Degradation of GSPT1 causes TP53-independent cell death in leukemia whilst sparing normal hematopoietic stem cells

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR-Cas9 screens implicated decreased translation initiation as protective to GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to GSPT1 degradation. We defined two Crbn amino acids that prevent Gspt1 degradation in mice, generated a knock-in mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant AML

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations

Identification of Combinatorial Patterns of Post-Translational Modifications on Individual Histones in the Mouse Brain

Post-translational modifications (PTMs) of proteins are biochemical processes required for cellular functions and signalling that occur in every sub-cellular compartment. Multiple protein PTMs exist, and are established by specific enzymes that can act in basal conditions and upon cellular activity. In the nucleus, histone proteins are subjected to numerous PTMs that together form a histone code that contributes to regulate transcriptional activity and gene expression. Despite their importance however, histone PTMs have remained poorly characterised in most tissues, in particular the brain where they are thought to be required for complex functions such as learning and memory formation. Here, we report the comprehensive identification of histone PTMs, of their combinatorial patterns, and of the rules that govern these patterns in the adult mouse brain. Based on liquid chromatography, electron transfer, and collision-induced dissociation mass spectrometry, we generated a dataset containing a total of 10,646 peptides from H1, H2A, H2B, H3, H4, and variants in the adult brain. 1475 of these peptides carried one or more PTMs, including 141 unique sites and a total of 58 novel sites not described before. We observed that these PTMs are not only classical modifications such as serine/threonine (Ser/Thr) phosphorylation, lysine (Lys) acetylation, and Lys/arginine (Arg) methylation, but also include several atypical modifications such as Ser/Thr acetylation, and Lys butyrylation, crotonylation, and propionylation. Using synthetic peptides, we validated the presence of these atypical novel PTMs in the mouse brain. The application of data-mining algorithms further revealed that histone PTMs occur in specific combinations with different ratios. Overall, the present data newly identify a specific histone code in the mouse brain and reveal its level of complexity, suggesting its potential relevance for higher-order brain functions

Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation

Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics

Electrical characterization of interface stability between magnesium and selenium-passivated n-type silicon (001)

We reported selenium passivation of silicon (001) surface and showed the ohmic nature of the interface between magnesium and selenium-passivated n -type silicon (001). In this paper, we present a detailed study on the thermal stability of this interface. Magnesium contacts on selenium-passivated silicon (001) are annealed from 200°C to 500°C at an interval of 50°C and then characterized by current-voltage measurements. Schottky barrier heights of the contacts are measured by capacitance-voltage and activation-energy methods. Schottky behaviour sets in only when the interface is subjected to annealing above 375°C. On the contrary, magnesium contacts on bare silicon (001) turn from ohmic to Schottky at much lower temperatures. The temperature difference is 50-150°C. We deduce that selenium passivation suppresses silicidation between magnesium and silicon, because it reduces the surface reactivity of silicon by eliminating dangling bonds and relaxing strained bonds

Ultrafast dynamical charge-lattice coupling in rare-earth nickelate thin films studied by time-resolved terahertz spectroscopy

Rare-earth nickelates exhibit a rich phase diagram formed by the complex interplay of intertwined and competing energetics of fundamental entities. To unwind the coupling and interaction mechanisms of fundamental entities underneath, time-resolved terahertz (THz) spectroscopy was implemented to understand non-equilibrium carrier and lattice dynamics of epitaxial thin films of La x Eu1-x NiO3 (x = 0, 0.25, 0.50, 1) systems, where x = 0 is insulating while remaining are metallic at room temperature. The THz transmittance of the insulating and metallic phases exhibit contrasting photo-induced phases associated with bi-exponential and mono-exponential relaxation mechanisms, respectively. A pronounced oscillatory feature superimposed on the mono-exponential relaxation manifests only in the metallic phase. As ascribed to the acoustic phonons, the 'x' dependent behavior of this feature reveals an inverse relation between the strength of electron-phonon coupling and the magnitude of conductivity. In the insulating state, in contrast, the relaxation time constants are associated with the recovery of charge-ordering and electron-phonon thermalization. This dynamical lattice-charge interaction study demonstrates use of this ultrafast phenomena in nickelate thin films in new generation ultrafast photo-acoustic devices as an alternate to conventional surface acoustic wave device.D S R thanks the Science and Engineering Research Board (SERB), Department of Science and Technology, New Delhi, for financial support under research Project No. CRG/2020/002338