Håkon Reikvam

The Crosstalk Between the Matrix Metalloprotease System and the Chemokine Network in Acute Myeloid Leukemia

Matrix metalloproteinases (MMPs) comprise a large family of zinc-dependent endopeptidases, which are best known for their ability to degrade essentially all components of the extracellular matrix (ECM). By breaking down ECM, MMPs may remove physical barriers, thus allowing cells to migrate and potentially invade other tissues. Recent evidence, however, shows that the proteolytic activities of MMPs also affect several fundamental physiological processes. Primary human acute myeloid leukemia (AML) cells often show constitutive release of several MMPs and chemokines, and there seems to be a crosstalk between the MMP system and the chemokine network. Firstly, the nuclear factor-κB (NF-κB) system represents a common regulator at the transcriptional level both for MMPs (e.g. MMP-1 and MMP-9) and for the constitutive release of several chemokines (CCL2-4/CXCL1/8) by primary human AML cells. Secondly, the crosstalk at the molecular level probably includes MMP-mediated structural alteration and activation of constitutively released chemokines involved in AML cell migration (e.g. CXCL12) and stimulation of bone marrow angiogenesis (e.g. CXCL8). Thirdly, at a functional level the two systems interact because the chemokine network plays a role in similar physiological processes as the MMPs, including AML cell proliferation and migration and local regulation of angiogenesis. Both the chemokine system and MMPs are currently being evaluated as targets in anti-angiogenesis/cancer therapy and may also have potential therapeutic implications in AML. This review introduces the different members of the MMP family and describes their interactions with the chemokine network and the possible involvement of MMPs together with chemokines in leukemogenesis and chemosensitivity in AML.

Heat shock protein 90 - a potential target in the treatment of human acute myelogenous leukemia.

Heat shock proteins (HSPs) are molecular chaperones that stabilize folding and conformation of normal as well as oncogenic proteins. These chaperones thereby prevent the formation of protein aggregates. HSPs are often overexpressed in human malignancies, including AML. HSP90 is the main chaperon required for the stabilization of multiple oncogenic kinases involved in the development of acute myelogenous leukemia (AML). HSP90 client proteins are involved in the regulation of apoptosis, proliferation, autophagy and cell cycle progression; several of these proteins are in addition considered as possible therapeutic targets for the treatment of AML. HSP90 inhibition thereby offers the possibility to modulate several intracellular regulatory pathways through targeting of a single molecule. Several direct inhibitors of HSP90 have been developed, and they are classified into four groups: benzoquinon ansamycines and their derivatives, radicicol and its derivates, small synthetic inhibitors and a final group of other inhibitors. The HSP90 activity is regulated by posttranscriptional modulation; HSP90 inhibition can thereby be indirectly achieved through increased acetylation caused by histone deacetylase inhibitors. Many of these agents have entered phase I/II clinical trials, and the results from these initial studies have documented that HSP90 inhibition can mediate antileukemic effects in vivo. However, one would expect immunosuppressive side effects because HSP90 inhibitors have both direct and indirect inhibitory effects on T cell activation. Thus, future clinical studies are needed to clarify the efficiency and toxicity of HSP90 inhibitors in the treatment of human AML, including studies where HSP90 inhibitors are combined with conventional chemotherapy.

Acute Myeloid Leukemia with the t(8;21) Translocation: Clinical Consequences and Biological Implications

The t(8;21) abnormality occurs in a minority of acute myeloid leukemia (AML) patients. The translocation results in an in-frame fusion of two genes, resulting in a fusion protein of one N-terminal domain from the AML1 gene and four C-terminal domains from the ETO gene. This protein has multiple effects on the regulation of the proliferation, the differentiation, and the viability of leukemic cells. The translocation can be detected as the only genetic abnormality or as part of more complex abnormalities. If t(8;21) is detected in a patient with bone marrow pathology, the diagnosis AML can be made based on this abnormality alone. t(8;21) is usually associated with a good prognosis. Whether the detection of the fusion gene can be used for evaluation of minimal residual disease and risk of leukemia relapse remains to be clarified. To conclude, detection of t(8;21) is essential for optimal handling of these patients as it has both diagnostic, prognostic, and therapeutic implications.

Venous thromboembolism in patients with essential thrombocythemia and polycythemia vera

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPNs), which generally follow a benign and indolent clinical course. However, venous thromboses are common and constitute the main cause of morbidity and mortality. The discovery of the JAK2V617F mutation and other biomarkers has advanced our understanding of these diseases. There is a strong association between the presence of the JAK2V617F mutation and the development of thrombosis in ET. If venous thrombosis presents with unusual manifestations, the diagnosis of a MPN, such as PV or ET, should be part of the differentials. Treatment of venous thrombosis in MPN follows the same principle as in other patients with venous thrombosis, but careful attention to primary and secondary prophylaxis in addition to heparin-induced thrombocytopenia should be given. Cytoreductive therapy is indicated in high-risk subgroups of PV and ET patients, and alternative therapeutic agents have different effects on risk of venous thrombosis. New therapeutic approaches are emerging, and JAK2 inhibitors, histone deacetylase inhibitors and next-generation anticoagulants are in various stages of clinical development for the treatment of MPN, but their exact role in thrombosis prevention and treatment remains unclear.

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.

The chemokine network in acute myelogenous leukemia: molecular mechanisms involved in leukemogenesis and therapeutic implications.

