mRNA Display Identifies Potent, Paralog-Selective Peptidic Ligands for ARID1B.

The ARID1A and ARID1B subunits are mutually exclusive components of the BAF variant of SWI/SNF chromatin remodeling complexes. Loss of function mutations in ARID1A are frequently observed in various cancers, resulting in a dependency on the paralog ARID1B for cancer cell proliferation. However, ARID1B has never been targeted directly, and the high degree of sequence similarity to ARID1A poses a challenge for the development of selective binders. In this study, we used mRNA display to identify peptidic ligands that bind with nanomolar affinities to ARID1B and showed high selectivity over ARID1A. Using orthogonal biochemical, biophysical, and chemical biology tools, we demonstrate that the peptides engage two different binding pockets, one of which directly involves an ARID1B-exclusive cysteine that could allow covalent targeting by small molecules. Our findings impart the first evidence of the ligandability of ARID1B, provide valuable tools for drug discovery, and suggest opportunities for the development of selective molecules to exploit the synthetic lethal relationship between ARID1A and ARID1B in cancer.

Challenges for the Discovery of Non-Covalent WRN Helicase Inhibitors.

The Werner Syndrome RecQ helicase (WRN) is a synthetic lethal target of interest for the treatment of cancers with microsatellite instability (MSI). Different hit finding approaches were initially tested. The identification of WRN inhibitors proved challenging due to a high propensity for artefacts via protein interference, i. e., hits inhibiting WRN enzymatic activities through multiple, unspecific mechanisms. Previously published WRN Helicase inhibitors (ML216, NSC19630 or NSC617145) were characterized in an extensive set of biochemical and biophysical assays and could be ruled out as specific WRN helicase probes. More innovative screening strategies need to be developed for successful drug discovery of non-covalent WRN helicase inhibitors.

Protein destabilization underlies pathogenic missense mutations in ARID1B.

ARID1B is a SWI/SNF subunit frequently mutated in human Coffin-Siris syndrome (CSS) and it is necessary for proliferation of ARID1A mutant cancers. While most CSS ARID1B aberrations introduce frameshifts or stop codons, the functional consequence of missense mutations found in ARID1B is unclear. We here perform saturated mutagenesis screens on ARID1B and demonstrate that protein destabilization is the main mechanism associated with pathogenic missense mutations in patients with Coffin-Siris Syndrome.

Systematic dissection of transcriptional regulatory networks by genome-scale and single-cell CRISPR screens.

Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.

PAX8 and MECOM are interaction partners driving ovarian cancer.

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. These include MDS1-EVI1 (also called PRDM3) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulates a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, patient-derived xenografts (PDXs) and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in ovarian cancers and we show that the MECOM locus and PAX8 sustain in vivo tumor growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer.

Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms.

Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGFß pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis.

Dissecting Effects of Anti-cancer Drugs and Cancer-Associated Fibroblasts by On-Chip Reconstitution of Immunocompetent Tumor Microenvironments.

A major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2+ breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (>50 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) antagonized the effects of trastuzumab. These observations constitute a proof of concept that tumors-on-chip are powerful platforms to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level drug responses, and to dissect the roles of stromal components.