Molecular dynamics of genome editing with CRISPR-Cas9 and rAAV6 virus in human HSPCs to treat sickle cell disease.

gene correction with CRISPR-Cas9 and a recombinant adeno-associated virus serotype 6 (rAAV6) in autologous hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD) has now entered early-phase clinical investigation. To facilitate the progress of CRISPR-Cas9/rAAV6 genome editing technology, we analyzed the molecular changes in key reagents and cellular responses during and after the genome editing procedure in human HSPCs. We demonstrated the high stability of rAAV6 to serve as the donor DNA template.

Alloantigen-specific type 1 regulatory T cells suppress through CTLA-4 and PD-1 pathways and persist long-term in patients.

Type 1 regulatory T (Tr1) cells are inducible, interleukin (IL)-10FOXP3 regulatory T cells that can suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have optimized an in vitro protocol to generate a Tr1-enriched cell product called T-allo10, which is undergoing clinical evaluation in patients with hematological malignancies receiving a human leukocyte antigen (HLA)–mismatched allo-HSCT. Donor-derived T-allo10 cells are specific for host alloantigens, are anergic, and mediate alloantigen-specific suppression.

Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease.

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the β-globin gene (). Ex vivo β-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD.

Identification of dual positive CD19+/CD3+ T cells in a leukapheresis product undergoing CAR transduction: a case report.

Background: Chimeric antigen receptor (CAR) therapy and hematopoietic stem cell transplantation (HSCT) are therapeutics for relapsed acute lymphocytic leukemia (ALL) that are increasingly being used in tandem. We identified a non-physiologic CD19+/CD3+ T-cell population in the leukapheresis product of a patient undergoing CAR T-cell manufacturing who previously received a haploidentical HSCT, followed by infusion of a genetically engineered T-cell addback product. We confirm and report the origin of these CD19+/CD3+ T cells that have not previously been described in context of CAR T-cell manufacturing.

A reproducible immunopotency assay to measure mesenchymal stromal cell-mediated T-cell suppression.

Background Aims: The T-cell suppressive property of bone marrow-derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T-cell suppression.

Methods: At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center, we developed an in vitro quality control T-cell suppression immunopotency assay (IPA) that uses anti-CD3 and anti-CD28 antibodies to stimulate T-cell proliferation.

MAP3K8 kinase regulates myeloma growth by cell-autonomous and non-autonomous mechanisms involving myeloma-associated monocytes/macrophages.

Benefit from cytotoxic therapy in myeloma may be limited by the persistence of residual tumour cells within protective niches. We have previously shown that monocytes/macrophages acquire a proinflammatory transcriptional profile in the myeloma microenvironment. Here we report constitutive activation of MAP3K8 kinase-dependent pathways that regulate the magnitude and extent of inflammatory activity of monocytes/macrophages within myeloma niches.

MAGE-C2 promotes growth and tumorigenicity of melanoma cells, phosphorylation of KAP1, and DNA damage repair.

Melanoma-associated antigen-encoding (MAGE) genes are expressed in melanoma and other cancers but not in normal somatic cells. MAGE expression is associated with aggressive tumor growth, poor clinical outcome, and resistance to chemotherapy, but the mechanisms have not been completely elucidated. In this study, we show that downregulation of MAGE-C2 in A375 melanoma cells and low-passage cultures from human metastatic melanomas (MRA cells) results in increased apoptosis and decreased growth of tumor xenografts in athymic nude mice.

Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets.

Background Aims: Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC.

MAGE I transcription factors regulate KAP1 and KRAB domain zinc finger transcription factor mediated gene repression.

Class I MAGE proteins (MAGE I) are normally expressed only in developing germ cells but are aberrantly expressed in many cancers. They have been shown to promote tumor survival, aggressive growth, and chemoresistance but the underlying mechanisms and MAGE I functions have not been fully elucidated. KRAB domain zinc finger transcription factors (KZNFs) are the largest group of vertebrate transcription factors and regulate neoplastic transformation, tumor suppression, cellular proliferation, and apoptosis.

Identification of novel small molecules that inhibit protein-protein interactions between MAGE and KAP-1.

The Class I MAGE proteins are normally expressed only in developing germ cells but are often aberrantly expressed in malignancies, particularly melanoma, making them good therapeutic targets. MAGE proteins promote tumor survival by binding to the RBCC region of KAP-1 and suppressing p53. Although, suppression of MAGE expression, by RNA interference, relieves p53 suppression and inhibits tumor growth, its therapeutic uses are limited by lack of methods for systemic delivery of small interfering RNA.

Role of β-TrCP ubiquitin ligase receptor in UVB mediated responses in skin.

Skin cancers are the most common cancers in the United States. Exposure to UVB radiation is a major risk factor for skin cancer induction. SCF(β-TrCP) E3 ubiquitin ligase has been found to be involved in cell cycle, cell proliferation and transformation.

Inhibition of beta-TrCP function potentiates UVB-induced apoptosis in hTERT-immortalized normal human keratinocytes.

Chronic skin exposure to UV radiation manifests in a score of biochemical events, DNA damage and mutations which can potentially cause skin cancer. The ubiquitin proteasome pathway controls the degradation of a majority of regulatory eukaryotic proteins including those which play a key role in tumorigenesis. SCFbetaTrCP E3 ubiquitin ligases mediate ubiquitination and proteasomal degradation of phosphorylated substrates that play a key role in signal transduction.

Role of GLI2 transcription factor in growth and tumorigenicity of prostate cells.

Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including prostate cancer. The GLI2 transcription factor is a primary mediator of Hh signaling. However, its relative contribution to development of prostate tumors is poorly understood.

CRD-BP mediates stabilization of betaTrCP1 and c-myc mRNA in response to beta-catenin signalling.

Although constitutive activation of beta-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the beta-catenin/Tcf pathway promotes tumorigenesis are incompletely understood. Messenger RNA turnover has a major function in regulating gene expression and is responsive to developmental and environmental signals. mRNA decay rates are dictated by cis-acting elements within the mRNA and by trans-acting factors, such as RNA-binding proteins (reviewed in refs 2, 3).

Gli2 is targeted for ubiquitination and degradation by beta-TrCP ubiquitin ligase.

The Hedgehog (Hh) signaling pathway plays a crucial role in embryogenesis and has been linked to the development of several human malignancies. The transcription factor Gli2 plays a key role in the transduction of Hh signals by modulating transcription of some Hh target genes, yet the mechanisms that control Gli2 protein expression are largely unknown. Here we report that beta-transducin repeat-containing protein (beta-TrCP) E3 ubiquitin ligase is required for Gli2 degradation.

Activation of Wnt/beta-catenin/Tcf signaling in mouse skin carcinogenesis.

Although Wnt/beta-catenin/Tcf signaling pathway has been shown to be an important factor in the development of many malignancies including colorectal, ovarian, prostate, and many other cancers, little is known about its role in non-melanoma skin cancers. Here, we report the first evidence that beta-catenin/Tcf signaling pathway is constitutively activated in non-melanocytic skin tumors induced by two stage chemical carcinogenesis protocol. Mouse skin tumors showed cytoplasmic and nuclear accumulation of beta-catenin, and upregulation of beta-catenin/Tcf target genes (c-myc and c-jun).

Antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells.

Background: The tremendous impact of prostate cancer (PCA) on the US male population has led to an increased attention on its prevention and on therapeutic intervention. Short-term models are needed to quickly screen the efficacy of promising agents against PCA. We have established recently several rat PCA cell lines from primary PCA in rats induced by a MNU-testosterone protocol, but their usefulness as a model for screening PCA preventive and therapeutic agents remains to be established.

Mechanisms of energy restriction: effects of corticosterone on cell growth, cell cycle machinery, and apoptosis.

The restriction of energy intake has documented beneficial effects on numerous diseases including cancer, yet the mechanism(s) that accounts for these effects is unknown. Recently, we showed that the inhibitory activity against mammary carcinogenesis mediated by energy restriction (ER) is accompanied by an increase in the secretion of adrenal cortical steroids. However, ER caused a concomitant reduction in circulating levels of insulin-like growth factor-1, which also may be involved in inhibiting carcinogenesis.

Mouse homologue of HOS (mHOS) is overexpressed in skin tumors and implicated in constitutive activation of NF-kappaB.

NF-kappaB transcription factor is activated upon ubiquitination and subsequent proteolysis of its inhibitor IkappaB. The phosphorylation-dependent ubiquitination is mediated by SCF E3 ubiquitin ligase. In this study, we identified a novel murine F-box/WD40 repeat-containing protein, mHOS (a homologue of HOS/betaTrCP2).