Acute myelogenous leukemia (AML) is a bone marrow disease in which the leukemic cells show constitutive release of a wide range of CCL and CXCL chemokines and express several chemokine receptors. The AML cell release of various chemokines is often correlated and three release clusters have been identified: CCL2-4/CXCL1/8, CCL5/CXCL9-11, and CCL13/17/22/24/CXCL5. CXCL8 is the chemokine usually released at highest levels. Based on their overall constitutive release profile, patients can be classified into distinct subsets that differ in their T cell chemotaxis towards the leukemic cells. The release profile is modified by hypoxia, differentiation status, pharmacological interventions, and T cell cytokine responses. The best investigated single chemokine in AML is CXCL12 that binds to CXCR4. CXCL12/CXCR4 is important in leukemogenesis through regulation of AML cell migration, and CXCR4 expression is an adverse prognostic factor for patient survival after chemotherapy. Even though AML cells usually release high levels of several chemokines, there is no general increase of serum chemokine levels in these patients and the levels are also influenced by patient age, disease status, chemotherapy regimen, and complicating infections. However, serum CXCL8 levels seem to partly reflect the leukemic cell burden in AML. Specific chemokine inhibitors are currently being developed, although redundancy and pleiotropy of the chemokine system are obstacles in drug development.

Targeting the angiopoietin (Ang)/Tie-2 pathway in the crosstalk between acute myeloid leukaemia and endothelial cells: studies of Tie-2 blocking antibodies, exogenous Ang-2 and inhibition of constitutive agonistic Ang-1 release.

Background: The Tie-2 receptor can bind its agonistic ligand Angiopoietin-1 (Ang-1) and the potential antagonist Ang-2. Tie-2 can be expressed both by primary human acute myeloid leukaemia (AML) cells and endothelial cells, and Tie-2-blocking antibodies are now being evaluated in clinical trials for cancer treatment. Design and methods: We investigated the effects of Tie-2-blocking antibodies, exogenous Ang-2 and pharmacological agents on AML cell proliferation and the release of angioregulatory mediators. Results: Tie-2-blocking antibodies had a growth inhibitory effect on human AML cells co-cultured with microvascular endothelial cells, but this inhibition was not observed when leukaemic cells were co-cultured with fibroblasts or osteoblasts. AML cell viability in co-cultures was not altered by anti-Tie-2. Furthermore, anti-Tie-2 decreased hepatocyte growth factor (HGF) levels and increased CXCL8 levels in co-cultures, whereas the levels of endocan (a proteoglycan released by endothelial cells) were not altered. The only significant effects of exogenous Ang-2 were decreased levels of HGF and endocan. Constitutive AML cell release of agonistic Ang-1 was decreased by the proteasomal inhibitor bortezomib and the specific IκB-kinase/NFκB inhibitor BMS-345541. Conclusion: We conclude that various strategies for inhibition of Tie-2-mediated signalling should be considered in AML therapy, possibly in combination with other antiangiogenic strategies.

Targeted therapy in acute myeloid leukaemia: current status and future directions.

Background: The limit of acceptable toxicity for standard chemotherapeutic drugs used in acute myeloid leukaemia (AML) therapy has been reached. New therapeutic strategies are therefore needed. Objective: This review summarizes development in new strategies, and gives an overview of the clinical status on new drugs for non-promyelocytic AML in adults. Methods: Information was principally gathered from the databases ClinicalTrials.gov and PubMed.gov. Results/conclusion: The major improvements in AML treatment during the last two decades has not been the introduction of new therapeutic agents, but rather the more optimal use of well-known drugs (e.g., high-dose cytarabine therapy, the use of ATRA in maintenance therapy of acute promyelocytic leukaemia) and improvement in the diagnosis and treatment of potentially life-threatening complications in patients treated with allogeneic stem cell transplantation. However, further investigations based on specific targeted therapy and stratification of patients according to knowledge of the individual disease and health status will probably be necessary in future studies of new targeted therapy.

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.

Therapeutic Targeting the Cell Division Cycle 25 (CDC25) Phosphatases in Human Acute Myeloid Leukemia — The Possibility to Target Several Kinases through Inhibition of the Various CDC25 Isoforms

The cell division cycle 25 (CDC25) phosphatases include CDC25A, CDC25B and CDC25C. These three molecules are important regulators of several steps in the cell cycle, including the activation of various cyclin-dependent kinases (CDKs). CDC25s seem to have a role in the development of several human malignancies, including acute myeloid leukemia (AML); and CDC25 inhibition is therefore considered as a possible anticancer strategy. Firstly, upregulation of CDC25A can enhance cell proliferation and the expression seems to be controlled through PI3K-Akt-mTOR signaling, a pathway possibly mediating chemoresistance in human AML. Loss of CDC25A is also important for the cell cycle arrest caused by differentiation induction of malignant hematopoietic cells. Secondly, high CDC25B expression is associated with resistance against the antiproliferative effect of PI3K-Akt-mTOR inhibitors in primary human AML cells, and inhibition of this isoform seems to reduce AML cell line proliferation through effects on NFκB and p300. Finally, CDC25C seems important for the phenotype of AML cells at least for a subset of patients. Many of the identified CDC25 inhibitors show cross-reactivity among the three CDC25 isoforms. Thus, by using such cross-reactive inhibitors it may become possible to inhibit several molecular events in the regulation of cell cycle progression and even cytoplasmic signaling, including activation of several CDKs, through the use of a single drug. Such combined strategies will probably be an advantage in human cancer treatment.