Proteomics-Based Discovery of First-in-Class Chemical Probes for Programmed Cell Death Protein 2 (PDCD2).

Chemical probes are essential tools for understanding biological systems and for credentialing potential biomedical targets. Programmed cell death 2 (PDCD2) is a member of the B-cell lymphoma 2 (Bcl-2) family of proteins, which are critical regulators of apoptosis. Here we report the discovery and characterization of 10 e, a first-in-class small molecule degrader of PDCD2. We discovered this PDCD2 degrader by serendipity using a chemical proteomics approach, in contrast to the conventional approach for making bivalent degraders starting from a known binding ligand targeting the protein of interest. Using 10 e as a pharmacological probe, we demonstrate that PDCD2 functions as a critical regulator of cell growth by modulating the progression of the cell cycle in T lymphoblasts. Our work provides a useful pharmacological probe for investigating PDCD2 function and highlights the use of chemical proteomics to discover selective small molecule degraders of unanticipated targets.

Development of potent and selective degraders of PI5P4Kγ.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), a family of three members in mammals (α, ß and γ), have emerged as potential therapeutic targets due to their role in regulating many important cellular signaling pathways. In comparison to the PI5P4Kα and PI5P4Kß, which usually have similar expression profiles across cancer cells, PI5P4Kγ exhibits distinct expression patterns, and pathological functions for PI5P4Kγ have been proposed in the context of cancer and neurodegenerative diseases. PI5P4Kγ has very low kinase activity and has been proposed to inhibit the PI4P5Ks through scaffolding function, providing a rationale for developing a selective PI5P4Kγ degrader. Here, we report the development and characterization of JWZ-1-80, a first-in-class PI5P4Kγ degrader. JWZ-1-80 potently degrades PI5P4Kγ via the ubiquitin-proteasome system and exhibits proteome-wide selectivity and is therefore a useful tool compound for further dissecting the biological functions of PI5P4Kγ.

Cancer Cells Evade Stress-Induced Apoptosis by Promoting HSP70-Dependent Clearance of Stress Granules.

The formation of stress granules (SG) is regarded as a cellular mechanism to temporarily limit protein synthesis and prevent the unfolding of proteins in stressed cells. It has been noted that SG formation can promote the survival of stressed cells. Paradoxically, however, persistent SGs could cause cell death. The underlying molecular mechanism that affects the relationship between SG dynamics and cellular states is not fully understood. Here we found that SG dynamics in cancer cells differ significantly from those in normal cells. Specifically, prolonged stress caused the formation of persistent SGs and consequently resulted in apoptosis in the normal cells. By contrast, cancer cells resolved SGs and survived the prolonged stress. Regarding the mechanism, the knockdown of HSP70 or the inhibition of the HSP70s' ATPase activity caused defective SG clearance, leading to apoptosis in otherwise healthy cancer cells. On the other hand, the knockout of G3BPs to block the formation of SGs allowed cancer cells to escape from the HSP70 inhibition-induced apoptosis. Given the observation that SG dynamics were barely affected by the inhibition of autophagy or proteasome, we propose that SG dynamics are regulated mainly by HSP70-mediated refolding of the unfolded proteins or their removal from SGs. As a result, cancer cells evade stress-induced apoptosis by promoting the HSP70-dependent SG clearance.

Disrupted mossy fiber connections from defective embryonic neurogenesis contribute to SOX11-associated schizophrenia.

Abnormal mossy fiber connections in the hippocampus have been implicated in schizophrenia. However, it remains unclear whether this abnormality in the patients is genetically determined and whether it contributes to the onset of schizophrenia. Here, we showed that iPSC-derived hippocampal NPCs from schizophrenia patients with the A/A allele at SNP rs16864067 exhibited abnormal NPC polarity, resulting from the downregulation of SOX11 by this high-risk allele. In the SOX11-deficient mouse brain, abnormal NPC polarity was also observed in the hippocampal dentate gyrus, and this abnormal NPC polarity led to defective hippocampal neurogenesis-specifically, irregular neuroblast distribution and disrupted granule cell morphology. As granule cell synapses, the mossy fiber pathway was disrupted, and this disruption was resistant to activity-induced mossy fiber remodeling in SOX11 mutant mice. Moreover, these mutant mice exhibited diminished PPI and schizophrenia-like behaviors. Activation of hippocampal neurogenesis in the embryonic brain, but not in the adult brain, partially alleviated disrupted mossy fiber connections and improved schizophrenia-related behaviors in mutant mice. We conclude that disrupted mossy fiber connections are genetically determined and strongly correlated with schizophrenia-like behaviors in SOX11-deficient mice. This disruption may reflect the pathological substrate of SOX11-associated schizophrenia.

Differentiation of adult human retinal pigment epithelial cells into dopaminergic-like cells in vitro and in the recipient monkey brain.

BACKGROUND: Cell therapy is proposed to be a potential treatment for Parkinson's disease (PD). Although fetal retinal pigment epithelial (RPE) cells have been tested in trials for treating PD patients, controversy has been raised over the issue of whether such cells can be reprogrammed into dopamine-producing cells for therapeutic efficacy. Here, we aim to investigate whether adult human RPE cells can be reprogrammed into dopamine-producing cells both in vitro and in the recipient monkey brain. METHODS: The RPE layer was isolated from frozen posterior eyeball tissue after penetrating keratoplasty surgery. The tumorigenicity of RPE cells was examined by G-banding and a tumor formation assay in nude mice. Immunogenicity was measured using a one-way mixed lymphocyte reaction (MLR) assay. Dopamine-production in chemically reprogrammed RPE cells was measured by HPLC. Finally, RPE cells were grafted into the brains of monkeys with MPTP-induced PD in order to investigate the potential of such cells treating PD patients in the future. RESULTS: RPE cell lines have been successively established from adult human eye tissues. Such cells can be chemically reprogrammed into dopamine-producing cells in vitro. Moreover, after being grafted into the brain caudate putamen of monkeys with MPTP-induced PD, RPE cells became tyrosine hydroxylase-positive cells, and recipient PD monkeys showed significant improvement of clinical conditions. CONCLUSIONS: This preclinical study using a primate model indicates that human adult RPE cells could be a potential cell source for the treatment of PD in the future.

Phosphorylation of Threonine343 Is Crucial for OCT4 Interaction with SOX2 in the Maintenance of Mouse Embryonic Stem Cell Pluripotency.

OCT4 is required to maintain the pluripotency of embryonic stem cells (ESCs); yet, overdose-expression of OCT4 induces ESC differentiation toward primitive endoderm. The molecular mechanism underlying this differentiation switch is not fully understood. Here, we found that substitution of threonine343 by alanine (T343A), but not aspartic acid (T343D), caused a significant loss of OCT4-phosphorylation signal in ESCs. Loss of such OCT4-phosphorylation compromises its interaction with SOX2 but promotes interaction with SOX17. We therefore propose that threonine343-based OCT4-phosphorylation is crucial for the maintenance of ESC pluripotency. This OCT4-phosphorylation-based mechanism may provide insight into the regulation of lineage specification during early embryonic development.