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Supplemental Table 1 shows RNA-seq signature genes. Supplementary Table 2 shows mass spectrometry profiling data for AD80 in HUH7. Supplementary Table 3 shows in vitro profiling data on compounds.

Supplementary Data Figures 1 shows PK data on AD80. Supplementary Data Figure 2 shows PCA analysis. Supplementary Data Figure 3 shows Gene Set Enrichment of HUH7 cells in response to AD80 and sorafenib. Supplementary Data Figure 4 shows western blot data. Supplementary Data Figure 5 shows representative purification gels of p38 isoforms used in the study. Supplementary Data Figure 6 shows a p38 sequence alignment. Supplementary Data Figure 7 shows rescue data.

Supplemental Synthetic Methods

The enhancer of zeste homolog 2 (EZH2) is the main enzymatic subunit of the PRC2 complex, which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to promote transcriptional silencing. EZH2 is overexpressed in multiple types of cancer including triple-negative breast cancer (TNBC), and high expression levels correlate with poor prognosis. Several EZH2 inhibitors, which inhibit the methyltransferase activity of EZH2, have shown promise in treating sarcoma and follicular lymphoma in clinics. However, EZH2 inhibitors are ineffective at blocking proliferation of TNBC cells, even though they effectively reduce the H3K27me3 mark. Using a hydrophobic tagging approach, we generated MS1943, a first-in-class EZH2 selective degrader that effectively reduces EZH2 levels in cells. Importantly, MS1943 has a profound cytotoxic effect in multiple TNBC cells, while sparing normal cells, and is efficacious in vivo, suggesting that pharmacologic degradation of EZH2 can be advantageous for treating the cancers that are dependent on EZH2.

ObjectiveThe diversity of the tumour microenvironment (TME) of intrahepatic cholangiocarcinoma (iCCA) has not been comprehensively assessed. We aimed to generate a novel molecular iCCA classifier that incorporates elements of the stroma, tumour and immune microenvironment (‘STIM’ classification).DesignWe applied virtual deconvolution to transcriptomic data from ~900 iCCAs, enabling us to devise a novel classification by selecting for the most relevant TME components. Murine models were generated through hydrodynamic tail vein injection and compared with the human disease.ResultsiCCA is composed of five robust STIM classes encompassing both inflamed (35%) and non-inflamed profiles (65%). The inflamed classes, namedimmune classical(~10%) andinflammatory stroma(~25%), differ in oncogenic pathways and extent of desmoplasia, with theinflammatory stromashowing T cell exhaustion, abundant stroma andKRASmutations (pdesert-like class(~20%) harbours the lowest immune infiltration with abundant regulatory T cells (phepatic stem-likeclass (~35%) is enriched in ‘M2-like’ macrophages, mutations inIDH1/2andBAP1,andFGFR2fusions. The remaining class (tumour classical: ~10%) is defined by cell cycle pathways and poor prognosis. Comparative analysis unveils high similarity between aKRAS/p19murine model and theinflammatory stromaclass (p=0.02). The KRAS-SOS inhibitor, BI3406, sensitises aKRAS-mutant iCCA murine model to anti-PD1 therapy.ConclusionsWe describe a comprehensive TME-based stratification of iCCA. Cross-species analysis establishes murine models that align closely to human iCCA for the preclinical testing of combination strategies.

