Recruitment of FBXO22 for targeted degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.

Recruitment of FBXO22 for Targeted Degradation of NSD2.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

Structure-function analysis of the SHOC2-MRAS-PP1C holophosphatase complex.

Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .

Discovery and Optimization of Glucose Uptake Inhibitors.

Aerobic glycolysis, originally identified by Warburg as a hallmark of cancer, has recently been implicated in immune cell activation and growth. Glucose, the starting material for glycolysis, is transported through the cellular membrane by a family of glucose transporters (GLUTs). Therefore, targeting glucose transporters to regulate aerobic glycolysis is an attractive approach to identify potential therapeutic agents for cancers and autoimmune diseases. Herein, we describe the discovery and optimization of a class of potent, orally bioavailable inhibitors of glucose transporters, targeting both GLUT1 and GLUT3.

p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression.

There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2.

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.

Epigenetic reprogramming by tumor-derived EZH2 gain-of-function mutations promotes aggressive 3D cell morphologies and enhances melanoma tumor growth.

In addition to genetic alterations, cancer cells are characterized by myriad epigenetic changes. EZH2 is a histone methyltransferase that is over-expressed and mutated in cancer. The EZH2 gain-of-function (GOF) mutations first identified in lymphomas have recently been reported in melanoma (~2%) but remain uncharacterized. We expressed multiple EZH2 GOF mutations in the A375 metastatic skin melanoma cell line and observed both increased H3K27me3 and dramatic changes in 3D culture morphology. In these cells, prominent morphological changes were accompanied by a decrease in cell contractility and an increase in collective cell migration. At the molecular level, we observed significant alteration of the axonal guidance pathway, a pathway intricately involved in the regulation of cell shape and motility. Furthermore, the aggressive 3D morphology of EZH2 GOF-expressing melanoma cells (both endogenous and ectopic) was attenuated by EZH2 catalytic inhibition. Finally, A375 cells expressing exogenous EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. This study not only demonstrates the first functional characterization of EZH2 GOF mutants in non-hematopoietic cells, but also provides a rationale for EZH2 catalytic inhibition in melanoma.

Nuclear pore component Nup98 is a potential tumor suppressor and regulates posttranscriptional expression of select p53 target genes.

The p53 tumor suppressor utilizes multiple mechanisms to selectively regulate its myriad target genes, which in turn mediate diverse cellular processes. Here, using conventional and single-molecule mRNA analyses, we demonstrate that the nucleoporin Nup98 is required for full expression of p21, a key effector of the p53 pathway, but not several other p53 target genes. Nup98 regulates p21 mRNA levels by a posttranscriptional mechanism in which a complex containing Nup98 and the p21 mRNA 3'UTR protects p21 mRNA from degradation by the exosome. An in silico approach revealed another p53 target (14-3-3σ) to be similarly regulated by Nup98. The expression of Nup98 is reduced in murine and human hepatocellular carcinomas (HCCs) and correlates with p21 expression in HCC patients. Our study elucidates a previously unrecognized function of wild-type Nup98 in regulating select p53 target genes that is distinct from the well-characterized oncogenic properties of Nup98 fusion proteins.

p53-Dependent induction of PVT1 and miR-1204.

p53 is a tumor suppressor protein that acts as a transcription factor to regulate (either positively or negatively) a plethora of downstream target genes. Although its ability to induce protein coding genes is well documented, recent studies have implicated p53 in the regulation of non-coding RNAs, including both microRNAs (e.g. miR-34a) and long non-coding RNAs (e.g. lincRNA-p21). We have identified the non-protein coding locus PVT1 as a p53-inducible target gene. PVT1, a very large (>300 kb) locus located downstream of c-myc on chromosome 8q24, produces a wide variety of spliced non-coding RNAs as well as a cluster of six annotated microRNAs: miR-1204, miR-1205, miR-1206, miR-1207-5p, miR-1207-3p, and miR-1208. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and luciferase assays reveal that p53 binds and activates a canonical response element within the vicinity of miR-1204. Consistently, we demonstrate the p53-dependent induction of endogenous PVT1 transcripts and consequent up-regulation of mature miR-1204. Finally, we have shown that ectopic expression of miR-1204 leads to increased p53 levels and causes cell death in a partially p53-dependent manner.

Noncoding RNAs: the missing "linc" in p53-mediated repression.

The tumor suppressor protein p53 coordinates the cellular response to stress through regulation of gene expression. Now, Huarte et al. (2010) identify a long intergenic noncoding RNA as a new player in p53-mediated repression of genes involved in apoptosis.

Caspase 2 is both required for p53-mediated apoptosis and downregulated by p53 in a p21-dependent manner.

Upon treatment with some DNA damaging agents, human H1299 tumor-derived cells expressing inducible versions of wild-type or mutant p53 with inactive transactivation domain I (p53(Q22/S23)) undergo apoptosis. In cells expressing either version of p53, caspase 2 activation is required for release of cytochrome c and cell death. Furthermore, silencing of PIDD (a factor previously shown to be required for caspase 2 activation) by siRNA suppresses apoptosis by both wild-type p53 and p53(Q22/S23). Despite the finding that caspase 2 is essential for DNA damage-facilitated, p53-mediated apoptosis, induction of wild-type p53 (with or without DNA damage) resulted in a reduction of caspase 2 mRNA and protein levels. In this study we sought to provide a mechanism for the negative regulation of caspase 2 by p53 as well as provide insight as to why p53 may repress a key mediator of p53-dependent apoptosis. Mechanistically, we show that DNA binding and/or transactivation domains of p53 are crucial for mediating transrepression. Further, expression of p21 (in p53-null cells inducibly expressing p21) is sufficient to mediate repression of caspase 2. Deletion of p21 or E2F-1 not only abrogated repression of caspase 2, but also stimulated the expression of caspase 2 above basal levels, implicating the requirement for an intact p21/Rb/E2F pathway in the downregulation of caspase 2. As this p53/p21-dependent repression of caspase 2 can occur in the absence of DNA damage, caspase 2 repression does not simply seem to be a consequence of the apoptotic process. Downregulation of caspase 2 levels by p53 may help to determine cell fate by preventing cell death when unnecessary.

A role for caspase 2 and PIDD in the process of p53-mediated apoptosis.

When treated with some DNA-damaging agents, human tumor-derived H1299 cells expressing inducible versions of wild-type or mutant p53 with inactive transactivation domain I (p53(Q22/S23)) undergo apoptosis as evidenced by cytochrome c release, nuclear fragmentation, and sub-G1 DNA content. Apoptosis induced by p53(Q22/S23) is relatively slow, however, and key downstream effector caspases are not activated. Nevertheless, with either version of p53, caspase 2 activation is required for release of cytochrome c and cell death. Remarkably, although p53(Q22/S23) is known to be defective in transcriptional activation of numerous p53 target genes, it can induce expression of proapoptotic targets including PIDD and AIP1 at least to the same extent as wild-type p53. Furthermore, RNAi silencing of PIDD, previously shown to be required for caspase 2 activation, suppresses apoptosis by both wild-type p53 and p53(Q22/S23). Thus, the initial stage of DNA damage-facilitated, p53-mediated apoptosis occurs by a PIDD- and caspase 2-dependent mechanism, and p53's full transcriptional regulatory functions may be required only for events that are downstream of cytochrome c release.