We examined the leukemic stem cell potential of blasts at different stages of maturation in childhood acute lymphoblastic leukemia (ALL). Human leukemic bone marrow was transplanted intrafemorally ...into NOD/scid mice. Cells sorted using the B precursor differentiation markers CD19, CD20, and CD34 were isolated from patient samples and engrafted mice before serial transplantation into primary or subsequent (up to quaternary) recipients. Surprisingly, blasts representative of all of the different maturational stages were able to reconstitute and reestablish the complete leukemic phenotype in vivo. Sorted blast populations mirrored normal B precursor cells with transcription of a number of stage-appropriate genes. These observations inform a model for leukemia-propagating stem cells in childhood ALL.
Highlights • Humanized mice can be used to study human specific infectious agents in vivo. • Human HSC engrafted immunodeficient IL2rg null mice develop human immune systems. • Emerging new models of ...humanized mice overcome limitations of current models. • New technologies are being implemented to provide models for vaccination.
SHARPIN is a key regulator of NFKB and integrin signaling. Mice lacking Sharpin develop a phenotype known as chronic proliferative dermatitis (CPDM), typified by progressive epidermal hyperplasia, ...apoptosis of keratinocytes, cutaneous and systemic eosinophilic inflammation, and hypoplasia of secondary lymphoid organs. Rag1(-/-) mice, which lack mature B and T cells, were crossed with Sharpin(-/-) mice to examine the role of lymphocytes in CDPM. Although inflammation in the lungs, liver, and joints was reduced in these double mutant mice, dermatitis was not reduced in the absence of functional lymphocytes, suggesting that lymphocytes are not primary drivers of the inflammation in the skin. Type 2 cytokine expression is increased in CPDM. In an attempt to reduce this aspect of the phenotype, Il4ra(-/-) mice, unresponsive to both IL4 and IL13, were crossed with Sharpin(-/-) mice. Double homozygous Sharpin(-/-) , Il4ra(-/-) mice developed an exacerbated granulocytic dermatitis, acute system inflammation, as well as hepatic necrosis and mineralization. High expression of CHI3L4, normally seen in CPDM skin, was abolished in Sharpin(-/-) , Il4ra(-/-) double mutant mice indicating the crucial role of IL4 and IL13 in the expression of this protein. Cutaneous eosinophilia persisted in Sharpin(-/-) , Il4ra(-/-) mice, although expression of Il5 mRNA was reduced and the expression of Ccl11 and Ccl24 was completely abolished. TSLP and IL33 were both increased in the skin of Sharpin(-/-) mice and this was maintained in Sharpin(-/-) , Il4ra(-/-) mice suggesting a role for TSLP and IL33 in the eosinophilic dermatitis in SHARPIN-deficient mice. These studies indicate that cutaneous inflammation in SHARPIN-deficient mice is autoinflammatory in nature developing independently of B and T lymphocytes, while the systemic inflammation seen in CPDM has a strong lymphocyte-dependent component. Both the cutaneous and systemic inflammation is enhanced by loss of IL4 and IL13 signaling indicating that these cytokines normally play an anti-inflammatory role in SHARPIN-deficient mice.
Introduction
Primary myelofibrosis (PMF) is a clonal stem cell disorder characterized by myeloid dominant hematopoiesis and dysregulated proliferation of fibroblasts in the bone marrow. However, how ...these aberrant myeloid cells and fibroblasts are produced remains unclear.
Aim and methods
In this study, we examined in vivo engraftment kinetics of PMF patient-derived CD34+ cells in immunecompromised NOD/SCID/IL2rgKO (NSG) mice. Engrafted human cells were analyzed with flow cytometry, and proliferation of fibroblastic cells and bone marrow fibrosis were assessed with the histo-pathological examination.
Results
Transplantation of PMF patient-derived circulating CD34+ fractions into NSG newborns recapitulates clinical features of human PMF. Engraftment of human CD45+ leukocytes resulted in anemia and myeloid hyperplasia accompanied by bone marrow fibrosis by six months post-transplantation. Fibrotic bone marrow contained CD45-vimentin+ cells of both human and mouse origin, suggesting that circulating malignant CD34+ subsets contribute to myelofibrotic changes in PMF through direct and indirect mechanisms.
Conclusion
A patient-derived xenotransplantation (PDX) model of PMF allows in vivo examination of disease onset and propagation originating from immature CD34+ cells and will support the investigation of pathogenesis and development of therapeutic modalities for the disorder.
In recent years, advances in the humanized mouse system have led to significantly increased levels of human hematopoietic stem cell (HSC) engraftment. The remaining limitations in human HSC ...engraftment and function include lymphoid-skewed differentiation and inefficient myeloid development in the recipients. Limited human HSC function may partially be attributed to the inability of the host mouse microenvironment to provide sufficient support to human hematopoiesis. To address this problem, we created membrane-bound human stem cell factor (SCF)/KIT ligand (KL)–expressing NOD/SCID/IL2rgKO (hSCF Tg NSG) mice. hSCF Tg NSG recipients of human HSCs showed higher levels of both human CD45+ cell engraftment and human CD45+CD33+ myeloid development compared with NSG recipients. Expression of hSCF/hKL accelerated the differentiation of the human granulocyte lineage cells in the recipient bone marrow. Human mast cells were identified in bone marrow, spleen, and gastrointestinal tissues of the hSCF Tg NSG recipients. This novel in vivo humanized mouse model demonstrates the essential role of membrane-bound hSCF in human myeloid development. Moreover, the hSCF Tg NSG humanized recipients may facilitate investigation of in vivo differentiation, migration, function, and pathology of human mast cells.
Understanding how the extracellular matrix impacts the function of cancer stem cells (CSCs) is a significant but poorly understood problem. We report that breast CSCs produce a laminin (LM) 511 ...matrix that promotes self-renewal and tumor initiation by engaging the alpha 6B beta 1 integrin and activating the Hippo transducer TAZ. Although TAZ is important for the function of breast CSCs, the mechanism is unknown. We observed that TAZ regulates the transcription of the alpha 5 subunit of LM511 and the formation of a LM511 matrix. These data establish a positive feedback loop involving TAZ and LM511 that contributes to stemness in breast cancer.
Although physiological development of human lymphoid subsets has become well documented in humanized mice, in vivo development of human myeloid subsets in a xenotransplantation setting has remained ...unevaluated. Therefore, we investigated in vivo differentiation and function of human myeloid subsets in NOD/SCID/IL2rγ(null) (NSG) mouse recipients transplanted with purified lineage(-)CD34(+)CD38(-) cord blood hematopoietic stem cells. At 4-6 mo posttransplantation, we identified the development of human neutrophils, basophils, mast cells, monocytes, and conventional and plasmacytoid dendritic cells in the recipient hematopoietic organs. The tissue distribution and morphology of these human myeloid cells were similar to those identified in humans. After cytokine stimulation in vitro, phosphorylation of STAT molecules was observed in neutrophils and monocytes. In vivo administration of human G-CSF resulted in the recruitment of human myeloid cells into the recipient circulation. Flow cytometry and confocal imaging demonstrated that human bone marrow monocytes and alveolar macrophages in the recipients displayed intact phagocytic function. Human bone marrow-derived monocytes/macrophages were further confirmed to exhibit phagocytosis and killing of Salmonella typhimurium upon IFN-γ stimulation. These findings demonstrate the development of mature and functionally intact human myeloid subsets in vivo in the NSG recipients. In vivo human myelopoiesis established in the NSG humanized mouse system may facilitate the investigation of human myeloid cell biology including in vivo analyses of infectious diseases and therapeutic interventions.
The identification of activating NOTCH1 mutations in T cell acute lymphoblastic leukemia (T-ALL) led to clinical testing of γ-secretase inhibitors (GSIs) that prevent NOTCH1 activation. However, ...responses to these inhibitors have been transient, suggesting that resistance limits their clinical efficacy. Here we modeled T-ALL resistance, identifying GSI-tolerant 'persister' cells that expand in the absence of NOTCH1 signaling. Rare persisters are already present in naive T-ALL populations, and the reversibility of their phenotype suggests an epigenetic mechanism. Relative to GSI-sensitive cells, persister cells activate distinct signaling and transcriptional programs and exhibit chromatin compaction. A knockdown screen identified chromatin regulators essential for persister viability, including BRD4. BRD4 binds enhancers near critical T-ALL genes, including MYC and BCL2. The BRD4 inhibitor JQ1 downregulates expression of these targets and induces growth arrest and apoptosis in persister cells, at doses well tolerated by GSI-sensitive cells. Consistently, the GSI-JQ1 combination was found to be effective against primary human leukemias in vivo. Our findings establish a role for epigenetic heterogeneity in leukemia resistance that may be addressed by incorporating epigenetic modulators in combination therapy.
The rhomboid 5 homolog 2 (Rhbdf2) gene encodes an inactive rhomboid (iRhom) protease, iRhom2, one of a family of enzymes containing a long cytosolic N terminus and a dormant peptidase domain of ...unknown function. iRhom2 has been implicated in epithelial regeneration and cancer growth through constitutive activation of epidermal growth factor receptor (EGFR) signaling. However, little is known about the physiological substrates for iRhom2 or the molecular mechanisms underlying these functions. We show that iRhom2 is a short-lived protein whose stability can be increased by select mutations in the N-terminal domain. In turn, these stable variants function to augment the secretion of EGF family ligands, including amphiregulin, independent of metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) activity. In vivo, N-terminal iRhom2 mutations induce accelerated wound healing as well as accelerated tumorigenesis, but they do not drive spontaneous tumor development. This work underscores the physiological prominence of iRhom2 in controlling EGFR signaling events involved in wound healing and neoplastic growth, and yields insight into the function of key iRhom2 domains.
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