Purpose: We recently developed a chemical strategy for the diversification and disease-specific improvement of tool and FDA-approved kinase inhibitors, and sought to use this strategy for genotype-specific drug discovery in hepatocellular carcinoma (HCC). Experimental Procedures: Primary and murine-derived HCC organoids were used for drug screening. Expansive multiomic (phosphoproteomics, RNAseq, and ChIPseq) and target engagement analyses were used in combination with in vitro rescue and in vivo efficacy experiments. Data Summary: Currently there are few personalized therapeutic approaches for advanced HCC, which is a leading cause of cancer-related deaths worldwide. b-catenin (CTNNB1)-mutated HCC represents 30% of the disease and is a subset of Wnt-driven tumors particularly unresponsive to current therapies. Using focused chemical libraries derived from clinical multi-kinase inhibitor scaffolds, we screened HCC organoids to identify a CTNNB1-mutant selective small molecule that we dub WNTinib. WNTinib retained exquisite selectivity in human and murine models expressing oncogenic b-catenin, including patient samples. Our multidisciplinary approach revealed that WNTinib inhibits KIT/MAPK signaling at multiple nodes. Moreover, we demonstrate that lack of direct engagement on BRAF and p38a/b kinases differentiates WNTinib from existing clinical kinase inhibitors, is necessary to avoid compensatory feedback signaling, and thereby provides a durable and selective transcriptional block of mutant b-catenin and Wnt targets through re-activation of cytoplasmic enhancer of zest homolog 2 (EZH2). Finally, we demonstrate that the unique mechanism of WNTinib provides superior activity compared to clinical therapies in vivo, including in drug-resistant models of mutant b-catenin HCC. Conclusion: Our studies identify a novel inhibitor of the Wnt pathway and uncover a previously unknown mechanism to harness the KIT/MAPK/EZH2 pathway to potently and selectively antagonize CTNNB1-mutant HCC with an unprecedented wide therapeutic index. Our study also provides the rationale to explore WNTinib in proof-of-concept trials with enriched CTNNB1- mutant advanced HCC patients. Citation Format: Alexander Rialdi, Mary Duffy, Alex Scopton, Amaia Lujambio, Arvin Dar, Ernesto Guccione. A novel kinase inhibitor for B-catenin mutant hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PR05.

Background and Aims: Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative platin-based regimens, commonly cisplatin. Here, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in HB cell lines, organoids and murine models. Methods: Paired tumor and adjacent tissues from 31 resected HBs and a validation set of 50 HBs were analyzed at the transcriptomic, genomic and epigenomic level using RNAseq, SNP and methylation arrays, respectively. The main targetable driver in HB was identified by gene co-expression network analysis (GCN) and its overexpression was confirmed by qRT-PCR. The anti-tumor effect of driver inhibition with molecular therapies alone or in combination with cisplatin was assessed in cell lines, patient-derived HB organoids and in a HB xenograft murine model. Results: IGF2 overexpression (FC> 4 vs adjacent tissue) was identified as the top targetable HB driver (71%, 22/31). IGF2high tumors displayed progenitor cell features and were significantly enriched in molecular classes with aggressive phenotypes and CTNNB1 mutations, while IGF2low tumors were enriched in inflammatory and TGF-β signaling. IGF2high tumors correlated with shorter recurrence-free survival after resection (median 34 months vs not reached for IGF2low; p = 0.02). IGF2 overexpression correlated in most cases (86%) with fetal promoter hypomethylation (50%), 11p15.5 loss of heterozygosity (LOH, 57%) or overexpression of miR483 (55%). Xentuzumab (anti-IGF1/2 mAb) alone or combined with cisplatin reduced proliferation and clonogenic capacity in IGF2high cell lines. The combination of xentuzumab and cisplatin exhibited synergistic effects in terms of cell viability in organoids derived from IGF2high human HBs. In mice, this combination induced a significant decrease in the viable tumor volume and extended survival compared to cisplatin alone (n = 13-14 per arm, p = 0.04). Conclusion: IGF2 is an actionable driver in HB and its overexpression was associated with fetal promoter hypomethylation, LOH or miR483 overexpression. The combination of a mAb against IGF1/2 (xentuzumab) with cisplatin led to remarkable anti-tumoral effects in pre-clinical models, providing the rationale for exploring this regimen in IGF2high HB patients. Citation Format: Jordi Abril-Fornaguera, Laura Torrens, Juan Carrillo-Reixach, Alexander Rialdi, Ugne Balaseviciute, Carla Montironi, Philipp Haber, Álvaro Del Río-Álvarez, Montserrat Domingo-Sàbat, Laura Royo, Nicholas Akers, Catherine E. Willoughby, Judit Peix, Miguel Torres-Martin, Marc Puigvehi, Roser Pinyol, Stefano Cairo, Margaret Childs, Rudolf Maibach, Rita Alaggio, Piotr Czauderna, Bruce Morland, Bojan Losic, Vincenzo Mazzaferro, Ernesto Guccione, Daniela Sia, Carolina Armengol, Josep M. Llovet. Identification of IGF2 as genomic driver and actionable therapeutic target in hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4005.

