The RanBP2/RanGAP1-SUMO complex gates ß-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein ß-arrestin2 (ß-arr2) regulates tumor suppressor p53 by counteracting Mdm2. ß-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. ß-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. ß-arr2 can be SUMOylated, but no information is available on how SUMO may regulate ß-arr2 nucleocytoplasmic shuttling. While we found ß-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and ß-arr2, via a SUMO interaction motif (SIM), that is required for ß-arr2 cytonuclear trafficking. This SIM promotes association of ß-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective ß-arr2 nuclear entry. Mutation of the SIM inhibits ß-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a ß-arr2 SIM nuclear entry checkpoint, coupled with active ß-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.

Biomaterials and Culture Systems for Development of Organoid and Organ-on-a-Chip Models.

The development of novel 3D tissue culture systems has enabled the in vitro study of in vivo processes, thereby overcoming many of the limitations of previous 2D tissue culture systems. Advances in biomaterials, including the discovery of novel synthetic polymers has allowed for the generation of physiologically relevant in vitro 3D culture models. A large number of 3D culture systems, aided by novel organ-on-a-chip and bioreactor technologies have been developed to improve reproducibility and scalability of in vitro organ models. The discovery of induced pluripotent stem cells (iPSCs) and the increasing number of protocols to generate iPSC-derived cell types has allowed for the generation of novel 3D models with minimal ethical limitations. The production of iPSC-derived 3D cultures has revolutionized the field of developmental biology and in particular, the study of fetal brain development. Furthermore, physiologically relevant 3D cultures generated from PSCs or adult stem cells (ASCs) have greatly advanced in vitro disease modelling and drug discovery. This review focuses on advances in 3D culture systems over the past years to model fetal development, disease pathology and support drug discovery in vitro, with a specific focus on the enabling role of biomaterials.

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells.

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non-cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy.