Faculty Bibliography

Loss-of-function mutations in KEAP1 frequently occur in lung cancer and are associated with resistance to standard of care treatment, highlighting the need for the development of targeted therapies. We have previously shown that KEAP1 mutant tumors have increased glutamine consumption to support the metabolic rewiring associated with NRF2 activation. Here, using patient-derived xenograft models and antigenic orthotopic lung cancer models, we show that the novel glutamine antagonist DRP-104 impairs the growth of KEAP1 mutant tumors. We find that DRP-104 suppresses KEAP1 mutant tumor growth by inhibiting glutamine-dependent nucleotide synthesis and promoting anti-tumor CD4 and CD8 T cell responses. Using multimodal single-cell sequencing and ex vivo functional assays, we discover that DRP-104 reverses T cell exhaustion and enhances the function of CD4 and CD8 T cells culminating in an improved response to anti-PD1 therapy. Our pre-clinical findings provide compelling evidence that DRP-104, currently in phase 1 clinical trials, offers a promising therapeutic approach for treating patients with KEAP1 mutant lung cancer. Furthermore, we demonstrate that by combining DRP-104 with checkpoint inhibition, we can achieve suppression of tumor intrinsic metabolism and augmentation of anti-tumor T cell responses.

During postnatal development, both the maturing microbiome and the host immune system are susceptible to environmental perturbations such as antibiotic use. The impact of timing in which antibiotic exposure occurs was investigated by treating mice from days 5-9 with amoxicillin or azithromycin, two of the most commonly prescribed medications in children. Both early-life antibiotic regimens disrupted Peyer's patch development and immune cell abundance, with a sustained decrease in germinal center formation and diminished intestinal immunoglobulin A (IgA) production. These effects were less pronounced in adult mice. Through comparative analysis of microbial taxa, Bifidobacterium longum abundance was found to be associated with germinal center frequency. When re-introduced to antibiotic-exposed mice, B. longum partially rescued the immunological deficits. These findings suggest that early-life antibiotic use affects the development of intestinal IgA-producing B cell functions and that probiotic strains could be used to restore normal development after antibiotic exposure.

UNLABELLED:Adaptive immune responses are induced by vaccination and infection, yet little is known about how CD4+ T cell memory differs between these two contexts. Notable differences in humoral and cellular immune responses to primary mRNA vaccination were observed and associated with prior COVID-19 history, including in the establishment and recall of Spike-specific CD4+ T cells. It was unclear whether CD4+ T cell memory established by infection or mRNA vaccination as the first exposure to Spike was qualitatively similar. To assess whether the mechanism of initial memory T cell priming affected subsequent responses to Spike protein, 14 people who were receiving a third mRNA vaccination, referenced here as the booster, were stratified based on whether the first exposure to Spike protein was by viral infection or immunization (infection-primed or vaccine-primed). Using multimodal scRNA-seq of activation-induced marker (AIM)-reactive Spike-specific CD4+ T cells, we identified 220 differentially expressed genes between infection- and vaccine-primed patients at the post-booster time point. Infection-primed participants had greater expression of genes related to cytotoxicity and interferon signaling. Gene set enrichment analysis (GSEA) revealed enrichment for Interferon Alpha, Interferon Gamma, and Inflammatory response gene sets in Spike-specific CD4+ T cells from infection-primed individuals, whereas Spike-specific CD4+ T cells from vaccine-primed individuals had strong enrichment for proliferative pathways by GSEA. Finally, SARS-CoV-2 breakthrough infection in vaccine-primed participants resulted in subtle changes in the transcriptional landscape of Spike-specific memory CD4+ T cells relative to pre-breakthrough samples but did not recapitulate the transcriptional profile of infection-primed Spike-specific CD4+ T cells. Together, these data suggest that CD4+ T cell memory is durably imprinted by the inflammatory context of SARS-CoV-2 infection, which has implications for personalization of vaccination based on prior infection history. ONE SENTENCE SUMMARY/UNASSIGNED:SARS-CoV-2 infection and mRNA vaccination prime transcriptionally distinct CD4+ T cell memory landscapes which are sustained with subsequent doses of vaccine.

