Single Sequence Research Articles

Mode Changing in PSR B0844-35 and PSR B1758-29 with Enhanced Emission at the Profile Centers

We have studied the single pulse emission from two pulsars, PSR B0844-35 and PSR B1758-29, over a wide frequency range of 300–750 MHz using the upgraded Giant Metrewave Radio Telescope. The two pulsars have relatively wide profiles with multiple components, which are a result of the line of sight traversing near the center of the emission beam. In both pulsars, the single pulse sequences show the presence of two distinct emission states, where the profiles become much brighter at the center, with prominent core components during one of the modes, while in the other mode the single pulses show odd–even subpulse drifting with a periodicity of around 2P, P being the rotation period of the pulsar. The centrally bright mode was seen for 10% of the observing duration in PSR B0844-35, which usually lasted for short durations of around 10 pulses, but had two longer sequences of around 100 pulses. On the contrary, the centrally bright mode was dominant in PSR B1758-29 and was seen for around 60% of the observing duration. PSR B1758-29 also showed period amplitude modulations of 60P–70P in both modes. The mode changing in these two pulsars facilitates investigation of the sparking process in the inner acceleration region, dominated by nondipolar magnetic fields. The change in the surface magnetic field configurations likely results in the emission mode change.

Multiomic integration reveals neuronal-extracellular vesicle coordination of gliotic responses in degeneration.

In the central nervous system (CNS), including in the retina, neuronal-to-glial communication is critical for maintaining tissue homeostasis including signal transmission, transfer of trophic factors, and in the modulation of inflammation. Extracellular vesicle (EV)-mediated transport of molecular messages to regulate these processes has been suggested as a mechanism by which bidirectional communication between neuronal and glial cells can occur. In this work we employed multiomics integration to investigate the role of EV communication pathways from neurons to glial cells within the CNS, using the mouse retina as a readily accessible representative CNS tissue. Further, using a well-established model of degeneration, we aimed to uncover how dysregulation of homeostatic messaging between neurons and glia via EV can result in retinal and neurodegenerative diseases. EV proteomics, glia microRNA (miRNA) Open Array and small RNA sequencing, and retinal single cell sequencing were performed, with datasets integrated and analysed computationally. Results demonstrated that exogenous transfer of neuronal miRNA to glial cells was mediated by EV and occurred as a targeted response during degeneration to modulate gliotic inflammation. Taken together, our results support a model of neuronal-to-glial communication via EV, which could be harnessed for therapeutic targeting to slow the progression of retinal-, and neuro-degenerations of the CNS.

Abstract A040: Spatially resolved immune cell networks in renal cell carcinoma

Abstract Interactions between immune cells within the tumor immune microenvironment (TIME) dictate immune cell function and their ability to respond to immunotherapy. Here, we have developed and applied a targeted imaging mass cytometry (IMC) approach to spatially interrogate immune cell interactions at a sufficient scale to associate them with patient outcomes. IMC uses heavy metal-conjugated antibodies and time of flight mass-spectrometry to visualize multiplexed protein targets at single-cell resolution on formaldehyde fixed paraffin embedded tissues. Application of this method to a RCC patient cohort requires the development of a tailored approach. To identify clinically relevant RCC immune cell states, fresh surgical renal cell carcinoma (RCC) samples were procured at the University Health Network through the REnal cancer MicroEnvironment DiscoverY (REMEDY) project and have undergone single cell RNA sequencing (scRNA-seq) and single cell suspension mass cytometry (SMC). Immune cell populations identified through scRNA-seq informed the selection of 43 protein targets sufficient to capture the protein profile and spatial relationships of select cell populations. Heavy metal-conjugated antibodies for each of these protein targets were tested and developed into a novel multiplexed IMC panel to quantify immune cell populations in RCC and applied to the REMEDY cohort. An established analytic pipeline was applied to map clusters of IMC-defined immune cell populations, using both supervised and unsupervised methods of cell identification, and benchmarked to those identified by scRNA-seq and SMC. Going forward, this approach will be repeated on a larger cohort of over 500 RCC patients with known clinical outcomes to identify immune cell networks as signatures associated with disease progression. RCC is amongst the most immune infiltrated solid tumors having varied response to immune checkpoint inhibitors, highlighting the potential for immune cell interactions to effect patient overall response to immunotherapy. With this tailored application of IMC to RCC we aim to characterize cell-cell interactions within the TIME to elucidate mechanisms that enable these interactions to dictate cell function and clinical outcomes. Using this approach, we have characterized over 30 cell populations, including clinically relevant RCC immune cell states, and quantified their spatial relationships within the TIME. We have identified preliminary relationships of immune cell networks within and between patients and determined their association to clinical features. In this preliminary data we observed heterogeneity of immune cell populations between patients as well as intratumoral regional spatial heterogeneity, highlighting the importance of utilizing spatial technologies to characterize immune cell interactions more comprehensively. ​​In summary, we have developed scalable IMC measurements which capture spatially resolved immune cell networks at single-cell resolution within the RCC TIME that will enable the first association of immune cell networks to clinical outcomes. Citation Format: Jennifer Pfeil, Daniel Stueckmann, Shirley Hui, Lisa Martin, Sally Zhang, Maria Komisarenko, Keith Lawson, Gary Bader, Antonio Finelli, Hartland Jackson. Spatially resolved immune cell networks in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A040.

Abstract B002: Pharmacodynamics and antitumor mechanism of the BRG1/BRM (SMARCA4/2) inhibitor FHD-286 in a Phase 1 study in subjects with AML or MDS

Abstract BRG/Brahma-associated factors (BAF) chromatin remodeling complexes are critical to the regulation of cellular differentiation and are implicated in cancer and other diseases. FHD-286 is a first-in-class dual inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4) that is being developed for the treatment of advanced hematologic malignancies. We previously reported that extended exposure of acute myeloid leukemia (AML) cells to FHD-286 led to upregulation of the myeloid maturation marker CD11b, downregulation of BRG1, and decreased cell proliferation. Morphologic features of differentiation were also observed, suggesting that BAF functions to maintain AML cells in an undifferentiated state and that FHD-286 may inhibit AML cell growth by overcoming this differentiation block. Biomarkers identified from these nonclinical studies were analyzed in a Phase 1 dose escalation study of FHD-286 monotherapy in patients with relapsed or refractory AML or myelodysplastic syndrome (MDS) (Study FHD-286-C-002). Flow cytometric analysis was performed on serially collected peripheral blood mononuclear cells (PBMCs) from a total of 31 subjects across 4 dose cohorts (2.5, 5, 7.5 and 10 mg once-daily). Peripheral blasts showed time- and dose-dependent upregulation of myeloid maturation markers, including CD11b and CD64, and concomitant decreases in BCL2, CD34, and BRG1. At the highest dose, substantial biomarker modulation was evident beginning at Cycle 1 Day 15 (C1D15), whereas significant modulation was not observed before C2D1 at the lower dose levels. Single cell RNA sequencing of bone marrow aspirates revealed distinct populations of blasts expressing leukemic stem cell (LSC) markers versus those expressing myeloid differentiation markers. In the majority of on-treatment bone marrow samples, blast populations expressing LSC markers were reduced, while populations expressing myeloid differentiation markers were expanded in a generally dose-dependent manner. Evidence of differentiation was also observed by morphological assessment of patient bone marrow samples. Importantly, these effects were observed in subjects with a wide range of mutation profiles, including those considered high risk. In summary, analysis of patient samples from Study FHD-286-C-002 revealed dose-dependent impacts on stemness and maturation markers in peripheral and bone marrow blasts. These results suggest that FHD-286 forces stem-like blasts to mature, possibly reducing their capacity for self-renewal, and leading to a gradual reduction in circulating blasts. Consistent with this, decreases in bone marrow and peripheral blast counts were observed across cohorts. These findings suggest combining FHD-286 with cytoreductive agents may be a promising strategy to target both LSC-like and proliferative blasts, which is supported by nonclinical data showing enhanced antitumor activity with various combination partners. Citation Format: Mike Collins, Astrid Thomsen, GiNell Elliott, Jessica Wan, Kim Horrigan, Laure Delestre, Virginie Penard-Lacronique, Stephane De Botton, Warren Fiskus, Kapil N. Bhalla, Courtney DiNardo, Samuel Agresta, Jessica Piel, Martin Hentemann. Pharmacodynamics and antitumor mechanism of the BRG1/BRM (SMARCA4/2) inhibitor FHD-286 in a Phase 1 study in subjects with AML or MDS [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 B002.

Understanding the molecular mechanisms of TDP‐43 neurotoxicity in ALS and FTD to inspire targeted therapies

AbstractALS and FTD are fatal and incurable neurodegenerative diseases, characterized by accumulation of pathologic forms of RNA‐binding proteins, predominantly TDP‐43. The functional consequences and potential neurotoxic effects of the highly heterogeneous TDP‐43 aggregates observed in postmortem brains are debated. Combining structural protein analysis with cellular systems we uncovered a novel and unexpected mechanism that counteracts pathologic aggregation of TDP‐43. More recently, we described two distinct mechanisms of aggregation resulting in either nuclear of cytoplasmic TDP‐43 aggregates, resembling the neuropathological heterogeneity described in FTD patients and potentially unravels the origins of heterogeneous pathological species occurring in TDP‐43 proteinopathies. We also showed that FTD heterogeneity is associated with alternate pathological TDP‐43 conformations, reminiscent of prion strains. We used advanced microscopy techniques to explore the differences in the physical and seeding properties between these pathological TDP‐43 aggregates. We found that the subcellular environment and organization of TDP‐43 aggregates differs in patient brains in a subtype‐specific manner. When isolated and introduced in cells, these distinct TDP‐43 assemblies triggered neoaggregate formation with seeding potencies and morphologies that mimicked human brain pathology. To study neurodegeneration phenotypes in human neurons, we developed a new methodology for generating human neuronal networks with remarkable maturity, longevity and reproducibility. Combining high density multielectron arrays and single cell RNA sequencing, we demonstrated the potential of these cultures for modeling neurodegeneration. In this new model, we identified novel RNA targets of TDP‐43, which we found abnormally accumulated in neurons with TDP‐43 pathology in human brains of ALS and FTD patients. The implications of these studies for understanding the mechanisms of neurodegeneration and inspiring therapeutic approaches will be discussed.

Abstract A026: Administration of preoperative ketorolac prevents osteosarcoma recurrence by promoting T cell memory and inhibiting immunosuppression

Abstract Introduction: Surgery induces an inflammatory response that is important for wound healing, but can also promote the survival of cancer cells and future tumor recurrence. Surgical injury activates the cyclooxygenase pathway and the production of downstream mediators such as prostaglandin E2 (PGE2) which has tumor-promoting and immunosuppressive activity. PGE2 stimulates regulatory T cells (Tregs), inhibits antigen presentation, and suppresses natural killer cells. Using a murine model of metastatic osteosarcoma, we investigated if preoperative blockade of this inflammatory cascade with ketorolac, a nonsteroidal anti-inflammatory drug, can boost anticancer T cell immunity. We also assessed if ketorolac had a synergistic effect when administered with a Programmed cell death protein-1 (PD-1) inhibitor. Methods: C3H/HeJ mice were inoculated with LM8 metastatic osteosarcoma cells. Tumors were resected at 50-150 mm3 and 2 hours before surgery, mice received an intraperitoneal injection containing either PBS, ketorolac, or ketorolac and antibodies to PD-1. All mice who survived beyond 100 days were considered long-term survivors and rechallenged with LM8 cells to evaluate immunological memory. Peripheral blood and spleen were collected and processed to perform flow cytometric analysis and single-cell sequencing of immune cell populations. Results: Following resection of primary tumors, 24 out of 25 control mice succumbed to lung metastasis by day 36 after resection while 4 out of 25 mice administered preoperative ketorolac exhibited long-term survival (defined as >90 days after tumor resection). All long-term survivors who received preoperative ketorolac were resistant to a rechallenge with LM8 cells. There was no difference in survival between mice who did and did not receive PD-1 inhibition. Flow cytometry analysis showed that the ketorolac-treated mice had increased T cell activity with a higher percent of effector and effector memory CD4+ and CD8+ T cells. In addition, a lower percent of myeloid derived suppressor cells (MDSCs) and Tregs were found in the spleens of mice treated with preoperative ketorolac. Single cell sequencing revealed that ketorolac-treated mice exhibited an expansion in B cells, specifically follicular B cells that expressed genes related to antigen presentation and cell proliferation. Conclusions: Administration of preoperative ketorolac in mice with osteosarcoma resulted in long-term survival after tumor resection and prevention of tumor recurrence after secondary challenge. Further, treatment with ketorolac reduced two cell populations of immunosuppressive significance, while enhancing immune cell memory. Thus, targeting surgery-induced inflammation with preoperative ketorolac should be further explored as a strategy to bolster a tumor-specific, T cell mediated immune response. Citation Format: Rebecca Pankove, Ramya Ganesan, Swati S Bhasin, Marina Michaud, Manoj Bhasin, Vikas P Sukhatme. Administration of preoperative ketorolac prevents osteosarcoma recurrence by promoting T cell memory and inhibiting immunosuppression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A026.

