Large-scale identification of disease genes involved in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of diseases in which chromosomal aberrations, small insertions or deletions, or point mutations in certain genes have profound consequences for prognosis. However, the majority of AML patients present without currently known genetic defects. Retroviral insertion mutagenesis in mice has become a powerful tool for identifying new disease genes involved in the pathogenesis of leukemia and lymphoma. Here we have used the Graffi-1.4 strain of murine leukemia virus, which causes predominantly AML, in a screen to identify novel genes involved in the pathogenesis of this disease. We report 79 candidate disease genes in common integration sites (CISs) and 15 genes whose family members previously were found to be affected in other studies. The majority of the identified sequences (60%) were not found in lymphomas and monocytic leukemias in previous screens, suggesting a specific involvement in AML. Although most of the virus integrations occurred in or near the 5' or 3' ends of the genes, suggesting deregulation of gene expression as a consequence of virus integration, 18 CISs were located exclusively within the genes, conceivably causing gene disruption.     

Radiation-induced myeloid leukemia in murine models

The use of radiation therapy is a cornerstone of modern cancer treatment. The number of patients that undergo radiation as a part of their therapy regimen is only increasing every year, but this does not come without cost. As this number increases, so too does the incidence of secondary, radiation-induced neoplasias, creating a need for therapeutic agents targeted specifically towards incidence reduction and treatment of these cancers. Development and efficacy testing of these agents requires not only extensive in vitro testing but also a set of reliable animal models to accurately recreate the complex situations of radiation-induced carcinogenesis. As radiation-induced leukemic progression often involves genomic changes such as rearrangements, deletions, and changes in methylation, the laboratory mouse Mus musculus, with its fully sequenced genome, is a powerful tool in cancer research. This fact, combined with the molecular and physiological similarities it shares with man and its small size and high rate of breeding in captivity, makes it the most relevant model to use in radiation-induced leukemia research. In this work, we review relevant M. musculus inbred and F₁ hybrid animal models, as well as methods of induction of radiation-induced myeloid leukemia. Associated molecular pathologies are also included.     

Learning from mouse models of MLL fusion gene-driven acute leukemia

5–10% of human acute leukemias carry chromosomal translocations involving the mixed lineage leukemia (MLL) gene that result in the expression of chimeric protein fusing MLL to >80 different partners of which AF4, ENL and AF9 are the most prevalent. In contrast to many other leukemia-associated mutations, several MLL-fusions are powerful oncogenes that transform hematopoietic stem cells but also more committed progenitor cells. Here, I review different approaches that were used to express MLL fusions in the murine hematopoietic system which often, but not always, resulted in highly penetrant and transplantable leukemias that closely phenocopied the human disease. Due to its simple and reliable nature, reconstitution of irradiated mice with bone marrow cells retrovirally expressing the MLL-AF9 fusion became the most frequently in vivo model to study the biology of acute myeloid leukemia (AML). I review some of the most influential studies that used this model to dissect critical protein interactions, the impact of epigenetic regulators, microRNAs and microenvironment-dependent signals for MLL fusion-driven leukemia. In addition, I highlight studies that used this model for shRNA- or genome editing-based screens for cellular vulnerabilities that allowed to identify novel therapeutic targets of which some entered clinical trials. Finally, I discuss some inherent characteristics of the widely used mouse model based on retroviral expression of the MLL-AF9 fusion that can limit general conclusions for the biology of AML. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.     

A two-mutation model of radiation-induced acute myeloid leukemia using historical mouse data

From studies of the atomic bomb survivors, it is well known that ionizing radiation causes several forms of leukemia. However, since the specific mechanism behind this process remains largely unknown, it is difficult to extrapolate carcinogenic effects at acute high-dose exposures to risk estimates for the chronic low-dose exposures that are important for radiation protection purposes. Recently, it has become clear that the induction of acute myeloid leukemia (AML) in CBA/H mice takes place through two key steps, both involving the Sfpi1 gene. A similar mechanism may play a role in human radiation-induced AML. In the present paper, a two-mutation carcinogenesis model is applied to model AML in several data sets of X-ray- and neutron-exposed CBA/H mice. The models obtained provide good fits to the data. A comparison between the predictions for neutron-induced and X-ray-induced AML yields an RBE for neutrons of approximately 3. The model used is considered to be a first step toward a model for human radiation-induced AML, which could be used to estimate risks of exposure to low doses.     

