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Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34 +ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders. The ability to maintain blood stem cells (HSCs) in vitro would allow us to provide better therapies for blood diseases. Here, the authors report that polymer-organised extracellular proteins, coupled to soft environments mimicking bone marrow stiffness, allow stromal cells to maintain HSCs in vitro.

Mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that mediates phosphoinositide-3-kinase (PI3K)/AKT signalling. This pathway is involved in a plethora of cellular functions including protein and lipid synthesis, cell migration, cell proliferation and apoptosis. In this study, we proposed to delineate the role of mTORC1 in haemopoietic lineage commitment using knock out (KO) mouse and cell line models. Mx1 -cre and Vav -cre expression systems were used to specifically target Raptor fl/fl (mTORC1), either in all tissues upon poly(I:C) inoculation, or specifically in haemopoietic stem cells, respectively. Assessment of the role of mTORC1 during the early stages of development in Vav -cre + Raptor fl/fl mice, revealed that these mice do not survive post birth due to aberrations in erythropoiesis resulting from an arrest in development at the megakaryocyte-erythrocyte progenitor stage. Furthermore, Raptor -deficient mice exhibited a block in B cell lineage commitment. The essential role of Raptor (mTORC1) in erythrocyte and B lineage commitment was confirmed in adult Mx1-cre + Raptor fl/fl mice upon cre-recombinase induction. These studies were supported by results showing that the expression of key lineage commitment regulators, GATA1 , GATA2 and PAX5 were dysregulated in the absence of mTORC1-mediated signals. The regulatory role of mTOR during erythropoiesis was confirmed in vitro by demonstrating a reduction of K562 cell differentiation towards RBCs in the presence of established mTOR inhibitors. While mTORC1 plays a fundamental role in promoting RBC development, we showed that mTORC2 has an opposing role, as Rictor- deficient progenitor cells exhibited an elevation in RBC colony formation ex vivo . Collectively, our data demonstrate a critical role played by mTORC1 in regulating the haemopoietic cell lineage commitment.

Targeted deletion of Raptor, a component of mechanistic target of rapamycin complex 1 (mTORC1), reveals an essential role for mTORC1 in initiation/maintenance of leukemia in a CLL model, resulting from a failure for haemopoietic stem/progenitor cells (HSPCs) to commit to the B cell lineage. Induction of Raptor-deficiency in NSG mice transplanted with Mx1-Raptor CLL progenitor cells (PKCα-KR-transduced HSPCs) after disease establishment revealed a reduction in CLL-like disease load and a significant increase in survival in the mice. Interestingly in an aggressive CLL-like disease model, rapamycin treatment reduced disease burden more effectively than AZD2014 (dual mTORC1/2 inhibitor), indicating a skew towards mTORC1 sensitivity with more aggressive disease. Rapamycin, but not ibrutinib, efficiently targeted the eEF2/eEF2K translation elongation regulatory axis, downstream of mTORC1, resulting in eEF2 inactivation through induction of eEF2T56 phosphorylation. mTOR inhibitor treatment of primary patient CLL cells halted proliferation, at least in part through modulation of eEF2K/eEF2 phosphorylation and expression, reduced protein synthesis and inhibited expression of MCL1, Cyclin A and Cyclin D2. Our studies highlight the importance of translation elongation as a driver of disease progression and identify inactivation of eEF2 activity as a novel therapeutic target for blocking CLL progression.

B cell antigen receptor (BCR) signalling competence is critical for the pathogenesis of chronic lymphocytic leukaemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKCα function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKCβII upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKCα-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKCα-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKCα-KR expressing cells, coincident with upregulation of PKCβII: supported by activation of key signalling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKCβII inhibitor) treatment of PKCα-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKCβII in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKCα-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKCβ expression facilitates leukemogenesis and identify that BCR-mediated signalling is a key driver of CLL development in the PKCα-KR model.

The precise role of mechanistic target of rapamycin complex 1 (mTORC1) during chronic lymphocytic leukemia (CLL) pathogenesis remains to be elucidated. Targeted deletion of mTORC1 component Raptor in adult mice reveals that mTORC1 function is essential for initiation and maintenance of CLL. Raptor-deficient bone marrow-derived PKCa-KR transduced haemopoietic progenitors failed to generate a CLL-like disease in vitro, due to an inability to overcome the mTORC1-mediated block in B cell lineage commitment. Induction of Raptor-deficiency in NSG mice transplanted with Mx1-Raptor BM-derived PKCa-KR transduced cells after disease was established, revealed a reduced CLL-like disease load and a significant increase in survival in the mice. Interestingly in mice transplanted with an aggressive CLL-like disease, rapamycin treatment reduced disease burden more effectively than AZD2014 (dual mTORC1/2 inhibitor), indicating a skew towards mTORC1 sensitivity with more aggressive leukemic disease. Rapamycin efficiently targeted the translation elongation axis eEF2/eEF2K downstream of mTORC1, resulting in eEF2 inactivation through induction of eEF2T56 phosphorylation. Rapamycin treatment of primary CLL cells halted proliferation, modulated eEF2K/eEF2 phosphorylation and inhibited MCL1 expression. Our studies demonstrate that mTORC1 plays an essential role in leukemia progression in vitro and in vivo in our CLL mouse model, with evidence for increased rapamycin sensitivity in aggressive secondary CLL transplants. Furthermore, the suppression of translation elongation through inactivation of eEF2 may offer a novel therapeutic target for blocking CLL progression. Competing Interest Statement The authors have declared no competing interest.

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via allogeneic stem cell transplantation (alloSCT), to engraft and repopulate the blood system. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs (ST-HSC) and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs or pericytes). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF1α ; expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF1α ; expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.Competing Interest StatementThe authors have declared no competing interest.Footnotes* This version of the manuscript has been revised.* http://dx.doi.org/10.5525/gla.researchdata.1326

B cell antigen receptor (BCR) signaling competence is critical for pathogenesis of chronic lymphocytic leukemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKCα function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKCβII upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKCα-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKCα-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKCα-KR expressing cells, coincident with upregulation of PKCβII: supported by activation of key signaling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKCβII inhibitor) treatment of PKCα-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKCβII in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKCα-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKCβ expression facilitates leukemogenesis and identify that BCR-mediated signaling is a key driver of CLL development in the PKCα-KR model. PKCβ facilitates leukemogenesis of CLL, driven through an SP1-regulated transcriptional program and promotes BCR signaling. Thus far, PKCβ is the only kinase within the BCR signaling pathway, a key pathway in driving CLL pathogenesis, implicated in the generation of neoplastic B lineage cells.

The war on terrorism has raised profound questions of domestic governance-not primarily about power or policy, but about the capacities of government agencies, their personnel, procedures, work habits and styles, and ability to interface with each other. These are the \"workways of governance.\" As the aftermath of the tragic events of September 11, 2001, made very clear, shortcomings and defects in the workways of governance are all too often manifested only in times of crisis or scandal-after the damage is done. How much better it would be to design for government a version of the periodic physical examination, where tests of various types, together with the physician's observations and the patient's own testimony, combine to help define the patient's status and prospects. The Workways of Governance project, sponsored by the Governance Institute, was begun in 1997 in response to a growing concern that institutions of national government have evolved into structures and developed procedures and customs that constrain their workers and hamper their collective effectiveness. A unique feature of the project is the development of a periodic review of the quality of institutional life and work in government. Guidelines for evaluating and monitoring governmental entities will be established, and implemented annually or biennially. The resulting reports consist of both objectively measured data and subjective evaluation, with the ultimate aim of generating positive change toward improving the capacity of government. In this first volume of analysis arising from the Workways of Governance project, contributors apply these general guidelines to evaluate the leading institutions of the federal government, as listed in the first three articles of the Constitution: the House of Representatives, the Senate, the presidency (focusing on White House organization and staffing), the executive branch (focusing on the civil service), and the federal judiciary.

Treatment with tyrosine kinase inhibitors results in a survival benefit in patients with chronic myeloid leukemia (CML). However, relapse due to persistent leukemic stem cells (LSCs) requires additional selective targets for efficient eradication of the disease. Metabolomic analyses on patient-derived CML LSCs reveal that these have an increased dependency on oxidative metabolism that renders them sensitive to treatment with tigecycline, an FDA-approved inhibitor of mitochondrial translation. These findings uncover a new metabolic vulnerability in CML and provide a rational approach for further clinical evaluation. Treatment of chronic myeloid leukemia (CML) with imatinib mesylate and other second- and/or third-generation c-Abl-specific tyrosine kinase inhibitors (TKIs) has substantially extended patient survival 1 . However, TKIs primarily target differentiated cells and do not eliminate leukemic stem cells (LSCs) 2 , 3 , 4 . Therefore, targeting minimal residual disease to prevent acquired resistance and/or disease relapse requires identification of new LSC-selective target(s) that can be exploited therapeutically 5 , 6 . Considering that malignant transformation involves cellular metabolic changes, which may in turn render the transformed cells susceptible to specific assaults in a selective manner 7 , we searched for such vulnerabilities in CML LSCs. We performed metabolic analyses on both stem cell–enriched (CD34 + and CD34 + CD38 − ) and differentiated (CD34 − ) cells derived from individuals with CML, and we compared the signature of these cells with that of their normal counterparts. Through combination of stable isotope–assisted metabolomics with functional assays, we demonstrate that primitive CML cells rely on upregulated oxidative metabolism for their survival. We also show that combination treatment with imatinib and tigecycline, an antibiotic that inhibits mitochondrial protein translation, selectively eradicates CML LSCs both in vitro and in a xenotransplantation model of human CML. Our findings provide a strong rationale for investigation of the use of TKIs in combination with tigecycline to treat patients with CML with minimal residual disease.