The relative and synergistic contributions of genetics and environment to inter-individual immune response variation remain unclear, despite its implications for understanding both evolutionary biology and medicine. Here, we quantify interactive effects of genotype and environment on immune traits by investigating three inbred mouse strains rewilded in an outdoor enclosure and infected with the parasite, Trichuris muris. Whereas cytokine response heterogeneity was primarily driven by genotype, cellular composition heterogeneity was shaped by interactions between genotype and environment. Notably, genetic differences under laboratory conditions can be decreased following rewilding, and variation in T cell markers are more driven by genetics, whereas B cell markers are driven more by environment. Importantly, variation in worm burden is associated with measures of immune variation, as well as genetics and environment. These results indicate that nonheritable influences interact with genetic factors to shape immune variation, with synergistic impacts on the deployment and evolution of defense mechanisms.

OBJECTIVES/OBJECTIVE:To investigate the cutaneous microbiome spanning the entire psoriatic disease spectrum, and to evaluate distinguishing features of psoriasis (PsO) and psoriatic arthritis (PsA). METHODS:Skin swabs were collected from upper and lower extremities of healthy individuals and patients with PsO and PsA. Psoriatic patients contributed both lesional (L) and contralateral non-lesional (NL) samples. Microbiota were analysed using 16S rRNA sequencing. RESULTS:was higher in NL PsA samples compared with NL PsO samples (p<0.05), potentially serving as a biomarker for disease progression. CONCLUSIONS:These findings show differences in diversity, bacterial composition and microbe-microbe interactions between healthy and psoriatic skin, both L and NL. We further identified bacterial biomarkers that differentiate disease phenotypes, which could potentially aid in predicting the transition from PsO to PsA.

Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterised by accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms hampering the quality of life and are entry sites for bacterial infection - a major cause of morbidity and mortality in advanced disease. What drives the pathological processes that compromise the skin barrier remains unknown. Here, we report on increased transepidermal water loss and compromised expression of skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as IL-13, IL-22 and Oncostatin M) that activate STAT3 signalling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, repression of filaggrins could be counteracted by a cocktail of antibodies targeting these cytokines/receptors, by siRNA-mediated knockdown of JAK1/STAT3, and by JAK1 inhibitors. Notably, we show that treatment with a clinically approved JAK inhibitor, Tofacitinib, increases filaggrin expression in lesional skin from mycosis fungoides patients. Taken together, these findings indicate that malignant T cells secrete cytokines, which induce skin barrier defects through a JAK1/STAT3 dependent mechanism. As clinical grade JAK inhibitors largely abrogate the negative effect of malignant T cells on skin barrier proteins, our findings suggest that such inhibitors provide novel treatment options for CTCL patients with advanced disease and a compromised skin barrier.

Antibiotics, among the most used medications in children, affect gut microbiome communities and metabolic functions. These changes in microbiota structure can impact host immunity. We hypothesized that early-life microbiome alterations would lead to increased susceptibility to allergy and asthma. To test this, mouse pups between postnatal days 5-9 were orally exposed to water (control) or to therapeutic doses of azithromycin or amoxicillin. Later in life, these mice were sensitized and challenged with a model allergen, house dust mite (HDM), or saline. Mice with early-life azithromycin exposure that were challenged with HDM had increased IgE and IL-13 production by CD4⁺ T cells compared to unexposed mice; early-life amoxicillin exposure led to fewer abnormalities. To test that the microbiota contained the immunological cues to alter IgE and cytokine production after HDM challenge, germ-free mice were gavaged with fecal samples of the antibiotic-perturbed microbiota. Gavage of adult germ-free mice did not result in altered HDM responses, however, their offspring, which acquired the antibiotic-perturbed microbiota at birth showed elevated IgE levels and CD4⁺ cytokines in response to HDM, and altered airway reactivity. These studies indicate that early-life microbiota composition can heighten allergen-driven Th2/Th17 immune pathways and airway responses in an age-dependent manner.

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Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders. Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs. To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C ^stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C ^stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies. Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. [Formula presented] Disclosures: Frank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.
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