Faculty Bibliography

CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxA cluster, complemented by biochemical approaches. Among the candidates, we identified Myc-associated zinc-finger protein (MAZ) as a cofactor in CTCF insulation. MAZ colocalizes with CTCF at chromatin borders and, similar to CTCF, interacts with the cohesin subunit RAD21. MAZ KO disrupts gene expression and local contacts within topologically associating domains. Similar to CTCF motif deletions, MAZ motif deletions lead to derepression of posterior Hox genes immediately after CTCF boundaries upon differentiation, giving rise to homeotic transformations in mouse. Thus, MAZ is a factor contributing to appropriate insulation, gene expression and genomic architecture during development.

The contribution of epigenetic dysregulation to metastasis remains understudied. Through a meta-analysis of gene expression datasets followed by a mini-screen, we identified Plant Homeodomain Finger protein 8 (PHF8), a histone demethylase of the Jumonji C protein family, as a previously unidentified prometastatic gene in melanoma. Loss- and gain-of-function approaches demonstrate that PHF8 promotes cell invasion without affecting proliferation in vitro and increases dissemination but not subcutaneous tumor growth in vivo, thus supporting its specific contribution to the acquisition of metastatic potential. PHF8 requires its histone demethylase activity to enhance melanoma cell invasion. Transcriptomic and epigenomic analyses revealed that PHF8 orchestrates a molecular program that directly controls the TGFβ signaling pathway and, as a consequence, melanoma invasion and metastasis. Our findings bring a mechanistic understanding of epigenetic regulation of metastatic fitness in cancer, which may pave the way for improved therapeutic interventions.

The production of noncanonical mRNA transcripts is associated with cell transformation. Driven by our previous findings on the sensitivity of T cell acute lymphoblastic leukemia (T-ALL) cells to SF3B1 inhibitors, we identified that SF3B1 inhibition blocks T-ALL growth in vivo with no notable associated toxicity. We also revealed protein stabilization of the U2 complex component SF3B1 via deubiquitination. Our studies showed that SF3B1 inhibition perturbs exon skipping, leading to nonsense-mediated decay and diminished levels of DNA damage response-related transcripts, such as the serine/threonine kinase CHEK2, and impaired DNA damage response. We also identified that SF3B1 inhibition leads to a general decrease in R-loop formation. We further demonstrate that clinically used SF3B1 inhibitors synergize with CHEK2 inhibitors and chemotherapeutic drugs to block leukemia growth. Our study provides the proof of principle for posttranslational regulation of splicing components and associated roles and therapeutic implications for the U2 complex in T cell leukemia.

Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers^1,2, the mechanisms and consequences of tRNA deregulation in tumorigenesis are poorly understood. Here we use a CRISPR-Cas9 screen to focus on genes that have been implicated in tRNA biogenesis, and identify a mechanism by which altered valine tRNA biogenesis enhances mitochondrial bioenergetics in T cell acute lymphoblastic leukaemia (T-ALL). Expression of valine aminoacyl tRNA synthetase is transcriptionally upregulated by NOTCH1, a key oncogene in T-ALL, underlining a role for oncogenic transcriptional programs in coordinating tRNA supply and demand. Limiting valine bioavailability through restriction of dietary valine intake disrupted this balance in mice, resulting in decreased leukaemic burden and increased survival in vivo. Mechanistically, valine restriction reduced translation rates of mRNAs that encode subunits of mitochondrial complex I, leading to defective assembly of complex I and impaired oxidative phosphorylation. Finally, a genome-wide CRISPR-Cas9 loss-of-function screen in differential valine conditions identified several genes, including SLC7A5 and BCL2, whose genetic ablation or pharmacological inhibition synergized with valine restriction to reduce T-ALL growth. Our findings identify tRNA deregulation as a critical adaptation in the pathogenesis of T-ALL and provide a molecular basis for the use of dietary approaches to target tRNA biogenesis in blood malignancies.

Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. "Drug-tolerant persisters" (DTPs), a sub-population of cancer cells that survive via reversible, non-genetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKIs) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single cell RNA-sequencing reveal that HER2+ breast cancer cells cycle stochastically through a "pre-DTP" state, characterized by a G0-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting show that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation.

