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1.  A pan-BCL2 inhibitor renders bone marrow resident human leukemia stem cells sensitive to tyrosine kinase inhibition 
Cell stem cell  2013;12(3):316-328.
Summary
Leukemia stem cells (LSC) play a pivotal role in chronic myeloid leukemia (CML) tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), in part, through alternative splicing of self-renewal and survival genes. To elucidate splice isoform regulators of human BC LSC maintenance, we performed whole transcriptome RNA sequencing; splice isoform-specific qRT-PCR, nanoproteomics, stromal co-culture and BC LSC xenotransplantation analyses. Cumulatively, these studies show that alternative splicing of multiple pro-survival BCL2 family genes promotes malignant transformation of myeloid progenitors into BC LSC that are quiescent in the marrow niche and contribute to therapeutic resistance. Notably, a novel pan-BCL2 inhibitor, sabutoclax, renders marrow niche-resident BC LSC sensitive to TKIs at doses that spare normal progenitors. These findings underscore the importance of alternative BCL2 family splice isoform expression in BC LSC maintenance and suggest that combinatorial inhibition of pro-survival BCL2 family proteins and BCR-ABL may eliminate dormant LSC and obviate resistance.
doi:10.1016/j.stem.2012.12.011
PMCID: PMC3968867  PMID: 23333150
2.  NOTCH1 Signaling Promotes Human T-Cell Acute Lymphoblastic Leukemia Initiating Cell Regeneration in Supportive Niches 
PLoS ONE  2012;7(6):e39725.
Background
Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated.
Methodology and Principal Findings
We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34+ cells from NOTCH1Mutated T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1Wild-type CD34+ cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1Mutated CD34+ fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1Mutated T-ALL LIC-engrafted mice and resulted in depletion of CD34+CD2+CD7+ cells that harbor serial transplantation capacity.
Conclusions
These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies.
doi:10.1371/journal.pone.0039725
PMCID: PMC3387267  PMID: 22768113
3.  Phenothiazine Neuroleptics Signal to the Human Insulin Promoter as Revealed by a Novel High-Throughput Screen 
Journal of Biomolecular Screening  2010;15(6):663-670.
A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic β-cell. A cell line from human islets in which the expression of insulin and other β-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of β-cell differentiated function.
doi:10.1177/1087057110372257
PMCID: PMC3374493  PMID: 20547533
diabetes; chlorpromazine; ethopropazine
4.  Human β-cell Precursors Mature Into Functional Insulin-producing Cells in an Immunoisolation Device: Implications for Diabetes Cell Therapies 
Transplantation  2009;87(7):983-991.
Background
Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human β-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human β-cells and their progenitors and (2) the engraftment of encapsulated murine β-cells in allo- and autoimmune settings.
Methods
Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets.
Results
Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary β-cells ameliorated diabetes without stimulating a detectable T-cell response.
Conclusions
We demonstrate for the first time that human β-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of β-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.
doi:10.1097/TP.0b013e31819c86ea
PMCID: PMC2715156  PMID: 19352116
Encapsulation; Diabetes; Islet transplantation; Immunoisolation
5.  Islet Specific Wnt Activation in Human Type II Diabetes 
Experimental Diabetes Research  2009;2008:728763.
The Wnt pathway effector gene TCF7L2 has been linked to type II diabetes, making it important to study the role of Wnt signaling in diabetes pathogenesis. We examined the expression of multiple Wnt pathway components in pancreases from normal individuals and type II diabetic individuals. Multiple members of the Wnt signaling pathway, including TCF7L2, Wnt2b, β-catenin, pGSK3β, TCF3, cyclinD1, and c-myc, were undetectable or expressed at low levels in islets from nondiabetic individuals, but were also upregulated specifically in islets of type II diabetic patients. Culture of pancreatic tissue and islet isolation led to Wnt activation that was reversed by the Wnt antagonist sFRP, demonstrating that Wnt activation in that setting was due to soluble Wnt factors. These data support a model in which the Wnt pathway plays a dynamic role in the pathogenesis of type II diabetes and suggest manipulation of Wnt signaling as a new approach to β-cell-directed diabetes therapy.
doi:10.1155/2008/728763
PMCID: PMC2628766  PMID: 19165345

Results 1-5 (5)