Interferon-gamma Responses Research Articles

Abstract 6550: Biopsy analysis of trial S1616: Ipilimumab plus nivolumab versus ipilimumab alone in patients with anti-PD-1 refractory melanoma

Abstract Background: The phase II randomized trial S1616 (NCT03033576) showed that patients with melanoma refractory to anti-PD-1-based therapy had improved progression free survival (HR=0.63, p=0.037) and objective response (28% vs 9%) to the combination of ipilimumab with nivolumab compared to ipilimumab. Here, we report molecular and spatial proteomic features of biopsies collected from patients on S1616, both prior to and during therapy. Methods: Biopsies collected from patients from both arms at baseline (N=68 patients total) and early on-therapy (N=51; 43 with paired timepoints) were analyzed by whole exome sequencing (n=185 samples), RNA sequencing (n=105), histopathologic staining (n=149), and multiplexed ion beam imaging (n=45). Multiple biopsies were available for some patients. Mutations, gene expression, and tumor microenvironment were compared across timepoints and response to combination (N=18 responders [CR/PR], N=44 nonresponders [SD/PD]). Results: Baseline biopsies from patients responsive to combination had increased expression of genes (n=482, FDR<0.05) associated with coagulation and complement, fatty acid metabolism, oxidative phosphorylation, hypoxia, and interferon gamma response gene sets (FDR<0.05), compared to nonresponsive biopsies. Baseline biopsies from responders also had low levels of effector CD8 T cells (PD1+, TIM-3+, GZMB+, Ki67+) colocalized with tumor cells and myeloid populations expressing higher levels of MHC Class II. On-therapy biopsies from responders showed decreased detection of driver mutations by genomics, reduced gene expression of pathways enriched at baseline (oxidative phosphorylation, complement) by transcriptomics, and increased CD8 T cell to tumor cell ratios by histopathology, supporting observations of tumor regression. On-therapy biopsies from responders also had increased gene expression of genes related to inflammatory cytokine signaling. This correlated with increased proportions of effector CD8 T cells, compared to paired baseline or nonresponding biopsies, and increased organization of nonactivated CD8 T cells near mature endothelial structures (SMA+, CD31+ regions) adjacent to tumor. Biopsies from nonresponders did not demonstrate these dynamics and instead contained exhausted CD8 T cells (PD1+, TIM-3+, granzyme B-, Ki67-) colocalized with FOXP3+, CD4+ Tregs and CD163+, PD-L1+ M2 macrophages, both at baseline and on-therapy. Conclusion: In patients with melanoma refractory to anti-PD-1, addition of anti-CTLA-4 facilitates tumor-reactive CD8 T-cell infiltration and decreased suppressor cell dynamics, resulting in regression of some tumors with distinct transcriptome features. Conversely, biopsies from patients whose tumors progress on combination therapy lack expression of metabolic pathways and show CD8 T-cells restricted in proximity to M2 macrophages and Tregs. Citation Format: Katie M. Campbell, Zaid Bustami, Daniel G. Chen, Egmidio Medina, Cynthia R. Gonzalez, Nataly Naser Aldeen, Ignacio Baselga-Carretero, Agustin Vega-Crespo, Jessica Maxey, Jia M. Chen, Lawrence F. Kuklinski, Kari L. Kendra, Bartosz Chmielowski, Thach-Giao Truong, Nikhil I. Khushalani, Frances Collichio, Alexandra Ikeguchi, Adrienne I. Victor, Kim Margolin, Jeffrey A. Sosman, Sapna P. Patel, Siwen Hu-Lieskovan, James Moon, Shay Bellasea, Daniel K. Wells, Christine N. Spencer, Marshall A. Thompson, Michael Wu, Philip O. Scumpia, Ari VanderWalde, Antoni Ribas. Biopsy analysis of trial S1616: Ipilimumab plus nivolumab versus ipilimumab alone in patients with anti-PD-1 refractory melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6550.

Abstract 5397: Characteristics of cancer associated fibroblasts isolated from refractory colorectal cancer using RNA sequence

Abstract Primary tumor location of colorectal cancers (CRCs) is emerging as an important prognostic factor owing to distinct biological features. In fact, the patients with right-side CRCs (R-CRCs), in which primary tumors were resected in our department and followed by systemic chemotherapy, have a poorer prognosis in 2-year overall survival and progression-free survival than those with left-side CRCs (L-CRCs). Furthermore, cancer-associated fibroblasts (CAFs) promote tumor malignancy, but the precise transcriptional mechanisms regulating the acquisition of the CAF phenotype are not well understood. Here, we isolated the CAFs and normal counterparts from 10 CRCs which were performed radical resection, including 5 L-CRCs and 5 R-CRCs, and analyzed transcriptomic profile of CAFs by RNA-sequencing. Gene Set Enrichment Analysis (GSEA) was performed and inflammatory signatures, such as "HALLMARK IL2 STAT5 SIGNALING"(NOM p-value<0.001, FDR q-value=0.146), "HALLMARK IL6 JAK STAT3 SIGNALING"(NOM p-value=0.014, FDR q-value=0.162), "HALLMARK INTERFERON GAMMA RESPONSE"(NOM p-value=0.032, FDR q-value=0.1726), were enriched in the CAFs isolated from R-CRCs (R-CAFs). In addition to sidedness of CRCs, based on gene expression profiles from bulk tumors, CRCs were classified into four consensus molecular subtypes (CMS1-4). Among them, The fibrotic CMS4 subtype portends poor relapse-free survival. We identified 4 CMS4-CRCs, which have the characteristics of stromal activation and the worst prognosis among other CRCs. GSEA showed that the signature of "HALLMARK TNFA_SIGNALING_VIA_NFKB" (NOM p-value=0.023 FDR q-value=0.207), "HALLMARK GLYCOLYSIS" (NOM p-value=0.036 FDR q-value=0.239) were upregulated in the CAFs isolated from CMS4-CRCs. These results indicated that CAFs isolated from refractory CRCs have unique phenotypes and the elucidation of these mechanisms might lead to a promising treatment for refractory CRCs. Citation Format: Hiroaki Kasashima, Yasuhiro Fukui, Ken Yonemitsu, Kisyu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Shigeru Ree, Masakazu Yashiro, Kiyoshi Maeda. Characteristics of cancer associated fibroblasts isolated from refractory colorectal cancer using RNA sequence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5397.

