从Cereblon调节剂文库中鉴定出一种具有治疗潜力的强效选择性PPIL4降解剂

Molecular glue (MG)-based protein degradation offers the potential to directly degrade aberrant transcription factor drivers of childhood cancers. Here we report the screening of a novel library of 5056 CRBN-binding MG in acute leukemia (AL) and medulloblastoma (MB) cell lines. This identified SJ42872, a potent and selective degrader of the cyclophilin family protein and spliceosome protein peptidylprolyl isomerase-like 4 (PPIL4) with sparing of known MG neosubstrates such as GSPT1, CK1alpha, and IKZF1/3. SJ42872 degraded PPIL4 in dose-dependent manner (DC50 10-100 nM) with maximal degradation in less than one hour. SJ42872 displayed CRBN-dependent cytotoxicity in AL and MB cell lines, inducing cell cycle arrest and apoptosis. Inactivation of PPIL4 by CRISPR/Cas9 genome editing impaired cell growth in the ALL cell lines MHH-CALL-4 ( CRLF2- r BCR::ABL1- like) and NALM-6 ( DUX4- r). Mutagenesis of 4 potential degron motifs demonstrated that G278N abrogated degradation by SJ42872, and expression of PPIL4-G278N in AL cell lines conferred resistance to SJ42872. To elucidate the structural basis of selective PPIL4 degradation, we constructed an in-silico model of the PPIL4-SJ42872-CRBN complex, using a CK1alpha structure (PDB: 8G66) as a template. Our model revealed that the glutarimide moiety of SJ42872 forms interactions with key CRBN residues H378, W380, as well as a novel N351 interaction with the quinazolinone ring, while its piperazine ring forms H-bonds to D253 and E270 of PPIL4. Molecular dynamic simulations demonstrated SJ42872 maintained strong, persistent interactions with CRBN. Genome-wide CRISPR/Cas9 screening of SJ42872 in NALM-6 and REH ( ETV6::RUNX1 ) cells identified multiple pathways underlying the activity of SJ42872, including protein neddylation, mitochondrial gene expression and RNA transcription initiation, suggesting roles of PPIL4 in synthesis of RNA and proteins. Testing of SJ42872 in a cell line panel showed activity in multiple hematological malignancies. Although some activity in cord blood CD34+ cells and blood mononuclear cells was observed, a therapeutic index of SJ42872 >10 was observed, and there was no activity in normal microglial cells. Pharmacokinetic and pharmacodynamic analyses revealed rapid in vivo exposure in NSG mice, with high concentrations of SJ42872 detected within two hours post-intraperitoneal injection. Notably, near-complete degradation of PPIL4 was achieved in CRLF2- r patient-derived xenograft cells spleen and bone marrow in NSG mice treated with 60-100 mg/kg b.i.d. i.p., and complete degradation of PPIL4 was also observed in intracranially-engrafted MB cells in CD1 mice with 30-100 mg/kg i.p. Four-week efficacy studies of a CRLF2 -r PDX further demonstrated profound dose-dependent reductions in tumor growth and leukemia burden in vivo. These findings show the power of unbiased MG screening to identify new cancer cell vulnerabilities, and establish SJ42872 as promising therapeutic candidate for childhood leukemia and brain tumors.