Topoisomerase III-beta (Top3b) reduces nucleic acid torsional stress and intertwining generated during RNA and DNA metabolism while protecting the genome from pathological R-loops, which otherwise result in DNA breakage and genome instability. By studying Top3b knockout mice ( Top3b -KO), we find that the loss of Top3b accelerates the development of spontaneous atypical lymphoid hyperplasia and lymphomas arising in spleens and lymph nodes, organs with prominent Top3b expression. Aging Top3b -KO mice also display splenomegaly and systemic immune alterations including neutrophilia and lymphopenia consistent with chronic inflammation. At the molecular level, Top3b deficiency causes genome-wide R-loop accumulation in splenocytes as measured by CUT&Tag sequencing. Increased R-loops are associated with genomic breaks and activation of immune signaling pathways including innate and adaptive immune cell signaling, IL-4 signaling, FAK signaling, and cGAS-STING. In addition, knocking-out Top3b promotes the rapid development of syngeneic EL4 T cell lymphomas. In conclusion, our work implies that Top3b protects from lymphoma, tumorigenesis, and immune dysregulations.

Topoisomerase III-beta (Top3b) reduces nucleic acid torsional stress and intertwining generated during RNA and DNA metabolism while protecting the genome from pathological R-loops, which otherwise result in DNA breakage and genome instability. By studying Top3b knockout mice (Top3b-KO), we find that the loss of Top3b accelerates the development of spontaneous lymphoid tumors arising in spleens and lymph nodes, the organs with prominent Top3b expression. Aging Top3b-KO mice also display splenomegaly and systemic immune alterations including neutrophilia and lymphopenia suggestive of chronic inflammation. At the molecular level, Top3b deficiency causes genome-wide R-loop accumulation in splenocytes as measured by CUT&Tag sequencing. Increased R-loops is associated with genomic DNA breaks and activation of immune signaling pathways including the IL-6 signaling, interleukin-7 signaling and cGAS-STING. Moreover, knocking-out Top3b promotes the rapid development of syngeneic EL4 T-cell lymphomas. In conclusion, our work implies that, in addition to its role in preserving the nervous system, Top3b protects from tumorigenesis and immune dysregulations.

High fat (HF) diet is a major factor in the development of metabolic dysfunction-associated steatotic liver disease (MASLD) and steatohepatis (MASH), and mitochondria have been proposed to play a role in the pathogenesis of HF diet-induced MASH. Because Mitochondrial topoisomerase I (Top1MT) is exclusively present in mitochondria and Top1MT knock-out mice are viable, we were able to assess the role of Top1MT in the development of MASH. We show that after 16 weeks of HF diet, mice lacking Top1MT are prone to the development of severe MASH characterized by liver steatosis, lobular inflammation and hepatocyte damage. Mice lacking Top1MT also show prominent mitochondrial dysfunction, ROS production and mitochondrial DNA (mtDNA) release, accompanied by hepatic inflammation and fibrosis. In summary, our study demonstrates the importance of Top1MT in sustaining hepatocyte functions and suppressing MASH.

Supplementary Figure 3: Effects of gemcitabine treatment on IL6(R) expression in vitro, impact of IL-6R and YKL-40 inhibition in BTC cell lines in vitro, expression of IL6(R) in BTC patient tumors.

Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.

PDF file - 390K, Supplementary Figure 1. Histopathology of early lesions in DEN-injected Hmgn1+/+ and Hmgn1 tm1/tm1 at 25 weeks of age. Supplementary Table S1. Primers used in the study. Supplementary Table S2. List of differentially expressed genes (FC>2, p?0.01) in livers of Hmgn1+/+ and Hmgn1tm1/tm1 mice in groups A, B, and C. Supplementary Table S3. GO analysis of the differentially expressed genes

Supplementary Figure 2: Correlation of circulating biomarkers with CRP.

