Faculty Bibliography

Splicing alterations are common in disease, such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T cell acute lymphoblastic leukemia (T-ALL), that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease affecting proteasomal subunits, cell cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL.

RNA splicing is an essential process that governs many aspects of cellular proliferation, survival, and differentiation. Considering the importance of RNA splicing in gene regulation, alterations in this pathway have been implicated in many human cancers. Large-scale genomic studies have uncovered a spectrum of splicing machinery mutations that contribute to tumorigenesis. Moreover, cancer cells are capable of hijacking the expression of RNA-binding proteins (RBPs), leading to dysfunctional gene splicing and tumor-specific dependencies. Advances in next-generation RNA sequencing have revealed tumor-specific isoforms associated with these alterations, including the presence of neoantigens, which serve as potential immunotherapeutic targets. In this review, we discuss the various mechanisms by which cancer cells exploit RNA splicing to promote tumor growth and the current therapeutic landscape for splicing-based therapies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intestinal crypts have great capacity for repair and regeneration after intestinal stem cell (ISC) injury. Here, we define the cellular remodeling process resulting from ISC niche interruption by transient Notch pathway inhibition in adult mice. Although ISCs were retained, lineage tracing demonstrated a marked reduction in ISC function after Notch disruption. Surprisingly, Notch ligand-expressing Paneth cells were rapidly lost by apoptotic cell death. The ISC-Paneth cell changes were followed by a regenerative response, characterized by expansion of cells expressing Notch ligands Dll1 and Dll4, enhanced Notch signaling, and a proliferative surge. Lineage tracing and organoid studies showed that Dll1-expressing cells were activated to function as multipotential progenitors, generating both absorptive and secretory cells and replenishing the vacant Paneth cell pool. Our analysis uncovered a dynamic, multicellular remodeling response to acute Notch inhibition to repair the niche and restore homeostasis. Notably, this crypt regenerative response did not require ISC loss.

Single-cell sequencing approaches offer exploration of tissue architecture at unprecedented resolution. These tools are especially powerful when deconvoluting highly specialized microenvironments, such as stem cell (SC) niches. Here, we review single-cell studies that map the cellular and transcriptional makeup of stem and progenitor niches and discuss how these high-resolution analyses fundamentally advance our understanding of how niche factors shape SC biology and activity. In-depth characterization of the blueprint of SC-niche crosstalk, as well as understanding how it becomes dysregulated, will undoubtedly inform the development of more efficient therapies for malignancies and other pathologies.

A subset of B cell acute lymphoblastic leukemia (B-ALL) patients will relapse and succumb to therapy-resistant disease. The bone marrow microenvironment may support B-ALL progression and treatment evasion. Utilizing single-cell approaches, we demonstrate B-ALL bone marrow immune microenvironment remodeling upon disease initiation and subsequent re-emergence during conventional chemotherapy. We uncover a role for non-classical monocytes in B-ALL survival, and demonstrate monocyte abundance at B-ALL diagnosis is predictive of pediatric and adult B-ALL patient survival. We show that human B-ALL blasts alter a vascularized microenvironment promoting monocytic differentiation, while depleting leukemia-associated monocytes in B-ALL animal models prolongs disease remission in vivo. Our profiling of the B-ALL immune microenvironment identifies extrinsic regulators of B-ALL survival supporting new immune-based therapeutic approaches for high-risk B-ALL treatment.

Timed degradation of the cyclin-dependent kinase inhibitor p27^Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27^Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).

Intestinal mononuclear phagocytes (MPs) are composed of heterogeneous dendritic cell (DC) and macrophage subsets necessary for the initiation of immune response and control of inflammation. Although MPs in the normal intestine have been extensively studied, the heterogeneity and function of inflammatory MPs remain poorly defined. We performed phenotypical, transcriptional, and functional analyses of inflammatory MPs in infectious Salmonella colitis and identified CX3CR1⁺ MPs as the most prevalent inflammatory cell type. CX3CR1⁺ MPs were further divided into three distinct populations, namely, Nos2⁺CX3CR1^lo, Ccr7⁺CX3CR1^int (lymph migratory), and Cxcl13⁺CX3CR1^hi (mucosa resident), all of which were transcriptionally aligned with macrophages and derived from monocytes. In follow-up experiments in vivo, intestinal CX3CR1⁺ macrophages were superior to conventional DC1 (cDC1) and cDC2 in inducing Salmonella-specific mucosal IgA. We next examined spatial organization of the immune response induced by CX3CR1⁺ macrophage subsets and identified mucosa-resident Cxcl13⁺CX3CR1^hi macrophages as the antigen-presenting cells responsible for recruitment and activation of CD4⁺ T and B cells to the sites of Salmonella invasion, followed by tertiary lymphoid structure formation and the local pathogen-specific IgA response. Using mice we developed with a floxed Ccr7 allele, we showed that this local IgA response developed independently of migration of the Ccr7⁺CX3CR1^int population to the mesenteric lymph nodes and contributed to the total mucosal IgA response to infection. The differential activity of intestinal macrophage subsets in promoting mucosal IgA responses should be considered in the development of vaccines to prevent Salmonella infection and in the design of anti-inflammatory therapies aimed at modulating macrophage function in inflammatory bowel disease.

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 human disease. Here we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) by using primary human leukemia specimens and examine the dynamic responses of this architecture to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP-seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD 'fusion' event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small-molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia.