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Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti- Mycobacterium tuberculosis ( Mtb ) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance. Antimicrobial resistance is a global health threat and the development of alternative strategies to overcome it is of high interest. Here, the authors report proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery, and apply them for targeting a range of mycobacterial strains, including antibiotic-resistant ones.

Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF‐BP1, MULE) associates with both MYC and the MYC‐associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high‐throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC‐dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC‐activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. Synopsis New highly specific inhibitors of HUWE1 have been identified that can inhibit MYC function in a tumour cell‐specific manner, by preventing the HUWE1‐mediated block of a repressive complex of MYC with MIZ1 on MYC‐activated target genes. HUWE1 is essential for growth of colorectal tumors in vivo and is globally required for expression of MYC target genes. Small‐molecule inhibitors of HUWE1 inhibit expression of MYC target genes in a tumor‐cell specific manner. Inhibition of HUWE1 stabilizes MIZ1 and induces global accumulation of MIZ1 on MYC‐bound target genes. Accumulation of MIZ1 is required for repression of ribosomal protein genes upon HUWE1 inhibition. Graphical Abstract New highly specific inhibitors of HUWE1 have been identified that can inhibit MYC function in a tumour cell‐specific manner, by preventing the HUWE1‐mediated block of a repressive complex of MYC with MIZ1 on MYC‐activated target genes.

Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases. A structure-based design allows the development of a potent PROTAC to degrade BAF ATPase subunits SMARCA2 and SMARCA4 via recruitment of E3 ubiquitin ligase VHL and induce cancer cell death.

Aberrations in genes coding for subunits of the BRG1/BRM associated factor (BAF) chromatin remodeling complexes are highly abundant in human cancers. Currently, it is not understood how these mostly loss-of-function mutations contribute to cancer development and how they can be targeted therapeutically. The cancer-type-specific occurrence patterns of certain subunit mutations suggest subunit-specific effects on BAF complex function, possibly by the formation of aberrant residual complexes. Here, we systematically characterize the effects of individual subunit loss on complex composition, chromatin accessibility and gene expression in a panel of knockout cell lines deficient for 22 BAF subunits. We observe strong, specific and sometimes discordant alterations dependent on the targeted subunit and show that these explain intracomplex codependencies, including the synthetic lethal interactions SMARCA4–ARID2, SMARCA4–ACTB and SMARCC1–SMARCC2. These data provide insights into the role of different BAF subcomplexes in genome-wide chromatin organization and suggest approaches to therapeutically target BAF-mutant cancers. The authors generate cell lines deficient for 22 BAF subunits, studying effects on complex composition, chromatin accessibility and gene expression. They identify synthetic lethal interactions between SMARCA4–ARID2, SMARCA4–ACTB and SMARCC1–SMARCC2.

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains. A chemical probe BI-9321 for the PWWP1 domain of NSD3 and its inactive analog were identified. BI-9321 binds to the methyl-lysine binding site, reduces the association of NSD3 with chromatin and inhibits proliferation of acute myeloid leukemia cells.

Aberrations in genes coding for subunits of the BAF chromatin remodeling complexes are highly abundant in human cancers. Currently, it is not understood how these loss-of-function mutations contribute to cancer development and how they can be targeted therapeutically. The cancer-type-specific occurrence patterns of certain subunit mutations suggest subunit-specific effects on BAF complex function, possibly by the formation of aberrant residual complexes. Here, we systematically characterize the effects of individual subunit loss on complex composition, chromatin accessibility and gene expression in a panel of knock-out cell lines deficient for 22 BAF subunits. We observe strong, specific and sometimes discordant alterations dependent on the targeted subunit and show that these explain intra-complex co-dependencies, including the synthetic lethal interactions SMARCA4-ARID2, SMARCA4-ACTB and SMARCC1-SMARCC2. These data provide insights into the role of different BAF subcomplexes in genome-wide chromatin organization and suggest approaches to therapeutically target BAF mutant cancers.

In the version of this article originally published, several lines of text in the last paragraph of the right column on page 1 of the PDF were transposed into the bottom paragraph of the left column. The affected text of the left column should read “The ATP-dependent activities of the BAF (SWI/SNF) chromatin remodeling complexes affect the positioning of nucleosomes on DNA and thereby many cellular processes related to chromatin structure, including transcription, DNA repair and decatenation of chromosomes during mitosis 12,13 .” The affected text of the right column should read “SMARCA2/4 BD inhibitors are thus precluded from use for the treatment of SMARCA4 mutant cancers but could provide attractive ligands for PROTAC conjugation. Small molecules binding to other bromodomains have been successfully converted into PROTACs by conjugating them with structures capable of binding to the E3 ligases von Hippel−Lindau (VHL) or cereblon 5,6,10,11,25,26,27 .” The errors have been corrected in the PDF version of the paper.

Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE 1 ( HECTH 9, ARF ‐ BP 1, MULE ) associates with both MYC and the MYC ‐associated protein MIZ 1. We show here that HUWE 1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high‐throughput screening, we identify small molecule inhibitors of HUWE 1, which inhibit MYC ‐dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE 1 stabilizes MIZ 1. MIZ 1 globally accumulates on MYC target genes and contributes to repression of MYC ‐activated target genes upon HUWE 1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ 1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. image New highly specific inhibitors of HUWE 1 have been identified that can inhibit MYC function in a tumour cell‐specific manner, by preventing the HUWE 1‐mediated block of a repressive complex of MYC with MIZ 1 on MYC ‐activated target genes. HUWE1 is essential for growth of colorectal tumors in vivo and is globally required for expression of MYC target genes. Small‐molecule inhibitors of HUWE1 inhibit expression of MYC target genes in a tumor‐cell specific manner. Inhibition of HUWE1 stabilizes MIZ1 and induces global accumulation of MIZ1 on MYC‐bound target genes. Accumulation of MIZ1 is required for repression of ribosomal protein genes upon HUWE1 inhibition.

The hexosamine pathway provides UDP‐ N ‐acetylhexosamine donor substrates used in cytosolic and Golgi‐mediated glycosylation of proteins and for formation of glycosylphosphatidylinositol (GPI) anchors, which tether proteins to the outer plasma membrane. We have recently identified the murine glucosamine‐6‐phosphate (GlcN6P) acetyltransferase, EMeg32, as a developmentally regulated enzyme on the route to UDP‐ N ‐acetylglucosamine (UDP‐GlcNAc). Here we describe embryos and cells that have the EMeg32 gene inactivated by homologous recombination. Homozygous mutant embryos die at around embryonic day (E) 7.5 with a general proliferative delay of development. In vitro differentiated EMeg32 −/− ES cells show reduced proliferation. Mouse embryonic fibroblasts (MEFs) deficient for EMeg32 exhibit defects in proliferation and adhesiveness, which could be complemented by stable re‐expression of EMeg32 or by nutritional restoration of intracellular UDP‐GlcNAc levels. Reduced UDP‐GlcNAc levels predominantly translated into decreased O ‐GlcNAc modifications of cytosolic and nuclear proteins. Interestingly, growth‐impaired EMeg32 −/− MEFs withstand a number of apoptotic stimuli and express activated PKB/AKT. Thus, EMeg32‐dependent UDP‐GlcNAc levels influence cell cycle progression and susceptibility to apoptotic stimuli.