Abstract 4893: Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas

Abstract Introduction: Colorectal cancer (CRC) is second most common cause of cancer-related mortality in the USA and most patients with advanced tumors lack effective therapeutic options. Epigenetic silencing resulting in reduced expression of O6-methylguanine-DNA methyltransferase (MGMT) has been described in diverse tumor types including CRC and it is linked with reduced DNA repair capability and increased sensitivity to alkylating agents such as temozolomide. However, the potential role and clinical significance of MGMT overexpression in cancer cells has not been studied. Methods: We used spatially resolved multiplex quantitative immunofluorescence (mQIF) analysis to concurrently measure the levels of MGMT protein, γH2AX and CD8+ tumor infiltrating lymphocytes (TILs) in a cohort of 111 CRCs and paired non-tumor colorectal mucosa from the same patient represented in tissue microarray format. To assess the genomic and immunologic context of MGMT expression, we studied the methylome, mutation profiling and mRNA expression of 526 CRCs from TCGA. The immunologic landscape of tumors was studied using GSEA and multiple transcriptomic-based tumor immune infiltration scores such as TIMER, CIBERSORT, etc. Finally, the genomic alterations and immunogenicity of SW620 human CRC cells (with baseline MGMT hypermethylation) with or without MGMT transfection were studied using in vitro co-culture preparations coupled to ultra-deep DNAseq and flow cytometry analysis. Results: On mQIF analysis, cancer-cell MGMT protein upregulation was identified in 73.9% of CRCs and was associated with reduced levels of γH2Ax in malignant cells and lower CD8+ TILs. 26.1% of cases showed MGMT protein downregulation. The genomic analysis of the TCGA cohort revealed that the mean nonsynonymous mutational load was significantly lower in MGMT-high than low tumors with marked differences in the type of variants and frequency of oncogenic mutations. The transcriptomic analysis revealed that MGMT overexpressing tumors showed lower immune infiltration than the MGMT-low group and reduced representation of immune associated signatures including interferon gamma response and TNF-alpha signaling. In contrast, DNA repair, TP53 pathways, were enriched. The exogenous expression of MGMT in SW620 cells reduced the number of nonsynonymous mutations by 15.6% (141 vs 119 mean mutations) and was also associated with low T-cell activation (CD69+/CD8+ T-cells) and cancer-cell elimination on allogenic co-cultures with human PBMCs. Conclusion: MGMT protein upregulation is common in human CRCs and is associated with reduced DNA damage response, distinct genomic features, and adaptive immune evasion. To our knowledge, this study is the first to report the biological and clinical characteristics of CRCs with MGMT overexpression that could support diagnostic and therapeutic developments. Citation Format: Barani Kumar Rajendran, Janie Zhang, Shruti Desai, Joanna Gibson, Jassim DiPalermo, Patricia LoRusso, Yong Kong, Hongyu Zhao, Michael Cecchini, Kurt Alex Schalper. Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4893.

Abstract 5716: CDK7 as a drug target in advanced stage uveal melanoma

Abstract Uveal melanoma (UM) metastasizes predominantly to the liver, and most metastatic UM are BAP1-deficient. Targeted therapies, such as MEK inhibitors, have failed clinically. There are few FDA-approved therapies for metastatic UM, but these therapies are limited to only a subset of patients and have modest survival benefits. Hence, there is a need to identify novel and effective treatment strategies. Due to the low mutational burden in UM, targeting transcriptional may be an effective strategy. Here, we identified Cyclin Dependent Kinase 7 (CDK7), a key regulator of transcription, as an essential and druggable target in UM. CDK7 directly phosphorylates RNA polymerase II and is expressed in all UMs. siRNA-mediated knockdown of CDK7 decreased the growth of three BAP1-deficient UM cell lines. We tested a clinically relevant, highly specific CDK7 inhibitor, SY-5609, developed by Syros Pharmaceuticals and similarly showed that the cell lines were sensitive to the growth inhibitory effects of SY-5609. Furthermore, we observed synergistic effects on cell growth inhibition when combined with a MEK inhibitor (Trametinib). We performed RNA sequencing and identified several Hallmark Genesets to be positively enriched, including interferon-gamma response, TGF-beta signaling, and NFκB signaling after treatment with SY-5609. These results are validated using a multi-omic approach, utilizing Reverse Phase Protein Array (RPPA) data to validate transcriptome to proteome expression of these enriched pathways. Overall, our studies indicate that targeting CDK7 in uveal melanoma is a potential treatment strategy for BAP1-deficient UM and enhances MEK inhibitor efficacy. Citation Format: Francis J. Waltrich. CDK7 as a drug target in advanced stage uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5716.

