No abstract

T cells genetically modified with a CD19 chimeric antigen receptor (CD19CAR) are remarkably effective against B-cell malignancies in clinical trials. However, major concerns remain regarding toxicities, such as hypogammaglobulinemia, due to Bcell aplasia or severe cytokine release syndrome after overactivation of CAR T cells. To resolve these adverse events, we aimed to develop an inducible CAR system by using a tetracycline regulation system that would be activated only in the presence of doxycycline (Dox). In this study, the second-generation CD19CAR was fused into the third-generation Tet-On vector (Tet-CD19CAR) and was retrovirally transduced into primary CD8 þ T cells. Tet-CD19CAR T cells were successfully generated and had minimal background CD19CAR expression without Dox. Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD19 þ cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells. The Dox(þ) Tet-CD19CAR T cells also had significant antitumor activity in a xenograft model. However, without Dox, Tet-CD19CAR T cells lost CAR expression and CAR T-cell functions in vitro and in vivo, clearly segregating the "On" and "Off" status of Tet-CD19CAR cells by Dox administration. In addition to suicide-gene technology, controlling the expression and the functions of CAR with an inducible vector is a potential solution for CAR T-cell therapy-related toxicities, and may improve the safety profile of CAR T-cell therapy. This strategy might also open the way to treat other malignancies in combination with other CAR or TCR gene-modified T cells.

No abstract

The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney-specific expression is biologically relevant; however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to -1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho-expressing and -nonexpressing cells in transient reporter assays, but the activity was reduced to ∼20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30-40% methylated in Klotho-nonexpressing cells, but unmethylated in Klotho-expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5- to 3.0-fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho down-regulation.

Key Points• LSD1 is barely expressed in normal hematopoietic stem cells, but is overexpressed in leukemias especially those of a T-cell origin.• LSD1 overexpression forms preleukemic stem cells with an increased self-renewal potential in a transgenic mice model.Recent investigations indicate that epigenetic regulators act at the initial step of myeloid leukemogenesis by forming preleukemic hematopoietic stem cells (HSCs), which possess the increased self-renewal potential but retain multidifferentiation ability, and synergize with genetic abnormalities in later stages to develop full-blown acute myeloid leukemias. However, it is still unknown whether this theory is applicable to other malignancies. In this study, we demonstrate that lysine-specific demethylase 1 (LSD1) overexpression is a founder abnormality for the development of T-cell lymphoblastic leukemia/ lymphoma (T-LBL) using LSD1 transgenic mice. LSD1 expression is tightly regulated via alternative splicing and transcriptional repression in HSCs and is altered in most leukemias, especially T-LBL. Overexpression of the shortest isoform of LSD1, which is specifically repressed in quiescent HSCs and demethylates histone H3K9 more efficiently than other isoforms, increases self-renewal potential via upregulation of the HoxA family but retains multidifferentiation ability with a skewed differentiation to T-cell lineages at transcriptome levels in HSCs. Transgenic mice overexpressing LSD1 in HSCs did not show obvious abnormalities but developed T-LBL at very high frequency after g-irradiation. LSD1 overexpression appears to be the first hit in T-cell leukemogenesis and provides an insight into novel strategies for early diagnosis and effective treatment of the disease. (Blood. 2015;125(24):3731-3746)

The Notch signaling pathway has been recognized as a key factor for the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), because of the high incidence of activating mutations of Notch1. Notch inhibition could serve as a new treatment strategy for T-ALL; however, the attempts to perturb Notch signaling pathways have been unsuccessful so far. In this study, we found that proteasome inhibitors exert cytotoxic effects on T-ALL cells with constitutive activation of Notch1 to a similar extent as myeloma cells. The proteasome inhibitor bortezomib repressed the transcription of Notch1 and downstream effectors including Hes1, GATA3, RUNX3 and nuclear factor-κB (NF-κB) (p65 and p50), coincided with downregulation of the major transactivator Sp1 and its dissociation from Notch1 promoter. Overexpression of the Notch1 intracellular domain (NICD) significantly ameliorated bortezomib-induced cytotoxicity against T-ALL cells. Drug combination studies revealed that bortezomib showed synergistic or additive effects with key drugs for the treatment of T-ALL such as dexamethasone (DEX), doxorubicin and cyclophosphamide, which were readily abolished by NICD overexpression. The synergy of bortezomib and DEX was confirmed in vivo using a murine xenograft model. Our findings provide a molecular basis and rationale for the inclusion of proteasome inhibitors in treatment strategies for T-ALL.

Multiple myeloma (MM) cells acquire dormancy and drug resistance via interaction with bone marrow stroma cells (BMSC) in a hypoxic microenvironment. Elucidating the mechanisms underlying the regrowth of dormant clones may contribute to further improvement of the prognosis of MM patients. In this study, we find that the CD180/MD-1 complex, a noncanonical lipopolysaccharide (LPS) receptor, is expressed on MM cells but not on normal counterparts, and its abundance is markedly upregulated under adherent and hypoxic conditions. Bacterial LPS and anti-CD180 antibody, but not other Toll-like receptor ligands, enhanced the growth of MM cells via activation of MAP kinases ERK and JNK in positive correlation with expression levels of CD180. Administration of LPS significantly increased the number of CD180/CD138 double-positive cells in a murine xenograft model when MM cells were inoculated with direct attachment to BMSC. Knockdown of CD180 canceled the LPS response and Promoter analyses identified IKZF1 (Ikaros) as a pivotal transcriptional activator of the gene. Both cell adhesion and hypoxia activated transcription of the gene by increasing Ikaros expression and its binding to the promoter region. Pharmacological targeting of Ikaros by the immunomodulatory drug lenalidomide ameliorated the response of MM cells to LPS in a CD180-dependent manner and Thus, the CD180/MD-1 pathway may represent a novel mechanism of growth regulation of MM cells in a BM milieu and may be a therapeutic target of preventing the regrowth of dormant MM cells. This study describes a novel mechanism by which myeloma cells are regulated in the bone marrow, where drug resistance and dormancy can evolve after treatment, with potential therapeutic implications for treating this often untreatable blood cancer. .

Our results indicated that the Nrf2/AOX1 pathway was important for enhancing airway epithelial barrier integrity. Because the airway epithelium of asthmatics is susceptible to reduced barrier integrity, this pathway might be a new therapeutic target for asthma.