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Summary: Individuals with Down syndrome (DS; trisomy 21) display hyperactivation of interferon (IFN) signaling and chronic inflammation, which could potentially be explained by the extra copy of four IFN receptor (IFNR) genes encoded on chromosome 21. However, the clinical effects of IFN hyperactivity in DS remain undefined. Here, we report that a commonly used mouse model of DS overexpresses IFNR genes and shows hypersensitivity to IFN ligands in diverse immune cell types. When treated repeatedly with a TLR3 agonist to induce chronic inflammation, these animals overexpress key IFN-stimulated genes, induce cytokine production, exhibit liver pathology, and undergo rapid weight loss. Importantly, the lethal immune hypersensitivity and cytokine production and the ensuing pathology are ameliorated by JAK1 inhibition. These results indicate that individuals with DS may experience harmful hyperinflammation upon IFN-inducing immune stimuli, as observed during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, pointing to JAK1 inhibition as a strategy to restore immune homeostasis in DS.

Cytokine storms are drivers of pathology and mortality in myriad viral infections affecting the human population. In SARS-CoV-2-infected patients, the strength of the cytokine storm has been associated with increased risk of acute respiratory distress syndrome, myocardial damage, and death. However, the therapeutic value of attenuating the cytokine storm in COVID-19 remains to be defined. Here, we report results obtained using a novel mouse model of lethal sterile anti-viral immune responses. Using a mouse model of Down syndrome (DS) with a segmental duplication of a genomic region encoding four of the six interferon receptor genes (Ifnrs), we demonstrate that these animals overexpress Ifnrs and are hypersensitive to IFN stimulation. When challenged with viral mimetics that activate Toll-like receptor signaling and IFN anti-viral responses, these animals overproduce key cytokines, show exacerbated liver pathology, rapidly lose weight, and die. Importantly, the lethal immune hypersensitivity, accompanying cytokine storm, and liver hyperinflammation are blocked by treatment with a JAK1-specific inhibitor. Therefore, these results point to JAK1 inhibition as a potential strategy for attenuating the cytokine storm and consequent organ failure during overdrive immune responses. Additionally, these results indicate that people with DS, who carry an extra copy of the IFNR gene cluster encoded on chromosome 21, should be considered at high risk during the COVID-19 pandemic.One Sentence SummaryInhibition of the JAK1 kinase prevents pathology and mortality caused by a rampant innate immune response in mice.

Gemcitabine and cisplatin (GC) combination neoadjuvant chemotherapy (NAC) is standard of care for patients with muscle-invasive bladder cancer (MIBC). This combination is preferred due to its favorable toxicity profile. Approximately 25% of NAC treated patients will experience complete pathological response (pT0) at time of radical cystectomy (RC). NAC-treated patients with residual disease at RC have poorer prognosis compared to those who achieved pT0. Here we systematically defined the in vitro functional determinants of resistance to gemcitabine and cisplatin in bladder cancer. To accomplish this, we performed molecular profiling and functional genomic screening on a panel of bladder cancer cell lines (KU-19-19, T24, TCCSUP, 253J and 5637), each having the parental cell line with matched gemcitabine-, cisplatin-, and dual GC-resistant derivatives. We performed whole exome sequencing, RNA sequencing, and phospho-proteomic profiling on these 20 cell lines. To identify chemogenetic interactions that govern sensitivity or resistance to these agents, we performed whole genome CRISPR knockout screening in the GC-resistant cells both in the presence and absence of GC. We identified several novel genes that when inactivated are capable of sensitizing GC resistant cell lines to the two agents. The majority of shared chemosensitizing genes fall into several DNA damage response pathways. This is consistent with the established mechanism of action of GC chemotherapy and observations that have been made from clinical cohorts, where loss-of-function mutations in these pathways correlate with response to platinum-based chemotherapy. Notably, our work has identified several druggable candidates that could serve as potential targets for enhancing responses rates to GC. Interestingly, comparing these candidate genes and pathways with those differentially expressed in the RNA sequencing and proteomic profiling showed limited overlap. This work provides a comprehensive view of the functional determinants of GC sensitivity in bladder cancer. In support of and expanding on previous studies, we identified additional DNA damage response genes that when lost enhance sensitivity to GC. These genes should be further explored for their utility as predictive biomarkers of response to GC-based therapy in clinical cohorts. Importantly, a number of these could be targeted in combination with GC-based NAC to potentially improve response. Citation Format: Robert T. Jones, Tahlita C. Zuiverloon, Hedvig Vekony, Andrew Goodspeed, Teemu D. Laajala, Molishree Joshi, Colin Sempeck, Annie Jean, Megan Tu, Julia D. Wulfkuhle, Emanuel F. Petricoin, James C. Costello, Dan Theodorescu. Functional genomic screening in gemcitabine and cisplatin resistant bladder cancer cell lines identifies DNA repair pathways as mediators of chemosensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4091.

