Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Abstract 1119: Molecular characteristics of repotrectinib that enable potent inhibition of TRK fusion proteins and broad mutant selectivity

Abstract NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to first-generation TRK inhibitors (larotrectinib, entrectinib) but the emergence of NTRK mutations limits their efficacy. Next-generation TRK inhibitors (repotrectinib, selitrectinib) have compact macrocyclic structures designed to limit the susceptibility to mutations. However, a detailed understanding of mutant potency and precise binding interactions are lacking. TRK inhibitors were evaluated in cellular models expressing TRKA/B/C fusions with wild-type (WT) and resistance mutations: solvent front (SFM), gatekeeper (GKM), activation loop (xDFG), and compound mutations. In cell proliferation assays, differential potencies against wild type TRKA/B/C fusions were observed: larotrectinib (IC50 23.5 - 49.4 nM), entrectinib (IC50 0.3 - 1.3 nM), selitrectinib (IC50 1.8 - 3.9 nM), and repotrectinib (IC50 600 nM) against all TRK mutations and entrectinib had >400-fold decreased against all mutations except GKM where there was a range of potencies (IC50 < 0.2 - 60.4 nM). Repotrectinib and selitrectinib were less affected by resistance mutations however repotrectinib was approximately 10-fold more potent than selitrectinib against SFM and compound mutations and 100-fold more potent against GKM. For TRKA/B/C xDFG mutations, repotrectinib had moderate potency (IC50 11.8 - 67.6 nM) while selitrectinib was less potent (IC50 124 - 341 nM). Co-crystal structures of repotrectinib with TRKA and TRKA harboring a SFM provide insight into how subtle differences in macrocyclic inhibitor structure and conformational profiles affect potency and mutant selectivity. Analysis of the first protein structure of a kinase harboring a common SFM (TRKA G595R) revealed unexpected intramolecular interactions which provide insight into its prevalence. Repotrectinib, but not selitrectinib, caused tumor regression in LMNA-NTRKA xenograft models harboring GKM, SFM, or GKM+SFM compound mutations. In the clinic, tumor regression was observed with repotrectinib treatment of a larotrectinib-resistant cholangiocarcinoma patient with both LMNA-TRKA GKM and SFM mutations. Repotrectinib has shown responses in patients with TRK driven tumors with or without resistant mutations in the ongoing global registrational TRIDENT-1 study and has been granted 3 fast track designations. Taken together, the current data characterizes TRK inhibitor potency against resistance mutations and highlights structural characteristics of repotrectinib that enable potent inhibition of TRK proteins and evasion of drug resistance mediated by TRK mutations. Citation Format: Alexander Drilon, Dayong Zhai, Evan Rogers, Wei Deng, Xi Chen, Paul Sprengeler, Siegfried H. Reich, Brion W. Murray. Molecular characteristics of repotrectinib that enable potent inhibition of TRK fusion proteins and broad mutant selectivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1119

Molecular Characteristics of Repotrectinib That Enable Potent Inhibition of TRK Fusion Proteins and Resistant Mutations

chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to TRK inhibitors (larotrectinib and entrectinib) but often mutate, limiting the durability of response for patients. Next-generation inhibitors with compact macrocyclic structures (repotrectinib and selitrectinib) were designed to avoid resistance mutations. Head-to-head potency comparisons of TRK inhibitors and molecular characterization of binding interactions are incomplete, obscuring a detailed understanding of how molecular characteristics translate to potency. Larotrectinib, entrectinib, selitrectinib, and repotrectinib were characterized using cellular models of wild-type TRKA/B/C fusions and resistance mutant variants with a subset evaluated in xenograft tumor models. Crystal structures were determined for repotrectinib bound to TRKA (wild-type, solvent-front mutant). TKI-naïve and pretreated case studies are presented. Repotrectinib was the most potent inhibitor of wild-type TRKA/B/C fusions and was more potent than selitrectinib against all tested resistance mutations, underscoring the importance of distinct features of the macrocycle structures. Cocrystal structures of repotrectinib with wild-type TRKA and the TRKA SFM variant elucidated how differences in macrocyclic inhibitor structure, binding orientation, and conformational flexibility affect potency and mutant selectivity. The SFM crystal structure revealed an unexpected intramolecular arginine sidechain interaction. Repotrectinib caused tumor regression in xenograft models harboring GKM, SFM, xDFG, and GKM + SFM compound mutations. Durable responses were observed in TKI-naïve and -pretreated patients with cancers treated with repotrectinib (NCT03093116). This comprehensive analysis of first- and second-generation TRK inhibitors informs the clinical utility, structural determinants of inhibitor potency, and design of new generations of macrocyclic inhibitors

Translation control of the immune checkpoint in cancer and its therapeutic targeting

Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCTg;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies