Background Medical treatment in Cushing’s disease (CD) is limited due to poor understanding of its pathogenesis. Pathogenic variants of ubiquitin specific peptidase 8 (USP8) have been confirmed as causative in around half of corticotroph tumors. We aimed to further characterize the molecular landscape of those CD tumors lacking USP8 mutations in a large cohort of patients. Methods Exome sequencing was performed on 18 paired tumor–blood samples with wild-type USP8 status. Candidate gene variants were screened by Sanger sequencing in 175 additional samples. The most frequent variant was characterized by further functional in vitro assays. Results Recurrent somatic hotspot mutations in another deubiquitinase, USP48, were found in 10.3% of analyzed samples. Several possibly damaging variants were found in TP53 in 6 of 18 samples. USP48 variants were associated with smaller tumors and trended toward higher frequency in female patients. They also changed the structural conformation of USP48 and increased its catalytic activity toward its physiological substrates histone 2A and zinc finger protein Gli1, as well as enhanced the stimulatory effect of corticotropin releasing hormone (CRH) on pro-opiomelanocortin production and adrenocorticotropic hormone secretion. Conclusions USP48 pathogenic variants are relatively frequent in USP8 wild-type tumors and enhance CRH-induced hormone production in a manner coherent with sonic hedgehog activation. In addition, TP53 pathogenic variants may be more frequent in larger CD tumors than previously reported.

Fbw7, the substrate recognition subunit of SCF(Fbw7) ubiquitin ligase, mediates the turnover of multiple proto-oncoproteins and promotes its own degradation. Fbw7-dependent substrate ubiquitination is antagonized by the Usp28 deubiquitinase. Here, we show that Usp28 preferentially antagonizes autocatalytic ubiquitination and stabilizes Fbw7, resulting in dose-dependent effects in Usp28 knockout mice. Monoallelic deletion of Usp28 maintains stable Fbw7 but drives Fbw7 substrate degradation. In contrast, complete knockout triggers Fbw7 degradation and leads to the accumulation of Fbw7 substrates in several tissues and embryonic fibroblasts. On the other hand, overexpression of Usp28 stabilizes both Fbw7 and its substrates. Consequently, both complete loss and ectopic expression of Usp28 promote Ras-driven oncogenic transformation. We propose that dual regulation of Fbw7 activity by Usp28 is a safeguard mechanism for maintaining physiological levels of proto-oncogenic Fbw7 substrates, which is equivalently disrupted by loss or overexpression of Usp28.

Deregulated expression of MYC is a driver of colorectal carcinogenesis, suggesting that inhibiting MYC may have signifi cant therapeutic value. The PI3K and mTOR pathways control MYC turnover and translation, respectively, providing a rationale to target both pathways to inhibit MYC. Surprisingly, inhibition of PI3K does not promote MYC turnover in colon carcinoma cells, but enhances MYC expression because it promotes FOXO-dependent expression of growth factor receptors and MAPK-dependent transcription of MYC . Inhibition of mTOR fails to inhibit translation of MYC, because levels of 4EBPs are insuffi cient to fully sequester eIF4E and because an internal ribosomal entry site element in the 5′-untranslated region of the MYC mRNA permits translation independent of eIF4E. A small-molecule inhibitor of the translation factor eIF4A, silvestrol, bypasses the signaling feedbacks, reduces MYC translation, and inhibits tumor growth in a mouse model of colorectal tumorigenesis. We propose that targeting translation initiation is a promising strategy to limit MYC expression in colorectal tumors. SIGNIFICANCE:Inhibiting MYC function is likely to have a signifi cant therapeutic impact in colorectal cancers. Here, we explore several strategies to target translation initiation in order to block MYC expression. We show that a small-molecule inhibitor of eIF4A inhibits MYC expression and suppresses tumor growth in vivo . Cancer Discov; 5(7); 768-81.