Neoadjuvant platinum-based chemotherapy (NAC) followed by radical cystectomy is the preferred first-line option for treating patients diagnosed with muscle-invasive bladder cancer (MIBC). One of the two most commonly utilized chemotherapy regimens for MIBC consists of combination therapy with gemcitabine and cisplatin (GC). Importantly, cisplatin-based chemotherapy leads to complete response (pT0) in approximately 25% of patients, while the remaining 75% are left with residual disease. For MIBC patients, the presence of residual disease at cystectomy is associated with significantly poorer prognosis. Previous efforts to screen tumor-derived specimens have identified a number of mutational biomarkers associated with NAC response; however, to date these efforts have been limited by the number of patients profiled and the molecular and mutational heterogeneity intrinsic to bladder cancers. Moreover, these efforts have primarily relied on a single technology (e.g., whole-exome sequencing or transcriptome profiling), and were not designed to look at GC-resistance patterns in a way that integrates these data types. Therefore, we sought to systematically define the molecular phenotypes associated with chemoresistant bladder cancer cells, and to functionally interrogate these features to better define essential genes and pathways that govern chemoresponsiveness. To accomplish this, we have extensively characterized a panel of six bladder cancer cell lines (KU-19-19, T24, TCCSUP, 253J, 5637, and RT112), each of which has three derivative cell lines with acquired resistance to gemcitabine, cisplatin, and GC combination. For all of these cell lines we have performed whole-transcriptome mRNA sequencing, whole-exome sequencing, and reverse phase protein microarray. Additionally, we have performed functional genomic screening using whole-genome CRISPR knockout libraries, in the combination GC-resistant derivative cell lines. Using these data we identified multiple important chemoresistance genes and pathways, both previously known and novel. Interestingly, even though we found heterogeneous expression patterns seen across different –omic platforms, the functional genomic screens in GC-resistant cell lines converged on many genes and pathways that are essential to DNA damage response. These data suggest that functional genomic screens are perhaps more likely to identify genes and pathways that are directly linked to each of these drugs' specific mechanisms of action, as opposed to context-specific molecular differences seen across cell lines. Importantly, these results could enable improved prediction and personalization of therapy through identifying biomarkers that predict chemosensitivity and resistance. This study also suggests that identifying more generalizable strategies to overcome chemoresistance could increase the number of bladder cancer patients experiencing deeper and more complete responses to these common chemotherapeutic drugs. Citation Format: Robert T. Jones, Tahlita C. M. Zuiverloon, Hedvig Vekony, Andrew Goodspeed, Teemu D. Laajala, Molishree Joshi, Colin Sempeck, Annie Jean, Megan Tu, Julia D. Wulfkuhle, Emanuel F. Petricoin, James C. Costello, Dan Theodorescu. Multi-omic interrogation of gemcitabine and cisplatin-resistant bladder cancer cell lines identifies unique and shared mediators of chemosensitivity and resistance [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B11.