A chimeric antigen receptor uniquely recognizing MICA/B stress proteins provides an effective approach to target solid tumors.

BACKGROUND: The advent of chimeric antigen receptor (CAR) T cell therapies has transformed the treatment of hematological malignancies; however, broader therapeutic success of CAR T cells has been limited in solid tumors because of their frequently heterogeneous composition. Stress proteins in the MICA and MICB (MICA/B) family are broadly expressed by tumor cells following DNA damage but are rapidly shed to evade immune detection. METHODS: We have developed a novel CAR targeting the conserved α3 domain of MICA/B (3MICA/B CAR) and incorporated it into a multiplexed-engineered induced pluripotent stem cell (iPSC)-derived natural killer (NK) cell (3MICA/B CAR iNK) that expressed a shedding-resistant form of the CD16 Fc receptor to enable tumor recognition through two major targeting receptors. FINDINGS: We demonstrated that 3MICA/B CAR mitigates MICA/B shedding and inhibition via soluble MICA/B while simultaneously exhibiting antigen-specific anti-tumor reactivity across an expansive library of human cancer cell lines. Pre-clinical assessment of 3MICA/B CAR iNK cells demonstrated potent antigen-specific in vivo cytolytic activity against both solid and hematological xenograft models, which was further enhanced in combination with tumor-targeted therapeutic antibodies that activate the CD16 Fc receptor. CONCLUSIONS: Our work demonstrated 3MICA/B CAR iNK cells to be a promising multi-antigen-targeting cancer immunotherapy approach intended for solid tumors. FUNDING: Funded by Fate Therapeutics and NIH (R01CA238039).

A Survey of DDX21 Activity During Rev/RRE Complex Formation.

HIV-1 requires a specialized nuclear export pathway to transport unspliced and partially spliced viral transcripts to the cytoplasm. Central to this pathway is the viral protein Rev, which binds to the Rev response element in stem IIB located on unspliced viral transcripts and subsequently oligomerizes in a cooperative manner. Previous work identified a number of cellular DEAD-box helicases as in vivo binding partners of Rev, and siRNA experiments indicated a functional role for many in the HIV replication cycle. Two DEAD-box proteins, DDX1 and DDX3, had previously been shown to play a role in HIV pathogenesis. In this study, another protein identified in that screen, DDX21, is tested for protein and RNA binding and subsequent enzymatic activities in the context of the Rev/RRE pathway. We found that DDX21 can bind to the RRE with high affinity, and this binding stimulates ATPase activity with an enzymatic efficiency similar to DDX1. Furthermore, DDX21 is both an ATP-dependent and ATP-independent helicase, and both ATPase and ATP-dependent helicase activities are inhibited by Rev in a dose-dependent manner, although ATP-independent helicase activity is not. A conserved binding interaction between DDX protein's DEAD domain and Rev was identified, with Rev's nuclear diffusion inhibitory signal motif playing a significant role in binding. Finally, DDX21 was shown to enhance Rev binding to the RRE in a manner similar to that previously described for DDX1, although DDX3 does not. These data indicate that DDX1 and DDX21 have similar biochemical activities with regard to the Rev/RRE system, while DDX3 differs.

Genome-wide investigation of the role of the tRNA nuclear-cytoplasmic trafficking pathway in regulation of the yeast Saccharomyces cerevisiae transcriptome and proteome.

In eukaryotic cells, tRNAs are transcribed and partially processed in the nucleus before they are exported to the cytoplasm, where they have an essential role in protein synthesis. Surprisingly, mature cytoplasmic tRNAs shuttle between nucleus and cytoplasm, and tRNA subcellular distribution is nutrient dependent. At least three members of the ß-importin family, Los1, Mtr10, and Msn5, function in tRNA nuclear-cytoplasmic intracellular movement. To test the hypothesis that the tRNA retrograde pathway regulates the translation of particular transcripts, we compared the expression profiles from nontranslating mRNAs and polyribosome-associated translating mRNAs collected from msn5Δ, mtr10Δ, and wild-type cells under fed or acute amino acid depletion conditions. Our microarray data revealed that the methionine, arginine, and leucine biosynthesis pathways are targets of the tRNA retrograde process. We confirmed the microarray data by Northern and Western blot analyses. The levels of some of the particular target mRNAs were reduced, while others appeared not to be affected. However, the protein levels of all tested targets in these pathways were greatly decreased when tRNA nuclear import or reexport to the cytoplasm was disrupted. This study provides information that tRNA nuclear-cytoplasmic dynamics is connected to the biogenesis of proteins involved in amino acid biosynthesis.

Aurora-A overexpression in mouse liver causes p53-dependent premitotic arrest during liver regeneration.

Aurora-A, a serine-threonine kinase, is frequently overexpressed in human cancers, including hepatocellular carcinoma. To study the phenotypic effects of Aurora-A overexpression on liver regeneration and tumorigenesis, we generated transgenic mice overexpressing human Aurora-A in the liver. The overexpression of Aurora-A after hepatectomy caused an earlier entry into S phase, a sustaining of DNA synthesis, and premitotic arrest in the regenerating liver. These regenerating transgenic livers show a relative increase in binuclear hepatocytes compared with regenerating wild-type livers; in addition, multipolar segregation and trinucleation could be observed only in the transgenic hepatocytes after hepatectomy. These results together suggest that defects accumulated after first round of the hepatocyte cell cycle and that there was a failure to some degree of cytokinesis. Interestingly, the p53-dependent checkpoint was activated by these abnormalities, indicating that p53 plays a crucial role during liver regeneration. Indeed, the premitotic arrest and abnormal cell death, mainly necrosis, caused by Aurora-A overexpression were genetically rescued by p53 knockout. However, trinucleation of hepatocytes remained in the regenerating livers of the transgenic mice with a p53 knockout background, indicating that the abnormal mitotic segregation and cytokinesis failure were p53 independent. Moreover, overexpression of Aurora-A in transgenic liver led to a low incidence (3.8%) of hepatic tumor formation after a long latency period. This transgenic mouse model provides a useful system that allows the study of the physiologic effects of Aurora-A on liver regeneration and the genetic pathways of Aurora-A-mediated tumorigenesis in liver.

Cloning and functional analysis of hypothalamic homeobox gene Bsx1a and its isoform, Bsx1b.

The hypothalamus is a key regulatory unit of the neuroendocrine system and plays an essential role in energy balance and reproduction. Despite its important role, the molecular mechanisms underlying hypothalamic development are not fully understood. Here, we report molecular analyses of a newly identified murine homeobox gene, Bsx/Bsx1a, that is expressed in the developing and postnatal hypothalamus. We demonstrate that BSX1A is a DNA binding protein and a transcriptional activator. Transcriptional reporter assays identified the C-terminal region of BSX1A as an activation domain. We have isolated an alternative splice form of Bsx1a, designated Bsx1b, which retains the N-terminal region but lacks the homeodomain. Analyses of subcellular localization using transfected cell lines revealed that BSX1A and BSX1B localize in the nuclei and cytoplasm, respectively. Immunohistochemical analyses suggested that both BSX1A and BSX1B are expressed in the neonatal hypothalamus. Taking these data together, we propose that alternative RNA splicing is involved in hypothalamic development/function.