A fundamental as yet incompletely understood feature of Notch signal transduction is a transcriptional shift from repression to activation that depends on chromatin regulation mediated by transcription factor RBP-J and associated cofactors. Incorporation of histone variants alter the functional properties of chromatin and are implicated in the regulation of gene expression. Here, we show that depletion of histone variant H2A.Z leads to upregulation of canonical Notch target genes and that the H2A.Z-chaperone TRRAP/p400/Tip60 complex physically associates with RBP-J at Notch-dependent enhancers. When targeted to RBP-J-bound enhancers, the acetyltransferase Tip60 acetylates H2A.Z and upregulates Notch target gene expression. Importantly, the Drosophila homologs of Tip60, p400 and H2A.Z modulate Notch signaling response and growth in vivo. Together, our data reveal that loading and acetylation of H2A.Z are required to assure tight control of canonical Notch activation.

The PI3K/Akt signaling pathway, Notch, and other oncogenes cooperate in the induction of aggressive cancers. Elucidating how the PI3K/Akt pathway facilitates tumorigenesis by other oncogenes may offer opportunities to develop drugs with fewer side effects than those currently available. Here, using an unbiased in vivo chemical genetic screen in Drosophila, we identified compounds that inhibit the activity of proinflammatory enzymes nitric oxide synthase (NOS) and lipoxygenase (LOX) as selective suppressors of Notch-PI3K/Akt cooperative oncogenesis. Tumor silencing of NOS and LOX signaling mirrored the antitumor effect of the hit compounds, demonstrating their participation in Notch-PI3K/Akt-induced tumorigenesis. Oncogenic PI3K/Akt signaling triggered inflammation and immunosuppression via aberrant NOS expression. Accordingly, activated Notch tumorigenesis was fueled by hampering the immune response or by NOS overexpression to mimic a protumorigenic environment. Our lead compound, the LOX inhibitor BW B70C, also selectively killed human leukemic cells by dampening the NOTCH1-PI3K/AKT-eNOS axis.

Dorsal closure is an epithelial remodeling process taking place during Drosophila embryogenesis. JNK signaling coordinates dorsal closure. We identify and characterize acal as a novel negative dorsal closure regulator. acal represents a new level of JNK regulation. The acal locus codes for a conserved, long, non-coding, nuclear RNA. Long non-coding RNAs are an abundant and diverse class of gene regulators. Mutations in acal are lethal. acal mRNA expression is dynamic and is processed into a collection of 50 to 120 bp fragments. We show that acal lies downstream of raw, a pioneer protein, helping explain part of raw functions, and interacts genetically with Polycomb. acal functions in trans regulating mRNA expression of two genes involved in JNK signaling and dorsal closure: Connector of kinase to AP1 (Cka) and anterior open (aop). Cka is a conserved scaffold protein that brings together JNK and Jun, and aop is a transcription factor. Misregulation of Cka and aop can account for dorsal closure phenotypes in acal mutants.

SUMMARYEvidence suggests that Polycomb (Pc) is present at chromatin loop anchors in Drosophila. Pc is recruited to DNA through interactions with the GAGA binding factors GAF and Pipsqueak (Psq). Using HiChIP in Drosophila cells, we find that the psq gene, which has diverse roles in development and tumorigenesis, encodes distinct isoforms with unanticipated roles in genome 3D architecture. The BR-C, ttk, and bab domain (BTB)-containing Psq isoform (PsqL) colocalizes genome-wide with known architectural proteins. Conversely, Psq lacking the BTB domain (PsqS) is consistently found at Pc loop anchors and at active enhancers, including those that respond to the hormone ecdysone. After stimulation by this hormone, chromatin 3D organization is altered to connect promoters and ecdysone-responsive enhancers bound by PsqS. Our findings link Psq variants lacking the BTB domain to Pc-bound active enhancers, thus shedding light into their molecular function in chromatin changes underlying the response to hormone stimulus.

Analysis of tumorigenesis in Drosophila reveals a tumor-suppressor role for Hedgehog signaling in the context of oncogenic Notch signaling.

Innate lymphoid cells (ILCs) are highly plastic and predominantly mucosal tissue-resident cells that contribute to both homeostasis and inflammation depending on the microenvironment. The discovery of naïve-like ILCs suggests an ILC differentiation process that is akin to naïve T cell differentiation. Delineating the mechanisms that underlie ILC differentiation in tissues is crucial for understanding ILC biology in health and disease. Here, we showed that tonsillar ILCs expressing CD45RA lacked proliferative activity, indicative of cellular quiescence. CD62L distinguished two subsets of CD45RA + ILCs. CD45RA + CD62L + ILCs (CD62L + ILCs) resembled circulating naïve ILCs because they lacked the transcriptional, metabolic, epigenetic, and cytokine production signatures of differentiated ILCs. CD45RA + CD62L − ILCs (CD62L − ILCs) were epigenetically similar to CD62L + ILCs but showed a transcriptional, metabolic, and cytokine production signature that was more akin to differentiated ILCs. CD62L + and CD62L − ILCs contained uni- and multipotent precursors of ILC1s/NK cells and ILC3s. Differentiation of CD62L + and CD62L − ILCs led to metabolic reprogramming including up-regulation of genes associated with glycolysis, which was needed for their effector functions after differentiation. CD62L − ILCs with preferential differentiation capacity toward IL-22–producing ILC3s accumulated in the inflamed mucosa of patients with inflammatory bowel disease. These data suggested distinct differentiation potential of CD62L + and CD62L − ILCs between tissue microenvironments and identified that manipulation of these cells is a possible approach to restore tissue-immune homeostasis.