Abstract 888: Uncovering clinically relevant omics signatures from pan-cancer imaging and multi-omics data integration

Abstract Despite advancements in deep learning for histopathology, integrating these insights with multi-omics data to uncover clinically relevant omics pathway-level signatures remains a challenge. Our study addresses this gap by applying unsupervised learning techniques on pan-cancer multi-omics data, leveraging 3,080 Hematoxylin and Eosin (H&E) images from 1,010 patients in Clinical Proteomic Tumor Analysis Consortium (CPTAC) to uncover omics pathway-level signatures that drive discernable morphology phenotypes at the tissue level. First, imaging models were trained to predict clinical and mutation outcomes, and thereafter, integrated with transcriptomic and proteomic expression data using sparse canonical correlation analysis. Our findings reveal that images of TP53 mutated samples exhibited increased nuclear size and dense lymphoplasmacytic infiltration. These morphological changes correlated with neutrophil and macrophage signaling at the proteomic level, and IL-1 mediated signaling at the transcriptomic level, highlighting the complementary perspectives different omics can provide. To further elucidate immune interactions, we applied multi-omics deconvolution to identify 7 immune subtypes, characterizing each with gene set enrichment, germline DNA variations, and kinase activations. We show that imaging models trained to predict these subtypes differentiate tissue morphologies corresponding to immune enrichment (AUROC: 0.84). To further confirm the robustness of our approach, we trained models to predict co-regulated proteomic modules clustered by independent component analysis. We demonstrate a signature in pan-squamous tumors, consisting of T cell markers and interferon-gamma response proteins like CD4, CD48, and GBP5, which imaging models can successfully predict from histopathology. Manual review from pathologists confirmed lymphocytic T-cell density as a differentiator in samples with the highest and lowest T-cell signaling. Together, these approaches demonstrate that genomic characteristics discovered via unbiased mining of pan-cancer multi-omics data manifest as quantitative imaging phenotypes. These results underscore the potential of multi-omics and digital pathology to integratively uncover and confirm new cancer biology. Our ongoing work will explore predicting drug response and survival from morphology patterns related to multi-omics signatures. Citation Format: Joshua Wang, Runyu Hong, Jimin Tan, Wenke Liu, David Fenyȯ. Uncovering clinically relevant omics signatures from pan-cancer imaging and multi-omics data integration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 888.

Interferon-γ Responses to Chlamydia trachomatis Vaccine Candidate Proteins in Women With Different Chlamydia Outcomes.

Chlamydia trachomatis testing and treatment strategies have not decreased infection rates, justifying need for a chlamydia vaccine. A murine study showed that a vaccine consisting of major outer membrane protein (MOMP) and polymorphic membrane proteins (Pmps) E, F, G, and H elicited protective immunity; studies on human cellular immune responses to Pmps are sparse. Interferon gamma (IFN-γ) responses to these 5 proteins were measured by ELISPOT in peripheral blood mononuclear cells from women returning for treatment of a positive chlamydia test. Responses were compared in those with spontaneous chlamydia clearance versus persisting infection at baseline and no reinfection versus reinfection at a 3-month follow-up visit. IFN-γ response to 1 or more proteins was detected in 39% at baseline and 51.5% at follow-up, most often to PmpE and MOMP. IFN-γ responses to MOMP were detected less often at follow-up versus baseline in women with reinfection, but were maintained in those without reinfection. Women with spontaneous clearance had a higher magnitude of IFN-γ response to PmpE and MOMP. IFN-γ responses to these 5 C. trachomatis vaccine candidate proteins were heterogenous and primarily directed against MOMP and PmpE. Spontaneous chlamydia clearance and absence of reinfection may be clinical correlates of protection.

BpOmpW antigen administered with CAF01 adjuvant stimulates comparable T cell responses to Sigma adjuvant system

There are no licensed vaccines to protect vulnerable populations from the potentially fatal tropical infection, melioidosis, despite its causative agent, Burkholderia pseudomallei, being endemic in tropical and subtropical regions. A promising vaccine candidate, BpOmpW protected mice from melioidosis infection for up to 81 days and stimulated robust interferon gamma responses in CD4+, CD8+, NK and NKT cells. In order to progress to human studies, selection of an adjuvant with an acceptable human safety profile that stimulates appropriate correlates of protection is essential. Here we demonstrate that the CAF01 vaccine adjuvant elicits optimal immune correlates of protection when administered with our BpOmpW vaccine. Specifically, we demonstrate that CAF01 administered with BpOmpW elicits robust Th1 responses, with potent IFN-γ responses in CD4+ and CD8+ T cells and NKT cells, in addition to Th17 and Th2 responses. This formulation will be particularly effective in protecting susceptible populations including people with type 2 diabetes from melioidosis.

Long non-coding RNA GRASLND links melanoma differentiation and interferon-gamma response.

Melanoma is a highly malignant tumor, that stands as the most lethal form of skin cancer and is characterized by notable phenotypic plasticity and intratumoral heterogeneity. Melanoma plasticity is involved in tumor growth, metastasis and therapy resistance. Long non-coding RNAs (lncRNAs) could influence plasticity due to their regulatory function. However, their role and mode of action are poorly studied. Here, we show a relevance of lncRNA GRASLND in melanoma differentiation and IFNγ signaling. GRASLND knockdown revealed switching of differentiated, melanocytic melanoma cells towards a dedifferentiated, slow-proliferating and highly-invasive cell state. Interestingly, GRASLND is overexpressed in differentiated melanomas and associated with poor prognosis. Accordingly, we found GRASLND expressed in immunological "cold" tumors and it negatively correlates with gene signatures of immune response activation. In line, silencing of GRASLND under IFNγ enhanced the expression of IFNγ-stimulated genes, including HLA-I antigen presentation, demonstrating suppressive activity of GRASLND on IFNγ signaling. Our findings demonstrate that in differentiated melanomas elevated expression of GRASLND interferes with anti-tumor effects of IFNγ, suggesting a role of GRASLND in tumor immune evasion.

Suppression of interferon α and γ response by Huwe1-mediated Miz1 degradation promotes SARS-CoV-2 replication.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been demonstrated to limit the host interferon response; however, the underlying mechanism remains unclear. Here, we found that SARS-CoV-2 infection upregulated the E3 ubiquitin ligase Huwe1, which in turn facilitated the degradation of the transcription factor Miz1. The degradation of Miz1 hampered interferon alpha and gamma responses, consequently fostering viral replication and impeding viral clearance. Conversely, silencing or inhibiting Huwe1 enhanced the interferon responses, effectively curbing viral replication. Consistently, overexpressing Miz1 augmented the interferon responses and limited viral replication, whereas silencing Miz1 had the opposite effect. Targeting Huwe1 or overexpressing Miz1 elicited transcriptomic alterations characterized by enriched functions associated with bolstered antiviral response and diminished virus replication. Further study revealed Miz1 exerted epigenetic control over the transcription of specific interferon signaling molecules, which acted as common upstream regulators responsible for the observed transcriptomic changes following Huwe1 or Miz1 targeting. These findings underscore the critical role of the Huwe1-Miz1 axis in governing the host antiviral response, with its dysregulation contributing to the impaired interferon response observed during COVID-19.

The immunogenicity and reactogenicity of four COVID-19 booster vaccinations against SARS-CoV-2 variants following CoronaVac or ChAdOx1 nCoV-19 primary series.

The appropriate COVID-19 booster vaccine following inactivated or adenoviral vector COVID-19 vaccination is unclear. To investigate the immunogenicity of four COVID-19 booster vaccines. We prospectively enrolled healthy adults who received a two-dose CoronaVac or ChAdOx1 8-12 weeks earlier and allocated them to receive one of the following booster vaccine: inactivated (BBIBP-CorV), ChAdOx1 or mRNA (BNT162b2 at full [30 μg] and half [15 μg] dose) vaccines. We determined the reactogenicity and the humoral (anti-receptor binding domain IgG (anti-RBD-IgG), neutralizing antibodies (nAb) against Delta, Beta and Omicron variants) and cellular immunity measuring by interferon gamma (IFN-γ) responses post-booster. AR patients. Among the 352 participants (179 CoronaVac and 173 ChAdOx1 participants), 285 (81%) were female, and median age was 39 (IQR: 31-47) years. Two weeks post-booster, both 30 μg- and 15 μg- BNT162b2 induced the highest anti-RBD IgG concentration (BAU/mL); Coronavac-prime: 30 μg-BNT162b2, 5152.2 (95%CI 4491.7-5909.8); 15 μg-BNT162b2, 3981.1 (3397.2-4665.4); ChAdOx1, 1358.0 (1141.8-1615.1); BBIBP-CorV, 154.6 (92.11-259.47); ChAdOx1-prime: 30 μg-BNT162b2, 2363.8 (2005.6-2786.1; 15 μg-BNT162b2, 1961.9 (1624.6-2369.1); ChAdOx1, 246.4 (199.6-304.2); BBIBP-CorV, 128.1 (93.5-175.4). Similarly, both 30 μg- and 15 μg- BNT162b2 boosting induced the highest nAb titers against Beta, Delta and Omicron BA.1 variants and highest T-cell response at 2 weeks after boosting. While all BNT162b2 or heterologous ChAdOx1-boosted participants had nAb against Omicron, these were < 50% for BBIBP-CorV and 75% for homologous ChAdOx1-boosted participants. There was significant decrease in nAb ( > 4-fold) at 16-20 weeks post booster for all groups. Heterologous boosting with BNT162b2 following CoronaVac or ChAdOx1 primary series is most immunogenic. Additional studies are needed to verify the clinical efficacy and persistence of immunity following half-dose BNT162b2.

Experimental Data on PIRCHE and T-Cell Reactivity: HLA-DPB1-Derived Peptides Identified by PIRCHE-I Show Binding to HLA-A*02:01 in vitro and T-Cell Activation in vivo

Introduction: Human leukocyte antigen (HLA)-DPB1 mismatches during hematopoietic stem cell transplantation (HSCT) with an unrelated donor result in an increased risk for the development of graft-versus-host disease (GvHD). The number of CD8+ T-cell epitopes available for indirect allorecognition as predicted by the PIRCHE algorithm has been shown to be associated with GvHD development. As a proof of principle, PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro and in vivo. Methods: PIRCHE-I analysis was performed to identify HLA-DPB1-derived peptides that could theoretically bind to HLA-A*02:01. PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro by investigating binding affinities of HLA-DPB1-derived peptides to the HLA-A*02:01 in a competition-based binding assay. To investigate the capacity of HLA-DPB1-derived peptides to elicit a T-cell response in vivo, mice were immunized with these peptides. T-cell alloreactivity was subsequently evaluated using an interferon-gamma ELISpot assay. Results: The PIRCHE-I algorithm identified five HLA-DPB1-derived peptides (RMCRHNYEL, YIYNREEFV, YIYNREELV, YIYNREEYA, and YIYNRQEYA) to be presented by HLA-A*02:01. Binding of these peptides to HLA-A*02:01 was confirmed in a competition-based peptide binding assay, all showing an IC50 value of 21 μm or lower. The peptides elicited an interferon-gamma response in vivo. Conclusion: Our results indicate that the PIRCHE-I algorithm can identify potential immunogenic HLA-DPB1-derived peptides present in recipients of an HLA-DPB1-mismatched donor. These combined in vitro and in vivo observations strengthen the validity of the PIRCHE-I algorithm to identify HLA-DPB1 mismatch-related GvHD development upon HSCT.

