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On-target, cell-active chemical probes are of fundamental importance in both chemical and cell biology, whereas the application of poorly-characterised probes often leads to invalid conclusions.Human N-myristoyltransferase (NMT) has attracted increasing interest as a target in cancer and infectious diseases; here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis and cell cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM) or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies which used off-target compounds.

Over 30% of ER? breast cancer patients develop relapses and progress to metastatic disease despite treatment with endocrine therapies. The pioneer factor PBX1 translates epigenetic cues and mediates estrogen induced ER? binding. Here we demonstrate that PBX1 plays a central role in regulating the ER? transcriptional response to epidermal growth factor (EGF) signaling. PBX1 regulates a subset of EGF-ER? genes highly expressed in aggressive breast tumours. Retrospective stratification of luminal patients using PBX1 protein levels in primary cancer further demonstrates that elevated PBX1 protein levels correlate with earlier metastatic progression. In agreement, PBX1 protein levels are significantly upregulated during metastatic progression in ER?-positive breast cancer patients. Finally we reveal that PBX1 upregulation in aggressive tumours is partly mediated by genomic amplification of the PBX1 locus. Correspondingly, ER?-positive breast cancer patients carrying PBX1 amplification are characterized by poor survival. Notably, we demonstrate that PBX1 amplification can be identified in tumor derived-circulating free DNA of ER?-positive metastatic patients. Metastatic patients with PBX1 amplification are also characterized by shorter relapse-free survival. Our data identifies PBX1 amplification as a functional hallmark of aggressive ER?-positive breast cancers. Mechanistically, PBX1 amplification impinges on several critical pathways associated with aggressive ER?-positive breast cancer.

On-target, cell-active chemical probes are of fundamental importance in chemical and cell biology, whereas poorly characterized probes often lead to invalid conclusions. Human N-myristoyltransferase (NMT) has attracted increasing interest as target in cancer and infectious diseases. Here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis, and cell-cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM), or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies that used off-target compounds.

On-target, cell-active chemical probes are of fundamental importance in chemical and cell biology, whereas poorly characterized probes often lead to invalid conclusions. Human N-myristoyltransferase (NMT) has attracted increasing interest as target in cancer and infectious diseases. Here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis, and cell-cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM), or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies that used off-target compounds.