Cancer heterogeneity and evolution are not fully understood. Here, we show that mitochondrial DNA of the normal liver shapes tumor progression, histology, and immune environment prior to the acquisition of oncogenic mutation. Using conplastic mice, we show that mtDNA dictates the expression of the mitochondrial unfolded protein response (UPRmt) in the normal liver. Activation of oncogenic mutations in UPRmt-positive liver increases tumor incidence and histological heterogeneity. Further, in a subset of UPRmt-positive mice, invasive liver cancers develop. RNA sequencing (RNA-seq) analysis of the normal liver reveals that, in this subset, the PAPP-A/DDR2/SNAIL axis of invasion pre-exists along with elevated collagen. Since PAPP-A promotes immune evasion, we analyzed the immune signature and found that their livers are immunosuppressed. Further, the PAPP-A signature identifies the immune exhausted subset of hepatocellular carcinoma (HCC) in humans. Our data suggest that mtDNA of normal liver shapes the entire liver cancer portrait upon acquisition of oncogenic mutations. This work was supported by an RO1 AG059635 award from the NIH to D.G. Sí

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

The approved kinase inhibitors for hepatocellular carcinoma (HCC) are not matched to specific mutations within tumors. This has presented a daunting challenge; without a clear target or mechanism, no straightforward path has existed to guide the development of improved therapies for HCC. Here, we combine phenotypic screens with a class of conformation-specific kinase inhibitors termed type II to identify a multikinase inhibitor, AD80, with antitumoral activity across a variety of HCC preclinical models, including mouse xenografts. Mass spectrometry profiling found a number of kinases as putative targets for AD80, including several receptor and cytoplasmic protein kinases. Among these, we found p38 gamma and delta as direct targets of AD80. Notably, a closely related analog of AD80 lacking p38δ/γ activity, but retaining several other off-target kinases, lost significant activity in several HCC models. Moreover, forced and sustained MKK6 → p38→ATF2 signaling led to a significant reduction of AD80 activity within HCC cell lines. Together with HCC survival data in The Cancer Genome Atlas and RNA-seq analysis, we suggest p38 delta and gamma as therapeutic targets in HCC and an “AD80 inhibition signature” as identifying those patients with best clinical outcomes.

Inducing graft acceptance without chronic immunosuppression remains an elusive goal in organ transplantation. Using an experimental transplantation mouse model, we demonstrate that local macrophage activation through dectin-1 and toll-like receptor 4 (TLR4) drives trained immunity-associated cytokine production during allograft rejection. We conducted nanoimmunotherapeutic studies and found that a short-term mTOR-specific high-density lipoprotein (HDL) nanobiologic treatment (mTORi-HDL) averted macrophage aerobic glycolysis and the epigenetic modifications underlying inflammatory cytokine production. The resulting regulatory macrophages prevented alloreactive CD8+ T cell-mediated immunity and promoted tolerogenic CD4+ regulatory T (Treg) cell expansion. To enhance therapeutic efficacy, we complemented the mTORi-HDL treatment with a CD40-TRAF6-specific nanobiologic (TRAF6i-HDL) that inhibits co-stimulation. This synergistic nanoimmunotherapy resulted in indefinite allograft survival. Together, we show that HDL-based nanoimmunotherapy can be employed to control macrophage function in vivo. Our strategy, focused on preventing inflammatory innate immune responses, provides a framework for developing targeted therapies that promote immunological tolerance. Funding for this research was provided by: National Institutes of Health (R01 EB009638, R01 HL144072, P01 HL131478, EB009638, R01 HL118440, R01 HL125703, R01 AI139623) Netherlands Organisation for Scientific Research (ZonMW Veni 016156059, ZonMW Vidi 91713324, ZonMW Vici 91818622) Comunidad de Madrid (B2017/BMD-3731) American Heart Association (16SDG27250090) NIH Program of Excellence in Nanotechnology (HHSN368201000045C, K25 EB016673, P30 CA008748) Harold S. Geneen Charitable Trust Spanish Ministry of Science (SAF2016-80031-R) ERC Consolidator (310372) Sí