(Invited) Lab-in-a-Tube Design Enables a Rapid Diagnosis of Infectious Disease

Disease diagnosis increasingly relies on molecular tests that are often limited by infrastructure constraints. For example, tuberculosis (TB) remains a leading cause of death from infectious disease, but an estimated 4.2 million (39.6%) new TB cases were not diagnosed in 2021 and only 57% were confirmed by gold-standard sputum culture or PCR-based tests, partially due to limited accessibility. New point-of-care tests are thus urgently needed to address coverage disparities for chronic, malignant, and infectious diseases.We have developed two smartphone-based assays that can address two diagnostic shortfalls: an on-chip interferon-gamma release assay (IGRA) that detects a T-cell activation response induced by pathogen antigens in fingerstick blood samples, and a lab-in-tube assay that employs CRISPR to sensitively detect pathogen DNA in sputum. Both approaches translate current diagnostic approaches to point-of-care devices that require minimal equipment, user expertise, and time.Results from our on-chip IGRA also correlated with clinical IGRA results when we employed two proteins associated with TNF-alpha receptor signaling (4-1BB and OX-40) to analyze T-cell activation, indicating this approach could detect infections in individuals with compromised interferon-gamma responses; as observed in HIV patients, who are at increased risk for TB. On-chip IGRA results from a SARS-CoV-2-specific assay also corresponded with both infection and vaccination history, suggesting this data could be employed as a high-throughput, high-capacity test to evaluate the effect of vaccination or previous infection to confer a protective immune response, evaluate its change over time, and predict the response to new variant strains to guide healthcare decisions. Mitogen-induced results could also guide immunotherapy decisions, as reduced IGRA values in patients receiving immune checkpoint inhibitors predict poor treatment response, reduced progression-free survival, and increased risk for interstitial pneumonia, while their change after adoptive T-cell therapy can be an independent predictor for overall survival. Results from our lab-in-tube TB DNA assay also had robust diagnostic performance when compared to gold-standard clinical assays but lacked their more extensive personnel, workflow, and infrastructure requirements and were performed in a closed-tube system that protected user from exposure to infectious material. This lab-in-tube approach also permits parallel multiplex analyses to be performed in a single tube, and thus can be employed to detect mutations associated with drug-resistance to guide treatment decisions. For example, we found that an assay with an optimized guide RNA accurately detected a single-nucleotide polymorphism (rpoB S450L) responsible for the majority of rifampin-resistant TB cases.Both these self-contained handheld devices can employ lyophilized reagents to limit cold-chain concerns, can be readily adapted to diagnose other disease conditions by substituting other disease-specific antibodies or guide RNAs, use rechargeable batteries to avoid infrastructure limitations, and can directly transmit results to central sites to improve disease control efforts.

Treatment with β-Adrenoceptor Agonist Isoproterenol Reduces Non-parenchymal Cell Responses in LPS/D-GalN-Induced Liver Injury

There is an increasing evidence indicating the involvement of the sympathetic nervous system (SNS) in liver disease development. To achieve an extensive comprehension of the obscure process by which the SNS alleviates inflammatory damage in non-parenchymal liver cells (NPCs) during acute liver failure (ALF), we employ isoproterenol (ISO), a beta-adrenoceptor agonist, to mimic SNS signaling. ISO was administered to C57BL/6J mice to establish an acute liver failure (ALF) model using LPS/D-GalN, which was defined as ISO + ALF. Non-parenchymal cells (NPCs) were isolated from liver tissues and digested for tandem mass tag (TMT) labeled proteomics to identify differentially expressed proteins (DEPs). The administration of ISO resulted in a decreased serum levels of pro-inflammatory cytokines, e.g., TNF-α, IL-1β, and IL-6 in ALF mice, which alleviated liver damage. By using TMT analysis, it was possible to identify 1587 differentially expressed proteins (DEPs) in isolated NPCs. Notably, over 60% of the DEPs in the ISO + ALF vs. ALF comparison were shared in the Con vs. ALF comparison. According to enrichment analysis, the DEPs influenced by ISO in ALF mice were linked to biological functions of heme and fatty acid metabolism, interferon gamma response, TNFA signaling pathway, and mitochondrial oxidation function. Protein-protein interaction network analysis indicated Mapk14 and Caspase3 may serve as potentially valuable indicators of ISO intervention. In addition, the markers on activated macrophages, such as Mapk14, Casp1, Casp8, and Mrc1, were identified downregulated after ISO initiation. ISO treatment increased the abundance of anti-inflammatory markers in mouse macrophages, as evidenced by the immunohistochemistry (IHC) slides showing an increase in Arg + staining and a reduction in iNOS + staining. Furthermore, pretreatment with ISO also resulted in a reduction of LPS-stimulated inflammation signaling markers, Mapk14 and NF-κB, in human THP-1 cells. Prior treatment with ISO may have the potential to modify the biological functions of NPCs and could serve as an innovative pharmacotherapy for delaying the pathogenesis and progression of ALF.

Abstract B080: Non-genetic determinants driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer

Abstract The FDA approval of the KRAS G12C inhibitor (G12Ci) sotorasib and the advancement of similar drugs into the clinic marks a major milestone in treating KRAS G12C non-small cell lung cancer (NSCLC). However, not all patients respond (sotorasib – ORR = 37.1%, adagrasib - 43%, JDQ443 – 35%), motivating preclinical and clinical investigation into mechanisms of intrinsic and acquired resistance. For instance, clinical studies have reported on-target KRAS mutations and preclinical studies have demonstrated mitogen-activated protein kinase (MAPK) feedback reactivation including EGFR, SHP2, and WT RAS signaling. In response to targeted therapies, sub-populations of cells can enter quiescence or specific epigenetic-driven states that confer drug tolerance. However, epigenetic states defining drug-tolerant persister populations and contributing to adaptive resistance to KRAS G12Ci have not been reported. Using a high-complexity DNA-barcoding system and mathematical modeling, we observed that pre-existing resistant clones to KRAS G12Ci gain clonal fitness during treatment independent of growth rate. Using single-cell profiling and RNA-lineage tracing barcoded systems, we identified distinct subpopulations of cells defined by specific chromatin and transcriptional states, including activation of interferon-gamma genes (ISG) and increased chromatin accessibility for pioneer transcription factors, which may contribute to MAPK reactivation-driven resistance. These results suggest a potential role of pioneer transcription factor-activated interferon-gamma response in driving MAPK feedback reactivation in KRAS G12Ci- resistant tumor subpopulations. Citation Format: Chendi Li, Christopher J Graser, Qian Qin, Usman M Syed, Barbara Karakyriakou, Sarah E Clark, Anne Y Saiki, Paul E Hughes, Christopher Ott, Luca Pinello, Franziska Michor, Aaron N Hata. Non-genetic determinants driving sub-clonal resistance to KRAS G12C combination therapies in KRAS mutant non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B080.

Identification of age-specific biomarkers of spinal cord injury: A bioinformatics analysis of young and aged mice models.

Spinal cord injury (SCI) is a debilitating event that often results in long-term physical damage, disability, and a significant impact on a patient's quality of life. Past research has noted an age-dependent decline in regeneration post-SCI. This study seeks to identify potential biomarkers and enriched pathways in young and aged SCI mouse models. We retrieved the microarray data of spinal cord samples from SCI mice and control mice from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using the R software and the Linear Models for Microarray Data (limma) package. The Gene Set Enrichment Analysis (GSEA) determined enrichment differences among gene sets. The Weighted Gene Co-expression Network Analysis (WGCNA) pinpointed clinically significant modules and hub genes in SCI. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was employed to uncover significantly related pathways of crucial genes in SCI. We found 2560 DEGs in the young SCI group, comprised of 1733 upregulated RNAs and 827 downregulated RNAs. In the aged SCI group, 3048 DEGs were revealed including 1856 upregulated and 1192 downregulated genes. The GSEA revealed 12 enriched signaling pathways in the young SCI group, such as IL6/JAK/STAT3 signaling, interferon alpha response, and interferon gamma response, and 21 signaling pathways in the aged SCI group such as IL6/JAK/STAT3 signaling, E2F targets, and angiogenesis-related pathways. The WGCNA identified the turquoise module as significantly associated with the clinical traits of both young and aged SCI, and revealed 3181 hub genes. Ultimately, 1858 significant genes in SCI were found, with associated signaling pathways including epithelial-mesenchymal transition (EMT), interferon gamma response, and KARS signaling. Our study explored the RNA expression patterns and enriched signaling pathways in young and aged SCI mice. These findings may provide potential biomarkers for targeted SCI therapy.

Kinetics and Biology of Circulating Tumor Cells (CTCs) and Measurable Residual Disease (MRD): Two Dynamic High-Risk Clones in Multiple Myeloma (MM)

BACKGROUND CTCs and MRD are two small clones associated with a high risk of progression in smoldering MM (SMM) and post treatment relapse in active MM. Despite growing knowledge about their clinical significance and biological features, these questions remain unanswered: 1) What is the prognostic value of CTC and MRD kinetics? 2) Is MM dissemination and on-treatment resistance driven by specific genomic alterations? 3) Are CTCs and MRD genetically related? AIM Analyze the kinetics and molecular features of CTCs and MRD in SMM and MM patients. METHODS CTC kinetics were investigated using NGF in 164 SMM patients with ≥3 assessments collected every 6 months in the iMMunocell study. MRD kinetics were investigated using NGF in 277 MM patients with ≥4 assessments in BM aspirates after induction, transplant, consolidation, and yearly during maintenance (GEM2012MENOS65-GEM2014MAIN trials). Unsupervised clustering based on CTC and MRD kinetics was performed using CONNECTOR. Molecular features of BM tumor cells at diagnosis were compared to paired CTCs in 94 SMM and MM patients and to paired MRD in 96 MM patients. In 10 cases, genomic data from tripartite BM tumor cells, CTCs, and MRD was available. Tumor samples were isolated based on patient-specific aberrant phenotypes using FACS. Whole exome and RNA-seq were performed in 343 and 217 samples, respectively. RESULTS Based on the assessment of CTCs in 754 PB samples, SMM patients clustered into 3 groups: sustained undetectable (n = 20), stable (n = 71) and evolving (n = 73) CTC levels. With a median follow-up of 2 years, the respective 2y progression-free rates were 100%, 94% and 81% ( P =.003). Risk stratification based on CTC kinetics (C-index 0.68) outperformed a single baseline assessment (C-index 0.61) and, along with the IMWG 20/2/20 model, had independent prognostic value (HR 3.4; P = .01). Patients with high risk cytogenetics showed more frequently evolving CTC levels. Studying genetic drivers of tumor egress, we found CTCs to closely resemble BM tumor cells (88% concordance at copy number [CNA] and somatic mutational [SNV] level). No recurrent cell-specific SNV or CNA was identified. CTC's transcriptional profile revealed 268 DEGs enriched in interferon alpha, gamma response and cell-adhesion pathways. Next, the prognostic value of MRD dynamics was analyzed according to 1,759 assessments, identifying 3 patient clusters: sustained undetectable (n = 100), stable (n = 104), and evolving (n = 73) MRD levels. Respective 6-year rates of PFS after consolidation were 90%, 75% and 5% ( P &amp;lt;.001), and of OS were 94%, 95% and 66% ( P&amp;lt;.001). Risk stratification based on MRD kinetics (C-index 0.82) outperformed a single MRD assessment before maintenance (C-index 0.62), the R-ISS (C-index 0.57) and R2-ISS (C-index 0.61) at diagnosis. MRD cells showed heterogeneous genomic evolution (60% median concordance) diverging from BM tumor cells at diagnosis due to de novo CNA and SNV that emerged at a subclonal level, particularly after transplant. After induction, MM driver mutations were detected or remained clonal in 13/19 (68%) MRD, whereas after transplant, this was true for only 5/13 (38%) MRD. No recurrent cell-specific SNV or CNA was identified. The transcriptional signature of MRD was treatment dependent, with common molecular features of on-treatment resistance found in all 96 patient-paired samples analyzed after various treatment stages. The 334 DEGs were commonly enriched in KRAS signaling hallmarks. Genomic profiles of tripartite BM tumor cells, CTCs and MRD from 10 MM patients confirmed a patient-specific mutational landscape with genomic heterogeneity in MRD. For instance, one patient exhibited BRAF &amp; DIS3 mutations and del(13q) detected at baseline in BM tumor cells and CTCs, which persisted in MRD. After induction, de novo clonal SNVs, including a NEK11 mutation, emerged, followed by the appearance of 3 subclonal CNA after transplant, possibly contributing to resistance. CONCLUSIONS This is the largest dataset analyzing the kinetics and molecular features of CTCs and MRD in SMM and active MM. We showed superior prognostic value of CTC and MRD kinetics over single assessments. Tumor dissemination seems to be driven by transcriptional priming rather than acquired secondary genetic events, while on-treatment resistance is linked to genomic and transcriptional evolution, without recurrent genetic alterations and with common molecular signatures of resistance.