Background The origin of human Innate Lymphoid Cells (ILCs) remains less well characterised compared to mice. Single-cell RNA-sequencing recently revealed a population of ILC precursors (ILCP) expressing CD62L and CD45RA, which shows diversity in frequencies among blood, tonsil and intestine. Objective We set out to determine the differentiation capacity and epigenetic landscape of blood, tonsil and intestinal ILCP. Methods Blood, tonsil and intestinal ILCP were sort-purified on the basis of CD62L and CD45RA (CD62L+/−CD45RA+/−) and cultured on OP9-DL1 stromal cells under specific cytokine conditions. Additionally, genome-wide chromatin accessibility was measured using ATAC-sequencing. Results We show that all ILCP subsets in blood and tonsil were able to differentiate to IFN-γ-producing ILC1/NK cells. Whereas all blood ILCP subsets readily differentiated to IL-13-producing ILC2, no or very few ILC2 were generated from tonsil ILCP. In contrast, tonsil ILCP subsets showed higher ability to generate IL-22-producing ILC3, compared to blood ILCP. These data indicate that ILCP in blood are poised towards ILC1/ILC2 while ILCP in tonsil favours ILC1/ILC3 differentiation. To further dissect their development, we are currently analysing global epigenetic information of tonsil ILCP, aiming to delineate their chromatin landscape. Finally, we investigated the ILCP in intestinal samples and detected accumulation of CD62L−CD45RA+ ILCP in the inflamed mucosa of patients with inflammatory bowel disease (IBD) as compared to the non-inflamed tissue. We are currently analysing the potency of those cells. Conclusion Heterogeneity in ILC differentiation in blood and tissues could further reveal novel targets for IBD treatment.

Background: High unmet need of ovarian cancer (OC) suggests the discovery of new targeted therapeutics is crucial to improve patient prognosis. Unlike artificial model systems such as cell lines, primary cancer samples recapitulate the complexity of the original microenvironment consisting of cancer cells as well as stromal and immune cells; this is especially important when evaluating IO targets and signalling pathways. Supported by our previous success predicting therapy for late stage haematological cancer patients in the EXALT-I trial using AI-supported functional single cell quantification of drug action (Kornauth et. al. 2021) we set out to systematically reveal novel targets and pathways in OC using small molecule drugs (SMDs) as tools. Single cell phenotypic screening of OC MPAs (malignant pleural effusion and ascites) was enabled by the quantification of drug effects using an end-to-end scalable deep learning driven image analysis tool chain. This custom state-of-the-art AI software is critical to enable robust primary cell screening given the diversity of cells within each sample. This revealed anaplastic lymphoma kinase (ALK), as well as structurally related targets and pathway associated proteins, as being potential novel targets in a subset of OC patient samples. There is sparse literature evidence for therapeutic utilisation of the ALK pathway in OC, and the diversity of responses indicates a further novel patient selection method. Methods: MPAs from OC patients (n = 20) were collected and the sensitivity of the cancer cells to 85 SMDs was evaluated using high content microscopy. Individual cells were segmented and classified using convolutional neural networks and drug responses were estimated from the resulting cell counts. The integration of these results with whole exome and RNA sequencing guided target and pathway prioritisation. Results: Screening for novel sensitivities using SMDs as tools uncovered inhibitors of ALK and related targets as having strong cancer cell cytotoxic effects, recapitulated in solid tumour biopsies. Transcriptomic profiling revealed pathway correlations to ALK inhibitor sensitivity, however non-annotated polypharmacological effects of each drug cannot yet be excluded. Conclusions: Quantifying SMD sensitivity in a disease relevant model system identified ALK as a promising and overlooked target in OC, providing an upstream and potentially more specific target to the recently suggested MEK, PI3K and STAT3 (Papp et. al. 2018, Izar et al. 2020). While further work to confirm the target is required, this study supports a notion of patient-centric drug development using disease relevant models and deep learning. Our work introduces a novel patient-centric tool to advance understanding of the OC target landscape and provides a resource for the development of novel therapeutic approaches. Citation Format: Irene Gutierrez-Perez, Joost Van Ham, Valentin Aranha, Rin Okumura, Elisabeth Waltenberger, Isabella Alt, Claudia Baumgaertler, Maja Stulic, Edgar Petru, Christoph Minichsdorfer, Lukas Hefler, Judith Lafleur, Nikolaus Krall, Thorsten Füreder, Gregory Ian Vladimer, Robert Sehlke, Bojan Vilagos. Deep learning supported high content analysis of primary patient samples identifies ALK inhibition as a novel mechanism of action in a subset of ovarian cancers [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 1893.