Faculty Bibliography

The development of computational tools for the systematic prediction of metabolic vulnerabilities of cancer cells constitutes a central question in systems biology. Here, we present gmctool, a freely accessible online tool that allows us to accomplish this task in a simple, efficient and intuitive environment. gmctool exploits the concept of genetic Minimal Cut Sets (gMCSs), a theoretical approach to synthetic lethality based on genome-scale metabolic networks, including a unique database of synthetic lethals computed from Human1, the most recent metabolic reconstruction of human cells. gmctool introduces qualitative and quantitative improvements over our previously developed algorithms to predict, visualize and analyze metabolic vulnerabilities in cancer, demonstrating a superior performance than competing algorithms. A detailed illustration of gmctool is presented for multiple myeloma (MM), an incurable hematological malignancy. We provide in vitro experimental evidence for the essentiality of CTPS1 (CTPS synthase) and UAP1 (UDP-N-Acetylglucosamine Pyrophosphorylase 1) in specific MM patient subgroups.

Genomic context critically modulates regulatory function but is difficult to manipulate systematically. The murine insulin-like growth factor 2 (Igf2)/H19 locus is a paradigmatic model of enhancer selectivity, whereby CTCF occupancy at an imprinting control region directs downstream enhancers to activate either H19 or Igf2. We used synthetic regulatory genomics to repeatedly replace the native locus with 157-kb payloads, and we systematically dissected its architecture. Enhancer deletion and ectopic delivery revealed previously uncharacterized long-range regulatory dependencies at the native locus. Exchanging the H19 enhancer cluster with the Sox2 locus control region (LCR) showed that the H19 enhancers relied on their native surroundings while the Sox2 LCR functioned autonomously. Analysis of regulatory DNA actuation across cell types revealed that these enhancer clusters typify broader classes of context sensitivity genome wide. These results show that unexpected dependencies influence even well-studied loci, and our approach permits large-scale manipulation of complete loci to investigate the relationship between regulatory architecture and function.

Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases^1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe 'mammalian switching antibiotic resistance markers progressively for integration' (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.

Sox2 expression in mouse embryonic stem cells (mESCs) depends on a distal cluster of DNase I hypersensitive sites (DHSs), but their individual contributions and degree of interdependence remain a mystery. We analyzed the endogenous Sox2 locus using Big-IN to scarlessly integrate large DNA payloads incorporating deletions, rearrangements, and inversions affecting single or multiple DHSs, as well as surgical alterations to transcription factor (TF) recognition sequences. Multiple mESC clones were derived for each payload, sequence-verified, and analyzed for Sox2 expression. We found that two DHSs comprising a handful of key TF recognition sequences were each sufficient for long-range activation of Sox2 expression. By contrast, three nearby DHSs were entirely context dependent, showing no activity alone but dramatically augmenting the activity of the autonomous DHSs. Our results highlight the role of context in modulating genomic regulatory element function, and our synthetic regulatory genomics approach provides a roadmap for the dissection of other genomic loci.

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK-MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8⁺ T cells with reduced immunosuppressive regulatory T (T_reg) cells, while late MYC acquisition in slow progressors was associated with lower CD8⁺ T cell infiltration and more abundant T_reg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8⁺ T cells versus T_reg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8⁺ T/T_reg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8⁺ T cell cytotoxicity or depleting T_reg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

Effective public response to a pandemic relies upon accurate measurement of the extent and dynamics of an outbreak. Viral genome sequencing has emerged as a powerful approach to link seemingly unrelated cases, and large-scale sequencing surveillance can inform on critical epi-demiological parameters. Here, we report the analysis of 864 SARS-CoV-2 sequences from cases in the New York City metropolitan area during the COVID-19 outbreak in Spring 2020. The majority of cases had no recent travel history or known exposure, and genetically linked cases were spread throughout the region. Comparison to global viral sequences showed that early transmission was most linked to cases from Europe. Our data are consistent with numerous seeds from multiple sources and a prolonged period of unrecognized community spreading. This work highlights the complementary role of genomic surveillance in addition to traditional epidemiological indicators.

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (TXN), a major regulator of cellular redox status and, in addition, identified PRDM5 as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.

The authors would like to make a correction to their published paper [...].

Gene regulation through DNA methylation is a well described phenomenon that has a prominent role in physiological and pathological cell-states. This epigenetic modification is usually grouped in regions denominated CpG islands, which frequently co-localize with gene promoters, silencing the transcription of those genes. Recent genome-wide DNA methylation studies have challenged this paradigm, demonstrating that DNA methylation of regulatory regions outside promoters is able to influence cell-type specific gene expression programs under physiologic or pathologic conditions. Coupling genome-wide DNA methylation assays with histone mark annotation has allowed for the identification of specific epigenomic changes that affect enhancer regulatory regions, revealing an additional layer of complexity to the epigenetic regulation of gene expression. In this review, we summarize the novel evidence for the molecular and biological regulation of DNA methylation in enhancer regions and the dynamism of these changes contributing to the fine-tuning of gene expression. We also analyze the contribution of enhancer DNA methylation on the expression of relevant genes in acute myeloid leukemia and chronic myeloproliferative neoplasms. The characterization of the aberrant enhancer DNA methylation provides not only a novel pathogenic mechanism for different tumors but also highlights novel potential therapeutic targets for myeloid derived neoplasms.

In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the ZFP36L1 gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. In vitro reporter assay and 5'-azacitidine treatment confirmed the functional relevance of hyper-methylation of ZFP36L1 enhancer. Furthermore, in vitro rescue of ZFP36L1 expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of ZFP36L1 as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing ZFP36L1 as a novel candidate tumor suppressor gene.