Annular promyelocytes in acute myeloid leukemia

Granulocytes with ring-shaped nuclei (annular granulocytes; ring granulocytes) are normal bone marrow constituents in rodents. Studies in man have shown a small number of these cells in cases of myeloproliferative diseases. Myelocytes and metamyelocytes have also been described. Similar to rodents and some other animal species, the annular promyelocyte also exists in humans. The significance of these very rare cells in human haemopoiesis becomes an interesting question.     

Interleukin-17 in acute myeloid leukemia

There are several reports that angiogenesis plays important roles in hematological malignancies including acute myeloid leukemia (AML). Human interleukin-17 (IL-17) is a proinflammatory cytokine produced by activated CD4 T cells. IL-17 plays a potential role in T cell mediated angiogenesis. The role of IL-17 in pathologic angiogenesis has not been evaluated yet. The aim of the study was to determine plasma level of IL-17 in patients with AML. IL-17 levels were measured by ELISA in plasma samples taken from 68 adult patients with AML before chemotherapy was administered. In addition 20 out of 68 patients were reanalysed after achieving complete remission (CR). Ten samples from healthy volunteers were evaluated as the control. In this study we have demonstrated that serum level of IL-17 is not elevated in AML patients. These results suggest that angiogenesis in AML is not mediated by CD4 T cells. To our knowledge this is the first report about IL-17 serum level in acute leukemias. We are currently evaluating IL-17 levels in others haematological malignancies.     

An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Background

Xenotransplantation of patient-derived AML (acute myeloid leukemia) cells in NOD-scid Il2rγ ^null (NSG) mice is the method of choice for evaluating this human hematologic malignancy. However, existing models constructed using intravenous injection in adult or newborn NSG mice have inferior engraftment efficiency, poor peripheral blood engraftment, or are difficult to construct.

Methods

Here, we describe an improved AML xenograft model where primary human AML cells were injected into NSG newborn pups intrahepatically.

Results

Introduction of primary cells from AML patients resulted in high levels of engraftment in peripheral blood, spleen, and bone marrow (BM) of recipient mice. The phenotype of engrafted AML cells remained unaltered during serial transplantation. The mice developed features that are consistent with human AML including spleen enlargement and infiltration of AML cells into multiple organs. Importantly, we demonstrated that although leukemic stem cell activity is enriched and mediated by CD34⁺CD117⁺ subpopulation, CD34⁺CD117^? subpopulation can acquire CD34⁺CD117⁺ phenotype through de-differentiation. Lastly, we evaluated the therapeutic potential of Sorafenib and Regorafenib in this AML model and found that periphery and spleen AML cells are sensitive to these treatments, whereas BM provides a protective environment to AML.

Conclusions

Collectively, our improved model is robust, easy-to-construct, and reliable for pre-clinical AML studies.

     

Antiapoptotic microenvironment of acute myeloid leukemia

We showed previously that tumor-derived supernatant (TSN) from acute myeloid leukemia (AML) myeloblasts inhibits peripheral blood T cell activation and proliferation, rendering the T cells functionally incompetent. We show here that the AML TSN also significantly delays apoptosis of both resting and stimulated T cells, as judged by reduction in annexin V/propidium iodide staining. In addition, we show that this is not unique to T cells and that AML TSN inhibits apoptosis of peripheral B cells, neutrophils, and monocytes. Furthermore, it also enhances the survival of other AML myeloblasts with lower viability. Investigations into the mechanism demonstrate a reduction in the cleavage of procaspase-3, -8, and -9 and the caspase substrate, poly(ADP-ribose)polymerase (PARP). This may be due to Bcl-2, which is normally down-regulated in CD3/CD28-stimulated T cells, but is maintained in the presence of AML TSN. We conclude that AML cells generate an antiapoptotic microenvironment that favors the survival of malignant cells, but also inhibits apoptosis of other normal hemopoietic cells. Reversal of these immunosuppressive effects and restoration of normal immune responses in patients with AML would improve the success of immunotherapy protocols.     

Genetic profile of acute myeloid leukemia

Understanding genomic events and the cascade of their effects in cell function is crucial for identifying distinct subsets of acute myeloid leukemia and developing new therapeutic strategies. Conventional cytogenetics, fluorescence in situ hybridization investigations and molecular studies have provided much information over the past few years. This review will focus on major genomic mechanisms in acute myeloid luekemia and on the genes implicated in the pathogenesis of specific subtypes.     

Anti-leukemic activity of Lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia

Despite therapeutic advances, the long-term survival rates for acute myeloid leukemia (AML) are estimated to be 10% or less, pointing to the need for better treatment options. AML cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Thus, the in vitro and in vivo anti-tumor activities of lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, were investigated. In vitro assays were used to assess the ability of lintuzumab to mediate effector functions and to decrease the production of growth factors from AML cells. SCID mice models of disseminated AML with the multi-drug resistance (MDR)-negative HL60 and the MDR+, HEL9217 and TF1-α, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNF-α-induced pro-inflammatory cytokines and chemokines by AML cells. Lintuzumab promoted tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities against MDR- and MDR+ AML cell lines and primary AML patient samples. At doses from 3 to 30 mg/kg, lintuzumab significantly enhanced survival and reduced tumor burden in vivo, regardless of MDR status. Survival of the mice was dependent upon the activity of resident macrophages and neutrophils. The results suggest that lintuzumab may exert its therapeutic effects by modulating the cytokine milieu in the tumor microenvironment and through effector mediated cell killing. Given that lintuzumab induced meaningful responses in a phase 1 clinical trial, the preclinical antitumor activities defined in this study may underlie its observed therapeutic efficacy in AML patients.     

Anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia

Despite therapeutic advances, the long-term survival rates for acute myeloid leukemia (AML) are estimated to be 10% or less, pointing to the need for better treatment options. AML cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Thus, the in vitro and in vivo anti-tumor activities of lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, were investigated. In vitro assays were used to assess the ability of lintuzumab to mediate effector functions and to decrease the production of growth factors from AML cells. SCID mice models of disseminated AML with the multi-drug resistance (MDR)-negative HL60 and the MDR⁺, HEL9217 and TF1-α, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNFα-induced pro-inflammatory cytokines and chemokines by AML cells. Lintuzumab promoted tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities against MDR⁻ and MDR⁺ AML cell lines and primary AML patient samples. At doses from 3 to 30 mg/kg, lintuzumab significantly enhanced survival and reduced tumor burden in vivo, regardless of MDR status. Survival of the mice was dependent upon the activity of resident macrophages and neutrophils. The results suggest that lintuzumab may exert its therapeutic effects by modulating the cytokine milieu in the tumor microenvironment and through effector mediated cell killing. Given that lintuzumab induced meaningful responses in a phase 1 clinical trial, the preclinical antitumor activities defined in this study may underlie its observed therapeutic efficacy in AML patients.     

Dendritic cell vaccination in acute myeloid leukemia

The prognosis of patients with acute myeloid leukemia (AML) remains dismal, with a 5-year overall survival rate of only 5.2% for the continuously growing subgroup of AML patients older than 65 years. These patients are generally not considered eligible for intensive chemotherapy and/or allogeneic hematopoietic stem cell transplantation because of high treatment-related morbidity and mortality, emphasizing the need for novel, less toxic, treatment alternatives. It is within this context that immunotherapy has gained attention in recent years. In this review, we focus on the use of dendritic cell (DC) vaccines for immunotherapy of AML. DC are central orchestrators of the immune system, bridging innate and adaptive immunity and critical to the induction of anti-leukemic immunity. We discuss the rationale and basic principles of DC-based therapy for AML and review the clinical experience that has been obtained so far with this form of immunotherapy for patients with AML.     

FLT3 inhibitors in acute myeloid leukemia

FLT3 mutations are one of the most common findings in acute myeloid leukemia (AML). FLT3 inhibitors have been in active clinical development. Midostaurin as the first-in-class FLT3 inhibitor has been approved for treatment of patients with FLT3-mutated AML. In this review, we summarized the preclinical and clinical studies on new FLT3 inhibitors, including sorafenib, lestaurtinib, sunitinib, tandutinib, quizartinib, midostaurin, gilteritinib, crenolanib, cabozantinib, Sel24-B489, G-749, AMG 925, TTT-3002, and FF-10101. New generation FLT3 inhibitors and combination therapies may overcome resistance to first-generation agents.