Faculty Bibliography

Rewiring of metabolic pathways is a hallmark of tumorigenesis as cancer cells acquire novel nutrient dependencies to support oncogenic growth. A major genetic subtype of lung adenocarcinoma with KEAP1/NRF2 mutations, which activates the endogenous oxidative stress response, undergoes significant metabolic rewiring to support enhanced antioxidant production. We demonstrate that cancers with high antioxidant capacity exhibit a general dependency on exogenous non-essential amino acids (NEAAs) that is driven by the Nrf2-dependent secretion of glutamate through system x_c^- (XCT), which limits intracellular glutamate pools that are required for NEAA synthesis. This dependency can be therapeutically targeted by dietary restriction or enzymatic depletion of individual NEAAs. Importantly, limiting endogenous glutamate levels by glutaminase inhibition can sensitize tumors without alterations in the Keap1/Nrf2 pathway to dietary restriction of NEAAs. Our findings identify a metabolic strategy to therapeutically target cancers with genetic or pharmacologic activation of the Nrf2 antioxidant response pathway by restricting exogenous sources of NEAAs.

Staphylococcus aureus and its toxins have been linked to disease progression and mortality in advanced stages of cutaneous T-cell lymphoma (CTCL). CD8⁺ T cells play a crucial role in anti-cancer responses and high CD8⁺ T cell numbers in tumor lesions are associated with a favorable prognosis in CTCL. Here, we show that CD8⁺ T cells from both healthy donors and Sézary syndrome patients are highly susceptible to cell death induced by Staphylococcal alpha-toxin, whereas malignant T cells are not. Importantly, alpha-toxin almost completely blocks cytotoxic killing of CTCL tumor cells by peptide-specific CD8⁺ T cells, leading to their escape from induced cell death and continued proliferation. These findings suggest that alpha-toxin may favor the persistence of malignant CTCL cells in vivo by inhibiting CD8⁺ T cell cytotoxicity. Thus, we propose a novel mechanism by which colonization with Staphylococcus aureus may contribute to cancer immune evasion and disease progression in CTCL.

Abstract: T cell lymphomas comprise a distinct class of non-Hodgkin's lymphomas, which include mature T and natural killer (NK) cell neoplasms. While each malignancy within this group is characterized by unique clinicopathologic features, dysregulation in the Janus tyrosine family of kinases/Signal transducer and activator of transcription (JAK/STAT) signaling pathway, specifically aberrant STAT3 activation, is a common feature among these lymphomas. The mechanisms driving dysregulation vary among T cell lymphoma subtypes and include activating mutations in upstream kinases or STAT3 itself, formation of oncogenic kinases which drive STAT3 activation, loss of negative regulators of STAT3, and the induction of a pro-tumorigenic inflammatory microenvironment. Constitutive STAT3 activation has been associated with the expression of targets able to increase pro-survival signals and provide malignant fitness. Patients with dysregulated STAT3 signaling tend to have inferior clinical outcomes, which underscores the importance of STAT3 signaling in malignant progression. Targeting of STAT3 has shown promising results in pre-clinical studies in T cell lymphoma lines, ex-vivo primary malignant patient cells, and in mouse models of disease. However, targeting this pleotropic pathway in patients has proven difficult. Here we review the recent contributions to our understanding of the role of STAT3 in T cell lymphomagenesis, mechanisms driving STAT3 activation in T cell lymphomas, and current efforts at targeting STAT3 signaling in T cell malignancies.

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

Pathogenic Th17 cells play important roles in many autoimmune and inflammatory diseases. Their function depends on T cell receptor (TCR) signaling and cytokines that activate signal transducer and activator of transcription 3 (STAT3). TCR engagement activates stromal interaction molecule 1 (STIM1) and calcium (Ca²⁺) influx through Ca²⁺-release-activated Ca²⁺ (CRAC) channels. Here, we show that abolishing STIM1 and Ca²⁺ influx in T cells expressing a hyperactive form of STAT3 (STAT3C) attenuates pathogenic Th17 cell function and inflammation associated with STAT3C expression. Deletion of STIM1 in pathogenic Th17 cells reduces the expression of genes required for mitochondrial function and oxidative phosphorylation (OXPHOS) but enhances reactive oxygen species (ROS) production. STIM1 deletion or inhibition of OXPHOS is associated with a non-pathogenic Th17 gene expression signature and impaired pathogenic Th17 cell function. Our findings establish Ca²⁺ influx as a critical regulator of mitochondrial function and oxidative stress in pathogenic Th17 cell-mediated multiorgan inflammation.

Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.

Thymocyte selection associated high mobility group box 1 (Tox1), a transcription factor that is essential to establish the CD4+ lineage, is overexpressed in malignant cells from patients with CTCL. Knockdown of Tox1 leads to decreased malignant cell viability, while treatment with HDAC inhibitors results in normalization of Tox1expression. Another gene which is consistently overexpressed in patient samples is signal transducers and activators of transcription3 (Stat3), a transcription factor essential for the differentiation of Th17 and follicular helper T cells. Treating CTCL cell lines with a STAT3 inhibitor results in decreased cell number and increased apoptosis, highlighting the importance of this pathway for malignant cell survival. Importantly, the Koralov lab has developed a mouse model which constitutively expresses a hyperactive STAT3 allele, STAT3C, that recapitulates several key features of MF. To examine the contribution of TOX1overexpression to CTCL pathogenesis, we have introduced Tox1 cDNA downstream of a floxed stop cassette into the ubiquitously expressed Rosa26 locus of C57Bl/6J embryonic stem (ES) cells. We have screened the ES clones to validate the presence of the correctly targeted allele, and we have treated ES clones with TAT-Cre to delete the floxed stop cassette and drive subsequent expression of Tox1 cDNA. We have now generated R26Tox1^stopflmice using tetraploid complementation to generate 100% ES cell derived animals. These animals will be crossed to CD4Cre and CD4Cre STAT3C^stopflstrains, thus enabling us to study the contribution of Tox1 overexpression to T cell lymphomagenesis and allowing us to examine synergy between Tox1 overexpression and hyperactive STAT3 signaling in CTCL pathogenesis. We hope that these mice will pave the way to a better understanding of this enigmatic malignancy and allow us to develop a relevant small animal model of this disease.

The immunoglobulin heavy chain (Igh) locus features a dynamic chromatin landscape to promote class switch recombination (CSR), yet the mechanisms that regulate this landscape remain poorly understood. CHD4, a component of the chromatin remodeling NuRD complex, directly binds H3K9me3, an epigenetic mark present at the Igh locus during CSR. We find that CHD4 is essential for early B cell development but is dispensable for the homeostatic maintenance of mature, naive B cells. However, loss of CHD4 in mature B cells impairs CSR because of suboptimal targeting of AID to the Igh locus. Additionally, we find that CHD4 represses p53 expression to promote B cell proliferation. This work reveals distinct roles for CHD4 in B cell development and CSR and links the H3K9me3 epigenetic mark with AID recruitment to the Igh locus.