Supplementary Discussion from Regulation of α-Fetoprotein by Nuclear Factor-κB Protects Hepatocytes from Tumor Necrosis Factor-α Cytotoxicity during Fetal Liver Development and Hepatic Oncogenesis

Supplementary Tables 1-6, Figures 1-4 from Central Role of c-Myc during Malignant Conversion in Human Hepatocarcinogenesis

Supplementary Figures 1-3, Tables 1-3, Methods from Definition of Ubiquitination Modulator COP1 as a Novel Therapeutic Target in Human Hepatocellular Carcinoma

Supplementary Figure Legends 1-4 from Central Role of c-Myc during Malignant Conversion in Human Hepatocarcinogenesis

Supplementary Figure 5: Histopathology of the HuCCT-1 xenograft tumors from control group and groups treated with gemcitabine and gemcitabine in combination with tocilizumab.

PDF file - 279KB, Co-localization of CLPTM1L mutants (CLPTM1L-delta-Cterm and CLPTM1L-delta-Loop) with an ER marker, indicating a similar localization for WT and mutant CLPTM1L proteins to the endoplasmic reticulum.

Supplementary Data, Supplementary Figure Legends, Supplementary Tables

PDF file - 76KB, CLPTM1L protein levels are enhanced in pancreatic adenocarcinomas.

Background & aims: Several types of human stem cells from embryonic (ESCs) and induced pluripotent (iPSCs) to adult tissue-specific stem cells are commonly used to generate 3D liver organoids for modeling tissue physiology and disease. We have recently established a protocol for direct conversion of primary human hepatocytes (hPHs) from healthy donor livers into bipotent progenitor cells (hCdHs). Here we extended this culture system to generate hCdH-derived liver organoids for diverse biomedical applications. Methods: To obtain hCdHs, hPHs were cultured in reprogramming medium containing A83-01 and CHIR99021 for 7 days. Liver organoids were established from hCdHs (hCdHOs) and human liver cells (hLOs) using the same donor livers for direct comparison, as well as from hiPSCs. Organoid properties were analyzed by standard in vitro assays. Molecular changes were determined by RT-qPCR and RNA-seq. Clinical relevance was evaluated by transplantation into FRG mice, modeling of alcohol-related liver disease (ARLD), and in vitro drug-toxicity tests. Results: hCdHs were clonally expanded as organoid cultures with low variability between starting hCdH lines. Similar to the hLOs, hCdHOs stably maintained stem cell phenotype based on accepted criteria. However, hCdHOs had an advantage over hLOs in terms of EpCAM expression, efficiency of organoid generation and capacity for directed hepatic differentiation as judged by molecular profiling, albumin secretion, glycogen accumulation, and CYP450 activities. Accordingly, FRG mice transplanted with hCdHOs survived longer than mice injected with hLOs. When exposed to ethanol, hCdHOs developed stronger ARLD phenotype than hLOs as evidenced by transcriptional profiling, lipid accumulation and mitochondrial dysfunction. In drug-induced injury assays in vitro, hCdHOs showed a similar or higher sensitivity response than hPHs. Conclusion: hCdHOs provide a novel patient-specific stem cell-based platform for regenerative medicine, toxi-cology testing and modeling liver diseases.

Supplementary Figure 4: Pathway over-representation analysis of differentially expressed miRNAs across cellular compartments (parent cells, EV) and treatment groups (anti-IL6R (tocilizumab), anti-YKL40) in biliary tract cancer cells in vitro.

Upregulation of c-MYC in cancer stem cell enriched populations (S1); The doxycycline (Dox)-controlled mCherry marker expression in c-MYC inducible hepatoma cell lines (S2); The doxycycline (Dox) dose-dependent effects of c-MYC induction on CSC properties (S3); The doxycycline (Dox) dose-dependent effects of c-MYC induction on self-renewal potential (S4); c-MYC switch-on and switch-off effects on self-renewal of PLC cells (S5); Effect of p53 knockdown on self-renewal of hepatoma cell lines with doxycycline (Dox) regulated c-MYC expression (S6); p53 knockdown increases the growth rate of HepG2 cells with doxycycline (Dox) regulated c-MYC expression (S7).