most significantly identified metabolites associated with aGVHD identified a patient subset with high metabolite levels and increased frequencies of MDS/MDS-AML, steroid-requiring aGVHD and early TRM. On the other hand, a clustering analysis based on metabolites that were significantly altered for aGVHD, inflammation, and fluid retention comparison groups identified a patient subset with a highly significant association with TRM. Our study suggests that the systemic pretransplantation metabolic profiles can be used to identify patient subsets with an increased frequency of TRM.

Two acute myeloid leukemia patient subsets are identified based on the constitutive PI3K-Akt-mTOR signaling of their leukemic cells; a functional, proteomic, and transcriptomic comparison.

OBJECTIVES: Constitutive signaling through the phosphatidylinositol-3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) pathway is present in acute myeloid leukemia (AML) cells. The aim of the study was to compare constitutive PI3K-Akt-mTOR activation of primary AML cells for a large group of unselected patients. METHODS: We investigated expression and phosphorylation of 18 mediators in the PI3K-Akt-mTOR main track by flow cytometry for AML cells derived from 77 patients, and compared this with global gene expression profiles, proteomic, and transcriptomic profiles, and susceptibility to antileukemic agents. RESULTS: Patients were divided into two main subsets showing generally high or low constitutive pathway activation. The high activation subset was characterized by decreased frequency of cells showing monocytic differentiation, increased frequency of adverse karyotypes, decreased constitutive cytokine release, and increased expression of certain integrins. Finally, the two groups differed in their expression of genes encoding regulators of protein phosphorylation, whereas phosphoproteomic analyses showed differences especially with regard to transcriptional regulation. Antiproliferative effects of pathway inhibition were generally stronger for the low phosphorylation subset. CONCLUSION: The constitutive PI3K-Akt-mTOR activation differed between patients; this difference appears to be a part of complex phenotypic differences including cell communication, intracellular signaling through other pathways, and transcriptional regulation.

Resistance to the Antiproliferative In Vitro Effect of PI3K-Akt-mTOR Inhibition in Primary Human Acute Myeloid Leukemia Cells Is Associated with Altered Cell Metabolism.

Constitutive signaling through the phosphatidylinositol-3-kinase-Akt-mechanistic target of rapamycin (PI3K-Akt-mTOR) pathway is present in acute myeloid leukemia (AML) cells. However, AML is a heterogeneous disease, and we therefore investigated possible associations between cellular metabolism and sensitivity to PI3K-Akt-mTOR pathway inhibitors. We performed non-targeted metabolite profiling to compare the metabolome differences of primary human AML cells derived from patients susceptible or resistant to the in vitro antiproliferative effects of mTOR and PI3K inhibitors. In addition, the phosphorylation status of 18 proteins involved in PI3K-Akt-mTOR signaling and the effect of the cyclooxygenase inhibitor indomethacin on their phosphorylation status was investigated by flow cytometry. Strong antiproliferative effects by inhibitors were observed only for a subset of patients. We compared the metabolite profiles for responders and non-responders towards PI3K-mTOR inhibitors, and 627 metabolites could be detected. Of these metabolites, 128 were annotated and 15 of the annotated metabolites differed significantly between responders and non-responders, including metabolites involved in energy, amino acid, and lipid metabolism. To conclude, leukemia cells that are susceptible or resistant to PI3K-Akt-mTOR inhibitors differ in energy, amino acid, and arachidonic acid metabolism, and modulation of arachidonic acid metabolism alters the activation of mTOR and its downstream mediators.

In Vitro Characterization of Valproic Acid, ATRA, and Cytarabine Used for Disease-Stabilization in Human Acute Myeloid Leukemia: Antiproliferative Effects of Drugs on Endothelial and Osteoblastic Cells and Altered Release of Angioregulatory Mediators by Endothelial Cells.

The combined use of the histone deacetylase inhibitor valproic acid (VPA), the retinoic acid receptor- α agonist all-trans retinoic acid (ATRA), and the deoxyribonucleic acid polymerase- α inhibitor cytarabine (Ara-C) is now considered for disease-stabilizing treatment of acute myeloid leukemia (AML). Leukemogenesis and leukemia cell chemoresistance seem to be supported by neighbouring stromal cells in the bone marrow, and we have therefore investigated the effects of these drugs on primary human endothelial cells and the osteoblastic Cal72 cell line. The results show that VPA and Ara-C have antiproliferative effects, and the antiproliferative/cytotoxic effect of Ara-C was seen at low concentrations corresponding to serum levels found during low-dose in vivo treatment. Furthermore, in functional assays of endothelial migration and tube formation VPA elicited an antiangiogenic effect, whereas ATRA elicited a proangiogenic effect. Finally, VPA and ATRA altered the endothelial cell release of angiogenic mediators; ATRA increased levels of CXCL8, PDGF-AA, and VEGF-D, while VPA decreased VEGF-D and PDGF-AA/BB levels and both drugs reduced MMP-2 levels. Several of these mediators can enhance AML cell proliferation and/or are involved in AML-induced bone marrow angiogenesis, and direct pharmacological effects on stromal cells may thus indirectly contribute to the overall antileukemic activity of this triple drug combination.

Antileukaemic effect of PI3K-mTOR inhibitors in acute myeloid leukaemia-gene expression profiles reveal CDC25B expression as determinate of pharmacological effect.

Acute myeloid leukaemia (AML) is a heterogeneous malignancy. Intracellular signalling through the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway is important for regulation of cellular growth and metabolism, and inhibitors of this pathway is considered for AML treatment. Primary human AML cells, derived from 96 consecutive adult patients, were examined. The effects of two mTOR inhibitors (rapamycin, temsirolimus) and two PI3K inhibitors (GDC-0941, 3-methyladenine) were studied, and we investigated cytokine-dependent proliferation, regulation of apoptosis and global gene expression profiles. Only a subset of patients demonstrated strong antiproliferative effects of PI3K-mTOR inhibitors. Unsupervised hierarchical clustering analysis identified two main clusters of patients; one subset showing weak or absent antiproliferative effects (59%) and another group showing a strong growth inhibition for all drugs and concentrations examined (41%). Global gene expression analyses showed that patients with AML cell resistance against PI3K-mTOR inhibitors showed increased mRNA expression of the CDC25B gene that encodes the cell cycle regulator Cell Division Cycle 25B. The antileukaemic effect of PI3K-Akt-mTOR inhibition varies between patients, and resistance to these inhibitors is associated with the expression of the cell cycle regulator CDC25B, which is known to crosstalk with the PI3K-Akt-mTOR pathway and mediate rapamycin resistance in experimental models.

Soluble mediators released by acute myeloid leukemia cells increase capillary-like networks.

Increased bone marrow angiogenesis is seen in several hematological malignancies, including acute myeloid leukemia (AML). We used a co-culture assay of endothelial and vascular smooth muscle cells (vSMC) to investigate the effects of AML-conditioned medium on capillary networks. We investigated primary AML cells derived from 44 unselected patients and observed that for a large subset of patients, the constitutive cytokine release by the leukemic cells stimulated endothelial cell organization into capillary-like networks, while there were only minor or no effects for other patients. We analyzed the constitutive AML cell release of 31 cytokines for all the patients and performed a hierarchical cluster analysis of the cytokine profile which identified two major patient subsets that differed in their ability to enhance capillary-like networks; increased capillary-like networks was then associated with high constitutive release of several cytokines and especially high levels of several pro-angiogenic chemokines. Significantly increased network formation was not seen for any of the 11 acute lymphoblastic leukemia patients investigated. The cytokine response by activated normal T cells inhibited endothelial network formation in our in vitro model of angiogenesis and activated normal monocytes had only a minor influence on tube formation. Our study shows that AML-derived cytokines can induce the organization of endothelial cells into vessel-like structures.

Expression profile of heat shock proteins in acute myeloid leukaemia patients reveals a distinct signature strongly associated with FLT3 mutation status--consequences and potentials for pharmacological intervention.

Heat shock proteins (HSPs) are molecular chaperones that assist proteins in their folding to native structures. HSPs are regarded as possible therapeutic targets in acute myeloid leukaemia (AML). We used bioinformatical approaches to characterize the HSP profile in AML cells from 75 consecutive patients, in addition to the effect of the HSP90 inhibitor 17-DMAG. Patients harbouring a FLT3-internal tandem duplication (FLT3-ITD) were extensively overrepresented in the cluster with high HSP levels, indicating a strong dependence of HSPs in stabilizing FLT3-ITD encoded oncoproteins. FLT3 ligation further increased the levels of HSP90 and its co-chaperone HSP70. HSP90 inhibition had a stronger pro-apoptotic effect for AML cells with FLT3-ITD than for cells with wild-type FLT3, whereas the anti-proliferative effect of HSP90 inhibition was similar for the two patient subsets. HSP90 inhibition altered the constitutive cytokine release profile in an anti-angiogenic direction independent of FLT3 mutational status: (i) pro-angiogenic CXCL8, MMP-2 and MMP-9 showed a stronger decrease than anti-angiogenic CXCL9-11, (ii) the Tie-2 agonist Ang-1 showed a stronger decrease than the potentially antagonistic Ang-2, and (iii) VEGF and HGF levels were decreased. Finally, HSP90 inhibition counteracted the leukaemia-stimulating effect of endothelial cells. Our studies demonstrate that HSP90 inhibition mediates anti-leukaemic effects through both direct and indirect activity.

