Mirit I. Aladjem

Adrenocortical carcinoma (ACC) is a rare, aggressive, chemoresistant malignancy with limited effective systemic therapies and few recurrent actionable oncogenic drivers. Although IGF-2 is overexpressed in approximately 80% of ACCs, clinical inhibition of the IGF-1R/InsR axis has not improved outcomes. To identify clinically tractable combination strategies that enhance IGF-1R/InsR inhibition in ACC, we performed a quantitative high-throughput combinatorial screen of 2,803 mechanistically annotated oncology compounds with the IGF 1R/InsR inhibitor linsitinib in an ACC cell line. Downstream effects of IGF-1R/InsR signaling were assessed to identify actionable nodes for combination targeting. Candidate combinations were validated using short-term ACC patient-derived organoids (PDOs) and patient-derived xenograft (PDX) models. The screen identified MAPK pathway inhibitors—particularly MEK inhibition—as the top synergistic partners with IGF-1R/InsR inhibition. Genetic or pharmacologic inhibition of IGF-1R/InsR and MEK signaling suppressed ACC growth in vitro. Downstream profiling of IGF-1R/InsR signaling identified AKT as a major effector, supporting direct AKT targeting as a translatable substitute for IGF-1R/InsR inhibition in ACC. Accordingly, the AKT inhibitor capivasertib synergized with the MEK inhibitor cobimetinib in ACC cell lines and six ACC PDO models. In vivo, combined AKT and MEK inhibition reduced tumor growth more than either agent alone in an ACC xenograft and two ACC chemotherapy-resistant PDX models with acceptable tolerability. These data identify AKT as a critical survival pathway in ACC and support combined AKT and MEK inhibition with FDA-approved agents as a therapeutic approach for chemotherapy-resistant ACC.

Schlafen 11 (SLFN11) is a key determinant of sensitivity to DNA-damaging therapies in multiple cancers including leukemia, Ewing sarcoma, head and neck cancers, mesothelioma and small cell lung cancer (SCLC). Here, utilizing immunofluorescence microscopy and single-cell RNA sequencing we characterized SLFN11 expression across eight different human cancer cell lines and found cell-to-cell heterogenous expression both at the protein and RNA levels. Gene set enrichment and pathway analyses show that SLFN11 is coexpressed with a distinct stress-response state and enhanced genome maintenance programs while being inversely correlated with protein translation pathways. Publicly available single-cell RNA-sequencing datasets of human SCLC tumors confirmed the heterogeneous distribution of SLFN11 expression and confirmed its association with replication-related transcriptional programs. These findings indicate that heterogeneous SLFN11 expression reflects dynamic regulation at the single-cell level. They also underscore the limitations of bulk proteomic and transcriptome profiling and point to a potential reservoir of intrinsic chemotherapy resistance.

DNA replication in mammalian cells proceeds in chromosomal domains during defined windows of S phase; however, the mechanisms coordinating replication with restoration of chromatin states remain incompletely understood. Here, we identify p53-binding protein 1 (53BP1) condensates as regulators of heterochromatin replication and epigenetic restoration. Loss of 53BP1 delays mid-to-late S-phase progression, compromises recruitment of epigenetic restoration factors, and impairs timely completion of heterochromatin replication and restoration of heterochromatin-associated epigenetic marks. These defects were rescued by wild-type or condensate-competent 53BP1, but not by condensate-deficient mutants. Further, 53BP1 condensate formation and its role in heterochromatin replication depend on interaction with proliferating cell nuclear antigen, which also mediates 53BP1 relocalization from condensates to DNA damage foci upon genotoxic stress. Together, our findings reveal a previously unrecognized role for 53BP1 condensates in coordinating heterochromatin replication and epigenetic restoration, while serving as a reservoir for DNA damage response.

: Adrenocortical carcinoma (ACC) is a rare and highly aggressive endocrine malignancy originating from the adrenal cortex with limited effective treatment options. The underlying pathophysiology of ACC is uniquely characterized by abnormal steroid production and increased metabolic activity, highlighting the critical role of mitochondria in adrenal steroid hormone biosynthesis and tumor metabolism. In this study, we investigated the therapeutic potential of TR-107, a novel and highly selective small-molecule agonist targeting the mitochondrial protease ClpP. Pharmacologic hyperactivation of ClpP disrupts mitochondrial proteostasis and bioenergetics and has shown promising antitumor activity in various preclinical models. Our results demonstrated that TR-107 induces potent dose-dependent cytotoxic effects at nanomolar concentrations in ACC cell lines NCI-H295R and mACC3 as well as short-term ACC patient-derived organoid (PDO) models, markedly reducing cell viability and confluency in vitro . Metabolic analyses revealed that TR-107 significantly impaired oxygen consumption, indicating a disruption of oxidative phosphorylation and substantial attenuation of basal cellular respiration. Mechanistic studies showed dose-dependent increases in reactive oxygen species (ROS) levels and upregulation of proteins involved in mediating the ferroptotic rheostat. Pharmacokinetic assessment uncovered that TR-107 was not a substrate of the ABCB1 (MDR1/P-glycoprotein) efflux transporter, suggesting potential to overcome common multidrug resistance mechanisms. Given the importance of IGF-2 signaling in ACC, we further explored the combinatorial effects of TR-107 with IGF-1 receptor (IGF-1R) inhibitors and discovered that co-treatment produced synergistic reductions in cell viability across NCI-H295R, mACC3, and ACC PDOs. Collectively, these findings support the potential of mitochondrial ClpP hyperactivation as a promising therapeutic strategy for ACC and demonstrate that TR-107 exhibits significant antitumor activity as a monotherapy or in combination with IGF-1R inhibitors. These findings provide a strong rationale for advancing ClpP agonists into clinical development for the management of ACC.

Polycomb (PcG) bodies are nuclear foci formed by polycomb protein complexes that contain PcG-bound DNA and are implicated in gene regulation during development and differentiation, although their precise molecular function remains unclear. Using tyramide signal amplification sequencing, we provide a comprehensive view of genomic regions associated with PcG bodies, including specific centromeric and telomeric sites. These regions are enriched for the repressive marks H3K27me3 and H3K9me3, depleted of the active mark H3K4me3, and display low chromatin accessibility. We find a high density of replication origins around PcG bodies, consistent with the replication factor origin recognition complex–associated protein (ORCA)/LRWD1 localizing at these sites. ORCA interacts with the polycomb-repressive complex, stabilizes the H3K27 methyltransferase, and facilitates H3K27me3 deposition at specific chromatin sites. Loss of ORCA affects chromatin organization around PcG bodies, leading to decompaction of the repeat regions, and enhanced initiation from replication origins. Our results suggest that ORCA associates with PcG-bound chromatin to maintain a repressive environment that regulates replication origin firing timing.

Experimental validation of copy-number of the MYC locus in NCI-H889, DMS-114, NCI-H446 and RA022 patient derived cell lines and number of ecDNA and HSR positive cells in the RA022 patient derived cell lines

Fusion landscape and ecDNA RLF-MYCL fusions, related to Figure 3.

Heterogeneity in patient derived samples, related to Figure 5

Enhancer-enhancer interaction analysis derived from H3K27ac HiChIP in the NCI-H889 and NCI-H524 cell lines. Interaction strength of proximal enhancers was calculated from the “.hic” files generated by the JuiceBox suite.