Green housing reduces energy costs and may mitigate indoor allergens and pollutants, improving asthma morbidity. High asthma burden is seen in low-income neighborhoods. Past studies show improvements in respiratory symptoms when living in green homes.Develop partnership with Blue Sea Development Company to determine impacts of living in Melrose Commons V (MCV), a Leadership in Energy and Environmental Design (LEED) Platinum-certified affordable housing complex, on asthma in the South Bronx.Participants completed a home-based respiratory health questionnaire before moving into MCV. Follow-up occurred at 6, 12, and 18 months post-move. A home-based educational module was delivered on indoor environmental interventions to avoid asthma triggers. A pretest was given before the module and a posttest was given 9 months later, including an evaluation of behavioral practice changes.Outcomes included decreases in continuous daily respiratory symptoms (p.001), asthma symptoms disrupting sleep in the past month (p = .028), and urgent visits to a healthcare professional for asthma in the past 3 months (p = .038). Clinically relevant outcomes included fewer days with asthma symptoms; asthma episodes; days of work, school, or daycare missed; and emergency department visits. Education changes from pretest to posttest included increased knowledge about dust mites, roaches, mold, and chemical irritants (p = .007). Common behavioral changes included using hypoallergenic mattress covers, using green cleaning products, and eliminating bedroom carpets.Findings support the beneficial effect of LEED Platinum-certified buildings on respiratory health. Trends may be clinically and economically relevant. Advocacy efforts should promote the expansion of green housing and emphasize the development of healthy communities.

How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes read-through transcription for hundreds of kilobases. In these read-through regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Read-through transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.

The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.

The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3' end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery.

DNA methylation is a potential mechanism linking indoor air pollution to adverse health effects. Fetal and early-life environmental exposures have been associated with altered DNA methylation and play a critical role in progress of diseases in adulthood. We investigated whether exposure to indoor air pollution from solid fuels at different lifetime periods was associated with global DNA methylation and methylation at the IFG2/H19 imprinting control region (ICR) in a population-based sample of non-smoking women from Warsaw, Poland. Global methylation and IFG2/H19 ICR methylation were assessed in peripheral blood DNA from 42 non-smoking women with Luminometric Methylation Assay (LUMA) and quantitative pyrosequencing, respectively. Linear regression models were applied to estimate associations between indoor air pollution and DNA methylation in the blood. Compared to women without exposure, the levels of LUMA methylation for women who had ever exposed to both coal and wood were reduced 6.70% (95% CI: -13.36, -0.04). Using both coal and wood before age 20 was associated with 6.95% decreased LUMA methylation (95% CI: -13.79, -0.11). Further, the negative correlations were more significant with exposure to solid fuels for cooking before age 20. There were no clear associations between indoor solid fuels exposure before age 20 and through the lifetime and IFG2/H19 ICR methylation. Our study of non-smoking women supports the hypothesis that exposure to indoor air pollution from solid fuels, even early-life exposure, has the capacity to modify DNA methylation that can be detected in peripheral blood. © 2014 Elsevier Inc.

Emerging evidence indicates that maternal medical risk during pregnancy, such as gestational diabetes mellitus (GDM), preeclampsia, and obesity, predisposes the offspring to suboptimal development. However, the underlying biological/epigenetic mechanism in utero is still unknown. The current pilot study (N = 50) compared the levels of global methylation in the placenta and umbilical cord blood among women with and without each risk condition (GDM, preeclampsia, and obesity) and explored whether the levels of global methylation were associated with fetal/infant growth. Results show that global methylation levels in the placenta were lower in patients with gestational diabetes (P = .003) and preeclampsia (P = .05) but higher with obesity (P = .01). Suggestive negative associations were found between global methylation level in the placenta and infant body length and head circumference. While preliminary, it is possible that the placenta tissue, but not umbilical cord blood, may be epigenetically programmed by maternal GDM, preeclampsia, and obesity to carry out its own specific functions that influence fetal growth.