Future perspectives: therapeutic targeting of notch signalling may become a strategy in patients receiving stem cell transplantation for hematologic malignancies.

The human Notch system consists of 5 ligands and 4 membrane receptors with promiscuous ligand binding, and Notch-initiated signalling interacts with a wide range of other intracellular pathways. The receptor signalling seems important for regulation of normal and malignant hematopoiesis, development of the cellular immune system, and regulation of immune responses. Several Notch-targeting agents are now being developed, including natural receptor ligands, agonistic and antagonistic antibodies, and inhibitors of intracellular Notch-initiated signalling. Some of these agents are in clinical trials, and several therapeutic strategies seem possible in stem cell recipients: (i) agonists may be used for stem cell expansion and possibly to enhance posttransplant lymphoid reconstitution; (ii) receptor-specific agonists or antagonists can be used for immunomodulation; (iii) Notch targeting may have direct anticancer effects. Although the effects of therapeutic targeting are difficult to predict due to promiscuous ligand binding, targeting of this system may represent an opportunity to achieve combined effects with earlier posttransplant reconstitution, immunomodulation, or direct anticancer effects.

Primary human acute myelogenous leukemia cells release matrix metalloproteases and their inhibitors: release profile and pharmacological modulation.

OBJECTIVES: Angiogenesis seems important for both leukemogenesis and chemosensitivity in acute myelogenous leukemia (AML). Angiogenesis is regulated by the balance between pro- and antiangiogenic cytokines, which also indicates an important role of matrix metalloproteases (MMPs) and their natural inhibitors, tissue inhibitors of metalloproteases (TIMPs). We investigated the constitutive release of MMPs and TIMPs for a large group of consecutive AML patients. METHODS: AML cells were cultured in vitro either alone or together with microvascular endothelial cells, and levels of MMPs and TIMPs were determined in culture supernatants. RESULTS: AML cells showed constitutive release of several MMPs and TIMPs. For all patients, detectable MMP-10 release was observed, and most patients showed detectable release of at least one additional MMP, usually MMP-9 or MMP-2. A significant correlation was found between MMP-9 and TIMP-1 release and the release of several CCL and CXCL chemokines. MMP-9 release was higher for AML cells with monocytic differentiation corresponding to the FAB-subtype M4/M5 AML; it was mainly released in its inactive form, but endogenously active MMP-9 could be detected even in the presence of the constitutively released TIMP-1/2. Endothelial cells released relatively high levels of MMP-10, and these levels were further increased by coculture with AML cells. Patients achieving complete hematological remission after only one induction cycle showed relatively low constitutive MMP-2 release. CONCLUSION: We conclude that primary human AML cells show constitutive release of both MMPs and TIMPs, and this release may be important for leukemogenesis and possibly also for chemosensitivity.

Antiangiogenic therapy in acute myelogenous leukemia: targeting of vascular endothelial growth factor and interleukin 8 as possible antileukemic strategies.

Acute myelogenous leukemia (AML) is an aggressive disorder with an overall disease-free survival of 40-50% even for the younger patients under 60 years of age who can receive the most intensive treatment. The median age at the time of diagnosis is 60-65 years, and the large majority of elderly patients usually receive less intensive chemotherapy or only supportive therapy due to the high treatment-related mortality when using intensive therapy for elderly individuals. Thus, there is a need for new therapeutic approaches to improve the treatment in younger patients and to make AML-directed therapy with acceptable toxicity possible in elderly individuals. Angiogenesis seems to be important both for leukemogenesis and susceptibility to intensive chemotherapy, and antiangiogenic strategies are therefore considered for the treatment of AML. The two proangiogenic mediators vascular endothelial growth factor (VEGF) and interleukin 8, (IL-8, also referred to as CXCL8) seem to be important in human AML: VEGF is released at increased levels due to interactions between AML cells and neighboring nonleukemic cells, whereas IL-8 is released at high levels by native human AML cells. Thus, VEGF as a therapeutic target in AML is suggested both by experimental and clinical observations, whereas IL-8 as a target is mainly suggested by experimental evidence. In the present review we describe and discuss (i) the angioregulatory network of soluble mediators in AML, including both the systemic levels and local release by native human AML cells; and (ii) various therapeutic approaches to target VEGF and IL-8. Although single angioregulatory mediators can be targeted, it should be emphasized that the final effect of soluble mediators on angioregulation is determined by a complex angioregulatory network that varies between AML patients, and the final effect of targeting single mediators may therefore differ between patient subsets.