Image-based analysis as a method for mutation detection can be advantageous in settings when tumor tissue is limited or unavailable for direct testing. Here, we utilize two distinct and complementary machine learning methods of analyzing whole slide images (WSI) for predicting mutated BRAF. In the first method, WSI of melanomas from 256 patients were used to train a deep convolutional neural network (CNN) in order to develop a fully automated model that first selects for tumor-rich areas (Area Under the Curve AUC=0.96) then predicts for mutated BRAF (AUC=0.71). Saliency mapping was performed and revealed that pixels corresponding to nuclei were the most relevant to network learning. In the second method, WSI were analyzed using a pathomics pipeline that first annotates nuclei and then quantifies nuclear features, demonstrating that mutated BRAF nuclei were significantly larger and rounder nuclei compared to BRAF WT nuclei. Lastly, we developed a model that combines clinical information, deep learning, and pathomics that improves the predictive performance for mutated BRAF to AUC=0.89. Not only does this provide additional insights on how BRAF mutations affect tumor structural characteristics, machine learning-based analysis of WSI has the potential to be integrated into higher order models for understanding tumor biology.

SHP2 inhibitors (SHP2i) alone and in various combinations are being tested in multiple tumors with over-activation of the RAS/ERK pathway. SHP2 plays critical roles in normal cell signaling; hence, SHP2is could influence the tumor microenvironment. We found that SHP2i treatment depleted alveolar and M2-like macrophages, induced tumor-intrinsic CCL5/CXCL10 secretion and promoted B and T lymphocyte infiltration in Kras- and Egfr-mutant non-small cell lung cancer (NSCLC). However, treatment also increased intratumor gMDSCs via tumor-intrinsic, NF-kB-dependent production of CXCR2 ligands. Other RAS/ERK pathway inhibitors also induced CXCR2 ligands and gMDSC influx in mice, and CXCR2 ligands were induced in tumors from patients on KRASG12C-inhibitor trials. Combined SHP2(SHP099)/CXCR1/2(SX682) inhibition depleted a specific cluster of S100a8/9high gMDSCs, generated Klrg1+ CD8+ effector T cells with a strong cytotoxic phenotype but expressing the checkpoint receptor NKG2A, and enhanced survival in Kras- and Egfr-mutant models. Our results argue for testing RAS/ERK pathway/CXCR1/2/NKG2A inhibitor combinations in NSCLC patients.

Introduction: T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive leukemia with a high incidence in children, adolescents and young adults. Although multiple therapeutic options are available, almost one fifth of patients affected by T-ALL eventually succumb to the disease, suggesting an unrecognized biological complexity that might contribute to drug resistance. To better understand the differences between T-ALL subtypes and their clinical course, we systematically analyzed cohorts of patients with different risk profiles at the genetic and epigenetic level. We recently demonstrated that differences in three-dimensional (3D) chromatin architecture can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer-promoter interactions, impacting gene expression and leading to disease. As an example, we focused in particular on the Myc family of oncogenes, revealing disease-specific patterns of enhancer-promoter interactions.
Method(s): We initially profiled a large cohort of T-ALL patients falling under different risk categories in order to identify differences in their genetic and epigenetic features. We then systematically integrated matched in situ Hi-C, RNA-seq and CTCF ChIP-seq datasets to reveal widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in patients affected by TALL.
Result(s): Using primary acute leukemia patient samples, we have, for the first time, identified recurrent TAD disruptions in leukemia involving key oncogenes (e.g. NOTCH1, c-Myc) and their targets. For example, we identified a recurrent disruption of 3D chromatin topology in the c-Myc locus at a previously uncharacterized non-coding CTCF-bound region that insulates MYC from a downstream super-enhancer. This disruption enables chromatin interactions between the c-Myc oncogene and the downstream super-enhancer leading to an increase in c-Myc expression. In parallel, while focusing patients falling into a higher risk category, namely the early T cell progenitor acute lymphoblastic leukemia (ETP-ALL), we discovered a previously uncharacterized region of high activity encompassing a novel lncRNA interacting with the proto-oncogene N-Myc.
Conclusion(s):With the current study we demonstrated an inherent difference between subtypes of T-ALL based on their epigenetic profile, which in turn influences the expression of key oncogenes. By focusing on a new methods of regulating a known family of transcription factors, we provide a new mechanism which could open interesting ways for targeted therapy of patients at different risk levels