Kimberley Joanne Hatfield

The proteasome inhibitors bortezomib and PR-171 have antiproliferative and proapoptotic effects on primary human acute myeloid leukaemia cells.

Proteasome inhibitors represent a new class of antineoplastic drugs that are considered in the treatment of haematological malignancies. We compared the effects of the reversible proteasome inhibitor bortezomib (Velcade®) and the epoxomicin derivative PR‐171, an irreversible inhibitor, on primary human acute myeloid leukaemia (AML) cells. Both drugs inhibited autocrine‐ and cytokine‐dependent proliferation of primary AML blasts when tested at nanomolar levels (0·1–100 nmol/l). The antiproliferative effect was independent of basal chymotrypsin‐like proteasome activity (showing a 20‐fold variation between patients), genetic abnormalities, morphological differentiation and CD34 expression when testing a large group of consecutive patients (n = 54). The effect was retained in cocultures with bone marrow stromal cells. In addition, both drugs enhanced apoptosis. The effect of PR‐171 could be detected at lower concentrations than for bortezomib, especially when testing the influence on clonogenic AML cell proliferation. Both drugs had divergent effects on AML cells’ constitutive cytokine release. Furthermore, both drugs caused a decrease in proliferation and viability when tested in combination with idarubicin or cytarabine. An antiproliferative effect on primary human acute lymphoblastic leukaemia cells was also detected. We conclude that nanomolar levels of the proteasome inhibitors tested had dose‐dependent antiproliferative and proapoptotic effects on primary AML cells in vitro.

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.

Microvascular endothelial cells increase proliferation and inhibit apoptosis of native human acute myelogenous leukemia blasts

Interactions between acute myelogenous leukemia (AML) blasts and neighbouring endothelial cells in the bone marrow seem important both for disease development and susceptibility to chemotherapy. We investigated the effects of soluble mediators released by microvascular endothelial cells on native human AML cells. AML cells derived from 33 patients were cocultured with microvascular endothelial cells, separated by a semipermeable membrane. We investigated the effect of coculture on AML cell proliferation, viability/apoptosis and cytokine release. Coculture increased AML cell proliferation, and this growth enhancement included the clonogenic leukemia cell subset. Increased release of several soluble mediators was also detected (interleukin 3, interleukin 6, granulocyte‐macrophage and granulocyte colony‐stimulating factors) in cocultures. Our cytokine neutralization experiments suggest that an intercellular crosstalk involving several soluble mediators contribute to the increased leukemia cell proliferation. The presence of endothelial cells had an additional antiapoptotic effect on the AML cells. The endothelial cells did not have any growth‐enhancing effect on native human acute lymphoblastic leukemia cells. Our in vitro results suggest that the release of soluble mediators by microvascular endothelial cells supports leukemic hematopoiesis through paracrine mechanisms by direct enhancement of AML blast proliferation and by inhibition of leukemic cell apoptosis.

Implantable microenvironments to attract hematopoietic stem/cancer cells

The environments that harbor hematopoietic stem and progenitor cells are critical to explore for a better understanding of hematopoiesis during health and disease. These compartments often are inaccessible for controlled and rapid experimentation, thus limiting studies to the evaluation of conventional cell culture and transgenic animal models. Here we describe the manufacture and image-guided monitoring of an engineered microenvironment with user-defined properties that recruits hematopoietic progenitors into the implant. Using intravital imaging and fluorescence molecular tomography, we show in real time that the cell homing and retention process is efficient and durable for short- and long-term engraftment studies. Our results indicate that bone marrow stromal cells, precoated on the implant, accelerate the formation of new sinusoidal blood vessels with vascular integrity at the microcapillary level that enhances the recruitment hematopoietic progenitor cells to the site. This implantable construct can serve as a tool enabling the study of hematopoiesis.

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.

Primary human acute myeloid leukaemia cells increase the proliferation of microvascular endothelial cells through the release of soluble mediators

Bone marrow angiogenesis is suggested to play a role in the pathogenesis of acute myeloid leukaemia (AML) and endothelial cells may mediate chemosensitivity. This study investigated in vitro endothelial effects of coculture of microvascular endothelial cells (MVEC) with AML cells derived from 33 consecutive AML patients. A proliferation assay showed that (i) AML cells from the majority of patients examined increased endothelial cell proliferation, while cytokine neutralizing experiments had divergent effects on proliferation and (ii) the angiopoietin/Tie2 system was important for growth of AML cells, and angiopoietin‐1 induced phosphorylation of signal transducers and activators of transcription (STAT) proteins in AML cells. Finally, gene expression profiling of MVEC cocultured with AML cells was conducted in non‐contact cultures. Microarray analysis revealed that the majority of significantly expressed genes could be categorized into gene ontology terms involved in transcription, cellular organization and intracellular signalling. Our study indicates a role for the leukaemic‐endothelium crosstalk in leukaemogenesis with enhancement of endothelial cell growth and increased AML cell proliferation possibly mediated by angiopoietin‐1 and the STAT signalling pathway.

Release of angiopoietin-1 by primary human acute myelogenous leukemia cells is associated with mutations of nucleophosmin, increased by bone marrow stromal cells and possibly antagonized by high systemic angiopoietin-2 levels

The balance between proangiogenic Angiopoietin-1 (Ang-1) and the antagonistic Ang-2 is important both for leukemogenesis and chemosensitivity in human acute myelogenous leukemia (AML). We examined the release of Ang-1 and Ang-2 by AML cells cultured alone and in cocultures with stromal cells. Detectable Ang-1 release from AML cells was observed for most patients (62/91), whereas Ang-2 release was detected only for a minority (23/91). Coculture of AML and stromal cells led to increased Ang-1 levels. Furthermore, the role of the angiopoietin system was investigated by characterizing whether the differences in angiopoietin expression in AML patients can be related to nucleophosmin (NPM1) mutations. We compared the gene expression profiles of AML cells derived from 19 patients with FLT3 mutations and normal cytogenetics with and without NPM1 mutations and observed increased expression of Ang-1 in patients with NPM1 mutations. Finally, we found significantly higher Ang-2 levels in serum of AML patients compared with healthy controls. Our results suggest that AML cells are a major source of Ang-1 in leukemic bone marrow, especially in patients with NPM1 mutations, but the local levels are also influenced by stromal cells. Local Ang-2 release from AML cells is less common, but high systemic levels of Ang-2 may affect bone marrow angioregulation.

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.

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.