Genotype-Phenotype Mapping Via Single-Cell Multi-Omics Identifies Therapeutic Vulnerabilities in Vexas Syndrome

VEXAS (Vacuoles E1 enzyme X-linked Autoinflammatory Somatic) syndrome originates from hematopoietic stem cells (HSCs) carrying a somatic UBA1 p.M41 mutation. UBA1 p.M41 mutation leads to systemic inflammation and bone marrow failure, with poor survival. Other than allogenic stem cell transplantation, no curative therapy is available for VEXAS. This arises from the challenge of defining the phenotypic effects of the UBA1 p.M41 mutation in primary human samples, confounded due to the mixture of wildtype and mutated cells. Thus, it is essential to resolve the admixture of wildtype and UBA1 p.M41 mutated cells to understand the effect of the UBA1 p.M41 mutation in the disease-initiating HSCs. By doing so, we can identify potential mutant-specific therapeutic vulnerabilities to specifically eliminate UBA1 p.M41 HSCs. To map genotype-phenotype relationship in human VEXAS samples, we deployed and developed single-cell multi-omics including Genotyping of Transcriptomes (GoT) and Genotyping of Targeted loci with Chromatin Accessibility (GoT-ChA), allowing the capture of UBA1 genotype, cell identity, transcriptome, chromatin accessibility and signaling pathways at single cell resolution ( Fig.1a). We applied GoT to CD34+ enriched bone marrow mononuclear cells (BMMNCs) from VEXAS primary human samples ( n = 31,406 cells across 6 patients). Integrating UBA1 genotyping with gene expression data showed an enrichment of the mutation in myeloid-committed cells and depletion in B and T cells. Interestingly, this mutation is also enriched in natural killer (NK) cells and plasmacytoid dendritic cells (both originating from lymphoid progenitors), which have not been previously reported. This suggests that VEXAS HSPCs display a skewed differentiation towards myeloid, NK, and plasmacytoid dendritic cells, which may contribute to inflammation in VEXAS. High-resolution mapping of UBA1 p.M41 vs. UBA1 wt HSCs revealed myeloid priming of mutated HSCs. UBA1 p.M41 HSCs showed an increased inflammation signature (Interferon alpha and gamma response pathways) along with increased unfolded protein response (UPR), glycolysis and decreased autophagy pathways. We validated UPR pathway activity by showing the expected increase in XBP1 splicing in UBA1 p.M41 HSPCs using targeted capture of the unconventional splice site. Interestingly, transcriptome data also showed a global translational downregulation accompanied by specific ATF4 target gene enrichment ( Fig.1b). These results suggest a preferential induction of the PERK arm of the UPR. Surface protein expression analysis supported the transcriptomic data, showing upregulation of CD38, CD13 and HLA-DR, denoting myeloid bias and increased inflammatory response in UBA1 p.M41 HSCs. Next, we performed GoT-ChA on CD34 + cells from VEXAS patients (n = 27,894 cells across 3 patients) to study mutant-specific chromatin accessibility. Complementary to the transcriptome data, UBA1 p.M41 HSCs showed increased motif accessibility for TFs associated with myelopoiesis, indicative of myeloid priming. We also observed enrichment for ATF4 and CEPBD TF motifs - downstream of PERK pathway of UPR ( Fig.1b). To validate these findings, we developed GoT-ChA integrated with intracellular protein capture and demonstrated an increased expression of phosphorylated eIF2 and ATF4 proteins in the UBA1 p.M41 HSCs. Finally, to functionally validate the specific role of the PERK pathway on the fitness of UBA1 p.M41 HSCs, we performed assays using mobilized CD34 + cells in-vitro. UBA1 Inhibition in normal CD34 + cells led to ATF4 up-regulation, which correlated with cell survival. The addition of PERK inhibition reduced ATF4 expression and resulted in markedly reduced CD34+ viability. In contrast, inhibiting IRE1 pathway along with UBA1 inhibition did not affect HSC viability. Altogether, we demonstrated that human VEXAS HSCs show a cell-intrinsic inflammatory phenotype and are myeloid biased. Moreover, mutated HSCs tolerate proteome toxicity through activation of the PERK pathway as a part of UPR, providing a fitness advantage to the UBA1 p.M41 mutated HSCs. Targeting the PERK pathway may thus represent a new strategy for overcoming VEXAS syndrome via precision targeting of UBA1 p.M41 mutated HSCs.

Cell-Mediated Immunity (CMI) for SARS-CoV-2 Infection Among the General Population of North India: A Cross-Sectional Analysis From a Sub-sample of a Large Sero-Epidemiological Study.

Background Cell-mediated immunity (CMI), or specifically T-cell-mediated immunity, is proven to remain largely preserved against the variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including Omicron. The persistence of cell-mediated immune response in individuals longitudinally followed up for an extended period remains largely unelucidated. To address this, the current study was planned to study whether the effect of cell-mediated immunity persists after an extended period of convalescence or vaccination. Methods Whole blood specimens of 150 selected participants were collected and tested for Anti-SARS-CoV-2 Interferon-gamma (IFN-γ) response. Ex vivo SARS-CoV-2-specific interferon-gamma Enzyme-linked Immunospot (IFN-γ ELISpot) assay was carried out to determine the levels of virus-specific IFN-γproducing cells in individual samples. Findings Out of all the samples tested for anti-SARS-CoV-2 T-cell-mediated IFN-γ response, 78.4% of samples were positive. The median (interquartile range) spots forming units (SFU) per million levels of SARS-CoV-2-specific IFN-γ producing cells of the vaccinated and diagnosed participants was 336 (138-474) while those who were vaccinated but did not have the disease diagnosis was 18 (0-102); the difference between the groups was statistically significant. Since almost all the participants were vaccinated, a similar pattern of significance was observed when the diagnosedand the never-diagnosed participants were compared, irrespective of their vaccination status. Interpretations Cell-mediated immunity against SARS-CoV-2 persisted, irrespective of age and sex of the participant, for more than six months of previous exposure. Participants who had a history of diagnosed COVID-19 infection had better T-cell response compared to those who had never been diagnosed, in spite of being vaccinated.

Transcriptome Changes in Glioma Cells upon Infection with the Oncolytic Virus VV-GMCSF-Lact.

Oncolytic virotherapy is a rapidly evolving approach that aims to selectively kill cancer cells. We designed a promising recombinant vaccinia virus, VV-GMCSF-Lact, for the treatment of solid tumors, including glioma. We assessed how VV-GMCSF-Lact affects human cells using immortalized and patient-derived glioma cultures and a non-malignant brain cell culture. Studying transcriptome changes in cells 12 h or 24 h after VV-GMCSF-Lact infection, we detected the common activation of histone genes. Additionally, genes associated with the interferon-gamma response, NF-kappa B signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathways showed increased expression. By contrast, genes involved in cell cycle progression, including spindle organization, sister chromatid segregation, and the G2/M checkpoint, were downregulated following virus infection. The upregulation of genes responsible for Golgi vesicles, protein transport, and secretion correlated with reduced sensitivity to the cytotoxic effect of VV-GMCSF-Lact. Higher expression of genes encoding proteins, which participate in the maturation of pol II nuclear transcripts and mRNA splicing, was associated with an increased sensitivity to viral cytotoxicity. Genes whose expression correlates with the sensitivity of cells to the virus are important for increasing the effectiveness of cancer virotherapy. Overall, the results highlight molecular markers, biological pathways, and gene networks influencing the response of glioma cells to VV-GMCSF-Lact.

MiR-181a targets STING to drive PARP inhibitor resistance in BRCA- mutated triple-negative breast cancer and ovarian cancer

BackgroundPoly (ADP-ribose) polymerase inhibitors (PARPi) are approved for the treatment of BRCA-mutated breast cancer (BC), including triple-negative BC (TNBC) and ovarian cancer (OvCa). A key challenge is to identify the factors associated with PARPi resistance; although, previous studies suggest that platinum-based agents and PARPi share similar resistance mechanisms.MethodsOlaparib-resistant (OlaR) cell lines were analyzed using HTG EdgeSeq miRNA Whole Transcriptomic Analysis (WTA). Functional assays were performed in three BRCA-mutated TNBC cell lines. In-silico analysis were performed using multiple databases including The Cancer Genome Atlas, the Genotype-Tissue Expression, The Cancer Cell Line Encyclopedia, Genomics of Drug Sensitivity in Cancer, and Gene Omnibus Expression.ResultsHigh miR-181a levels were identified in OlaR TNBC cell lines (p = 0.001) as well as in tumor tissues from TNBC patients (p = 0.001). We hypothesized that miR-181a downregulates the stimulator of interferon genes (STING) and the downstream proinflammatory cytokines to mediate PARPi resistance. BRCA1 mutated TNBC cell lines with miR-181a-overexpression were more resistant to olaparib and showed downregulation in STING and the downstream genes controlled by STING. Extracellular vesicles derived from PARPi-resistant TNBC cell lines horizontally transferred miR-181a to parental cells which conferred PARPi-resistance and targeted STING. In clinical settings, STING levels were positively correlated with interferon gamma (IFNG) response scores (p = 0.01). In addition, low IFNG response scores were associated with worse response to neoadjuvant treatment including PARPi for high-risk HER2 negative BC patients (p = 0.001). OlaR TNBC cell lines showed resistance to platinum-based drugs. OvCa cell lines resistant to platinum showed resistance to olaparib. Knockout of miR-181a significantly improved olaparib sensitivity in OvCa cell lines (p = 0.001).ConclusionmiR-181a is a key factor controlling the STING pathway and driving PARPi and platinum-based drug resistance in TNBC and OvCa. The miR-181a-STING axis can be used as a potential marker for predicting PARPi responses in TNBC and OvCa tumors.