Modeling Acute Myeloid Leukemia Using StarPEG-Heparin Hydrogels.

Biomimetic semi-synthetic hydrogels formed from a combination of star-shaped poly(ethylene glycol) (starPEG) and the glycosaminoglycan, heparin, allows for the three-dimensional (3D) culture of various cells and tissues. In this chapter, we describe methods for the use of starPEG-heparin hydrogels to cultivate primary and immortalized human acute myeloid leukemia (AML) cells. The resulting 3D culture models allow for the study of AML development and response to chemotherapeutic agents.

Gene Expression Analyses in Models of Rosiglitazone-Induced Physiological and Pathological Mineralization Identify Novel Targets to Improve Bone and Vascular Health.

Clinical studies revealed detrimental skeletal and vascular effects of the insulin sensitizer rosiglitazone. We have shown earlier that rosiglitazone accelerates osteoblast differentiation from human mesenchymal stem cells (hMSC) at the expense of increased oxidative stress and cell death. In calcifying human vascular cells, rosiglitazone stimulates pathological mineralization, an effect diminished by the antioxidant resveratrol.

Cre recombinase expression cooperates with homozygous FLT3 internal tandem duplication knockin mouse model to induce acute myeloid leukemia.

Murine models offer a valuable tool to recapitulate genetically defined subtypes of AML, and to assess the potential of compound mutations and clonal evolution during disease progression. This is of particular importance for difficult to treat leukemias such as FLT3 internal tandem duplication (ITD) positive AML. While conditional gene targeting by Cre recombinase is a powerful technology that has revolutionized biomedical research, consequences of Cre expression such as lack of fidelity, toxicity or off-target effects need to be taken into consideration.

Targeting cell cycle and apoptosis to overcome chemotherapy resistance in acute myeloid leukemia.

Chemotherapy-resistant acute myeloid leukemia (AML), frequently driven by clonal evolution, has a dismal prognosis. A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockouts involving AraC metabolism and genes that regulate cell cycle arrest (cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53) as contributing to resistance. In human AML cohorts, reduced expression of CDKN2A conferred inferior overall survival and CDKN2A downregulation occurred at relapse in paired diagnosis-relapse samples, validating its clinical relevance.

Epigenetic Activation of Plasmacytoid DCs Drives IFNAR-Dependent Therapeutic Differentiation of AML.

Unlabelled: Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC).

Intact TP-53 function is essential for sustaining durable responses to BH3-mimetic drugs in leukemias.

Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status.

Hematopoietic stem and progenitor cell-restricted Cdx2 expression induces transformation to myelodysplasia and acute leukemia.

The caudal-related homeobox transcription factor CDX2 is expressed in leukemic cells but not during normal blood formation. Retroviral overexpression of Cdx2 induces AML in mice, however the developmental stage at which CDX2 exerts its effect is unknown. We developed a conditionally inducible Cdx2 mouse model to determine the effects of in vivo, inducible Cdx2 expression in hematopoietic stem and progenitor cells (HSPCs).

Distinct effects of ruxolitinib and interferon-alpha on murine JAK2V617F myeloproliferative neoplasm hematopoietic stem cell populations.

JAK2V617F is the most common mutation in patients with BCR-ABL negative myeloproliferative neoplasms (MPNs). The eradication of JAK2V617F hematopoietic stem cells (HSCs) is critical for achieving molecular remissions and cure. We investigate the distinct effects of two therapies, ruxolitinib (JAK1/2 inhibitor) and interferon-alpha (IFN-α), on the disease-initiating HSC population.

Analysis of telomerase target gene expression effects from murine models in patient cohorts by homology translation and random survival forest modeling.

Acute myeloid leukemia (AML) is an aggressive and rapidly fatal blood cancer that affects patients of any age group. Despite an initial response to standard chemotherapy, most patients relapse and this relapse is mediated by leukemia stem cell (LSC) populations. We identified a functional requirement for telomerase in sustaining LSC populations in murine models of AML and validated this requirement using an inhibitor of telomerase in human AML.

EphA2 Is a Therapy Target in EphA2-Positive Leukemias but Is Not Essential for Normal Hematopoiesis or Leukemia.

Members of the Eph family of receptor tyrosine kinases and their membrane bound ephrin ligands have been shown to play critical roles in many developmental processes and more recently have been implicated in both normal and pathological processes in post-embryonic tissues. In particular, expression studies of Eph receptors and limited functional studies have demonstrated a role for the Eph/ephrin system in hematopoiesis and leukemogenesis. In particular, EphA2 was reported on hematopoietic stem cells and stromal cells.

NK cells require IL-28R for optimal in vivo activity.

Natural killer (NK) cells are naturally circulating innate lymphoid cells that protect against tumor initiation and metastasis and contribute to immunopathology during inflammation. The signals that prime NK cells are not completely understood, and, although the importance of IFN type I is well recognized, the role of type III IFN is comparatively very poorly studied. IL-28R-deficient mice were resistant to LPS and cecal ligation puncture-induced septic shock, and hallmark cytokines in these disease models were dysregulated in the absence of IL-28R.

Autophagy is required for stem cell mobilization by G-CSF.

Granulocyte colony-stimulating factor (G-CSF) is widely used clinically to prevent neutropenia after cytotoxic chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Autophagy, a process of cytoplasmic component recycling, maintains cellular homeostasis and protects the cell during periods of metabolic stress or nutrient deprivation. We have observed that G-CSF activates autophagy in neutrophils and HSCs from both mouse and human donors.

Depletion of Jak2V617F myeloproliferative neoplasm-propagating stem cells by interferon-α in a murine model of polycythemia vera.

Interferon-α (IFNα) is an effective treatment of patients with myeloproliferative neoplasms (MPNs). In addition to inducing hematological responses in most MPN patients, IFNα reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNα on JAK2V617F mutant stem cells is debated.

Opposing actions of rosiglitazone and resveratrol on mineralization in human vascular smooth muscle cells.

Arteriosclerotic vascular disease is a major cardiac health problem in westernized countries and the primary cause of mortality in diabetic patients. Recent data have raised serious safety concerns with the antidiabetic rosiglitazone, a thiazolidinedione with peroxisome proliferator-activated receptor γ (PPAR-γ) agonistic activity, in regard to cardiovascular risks. A common feature of atherosclerosis is vascular mineralization.

Basic techniques in human mesenchymal stem cell cultures: differentiation into osteogenic and adipogenic lineages, genetic perturbations, and phenotypic analyses.

This unit describes basic techniques in human mesenchymal stem cell (hMSC) cultures. It includes protocols for the differentiation of hMSCs into osteogenic and adipogenic lineages, genetic perturbations, and phenotypic analyses. hMSCs can be differentiated with dexamethasone and β-glycerophosphate into mineralizing osteoblasts within 2 to 3 weeks, or with dexamethasone, indomethacin, and 3-isobutyl-1-methylxanthine into lipid vesicle-containing adipocytes within 1 to 2 weeks.

A new concept underlying stem cell lineage skewing that explains the detrimental effects of thiazolidinediones on bone.

Bone-marrow adipogenesis is an aging-related phenomenon and is correlated with osteoporosis. The latter is a prevalent bone disease in the elderly leading to increased fracture risk and mortality. It is widely hypothesized that the underlying molecular mechanism includes a shift in the commitment of mesenchymal stem cells (MSCs) from the osteogenic lineage to the adipogenic lineage.

Evidence for multiple peroxisome proliferator-activated receptor gamma transcripts in bone: fine-tuning by hormonal regulation and mRNA stability.

The expression, regulation and functional significance of multiple peroxisome proliferator-activated receptor gamma transcript variants in bone were studied. PPARG transcripts giving rise to PPARg-1 protein were expressed in human osteoblasts, whereas PPARG-2 transcript and protein remained virtually absent. PPARG expression underwent homologous regulation, was upregulated during differentiation and directly induced by the osteogenic hormone dexamethasone, suggesting a role for PPARg-1 in osteogenesis.

Comprehensive metabolic profiling and phenotyping of interspecific introgression lines for tomato improvement.

Tomato represents an important source of fiber and nutrients in the human diet and is a central model for the study of fruit biology. To identify components of fruit metabolic composition, here we have phenotyped tomato introgression lines (ILs) containing chromosome segments of a wild species in the genetic background of a cultivated variety. Using this high-diversity population, we identify 889 quantitative fruit metabolic loci and 326 loci that modify yield-associated traits.