Abstract B064: Single-cell RNA sequencing reveals tumorigenic trajectories of mismatch repair deficient cells

Abstract Mismatch repair (MMR) deficiency is commonly found in tumors with high mutational burden. Although the mutational signatures of these deficiencies are determined, the initial trajectories these cells follow to reach a tumorigenic state with high mutational load have not been studied due to lack of isogenic model systems. For this reason, we generated three isogenic MMR deficient cell lines from a normally untransformed cell line with few genomic abnormalities and low mutational burden. After continued culturing of these MMR-deficient cell lines for several months, we profiled them by single cell RNA sequencing using the 10X Chromium platform. Next, we identified different clusters of cells in MMR deficient cell lines that acquire tumorigenic properties. In these clusters, we identified gene expression signatures that are responsible from the tumorigenic properties of these clusters of cells. After isolation of the single clones that bear these gene expression signatures, we characterized how MMR deficiency can give rise to tumors with particular gene expression trajectories. In addition, we performed CRISPR/Cas9-based genome-wide screens to understand the vulnerabilities of these clusters of cells. Altogether, our results shed light on the early steps of tumorigenesis in MMR deficiency. Citation Format: Alexandra Vitor, Salvatore Benfatto, Frank Zenke, Sebastian Waszak, Balca Mardin. Single-cell RNA sequencing reveals tumorigenic trajectories of mismatch repair deficient cells [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 B064.

All‐in‐Human Target Discovery in Alzheimer’s Disease and Resilient Brains

AbstractBackgroundAlzheimer’s disease (AD) is characterized by neuropathological hallmarks including amyloid plaques and tau tangles and neurodegeneration in the frontal cortex. While there is a strong association of tau pathology with neurodegeneration and cognitive decline, around 30% of amyloid plaque‐carrying seniors remain cognitively healthy. We hypothesize that protective cellular mechanisms upstream of tau phosphorylation and neurodegeneration may underlie this resilience.MethodWe applied high resolution spatial transcriptomics and single nuclei RNA sequencing on postmortem frontal lobes from 10 demented and 10 non‐demented individuals with comparable degrees of amyloid pathology. We integrated the generated transcriptomics data with proteomics, genomics and drug action databases and interpret cellular transcriptome responses to pathology in the context of human specific protein‐protein interaction networks.ResultUsing spatial and single nuclei transcriptomics, we identify specific transcriptomic signatures and regulatory networks in cellular microenvironments around amyloid plaques and pTau pathology niches. Our results suggest that specific neuronal and glial subpopulations carry enriched vulnerability towards amyloid plaque and pTau depositions. We further find that certain cell death signaling pathways related to neurodegeneration are overrepresented in the context of pTau pathology. Finally, our multi‐omics database integration approach has led to the identification of potential drug targets for therapeutics addressing cellular mechanisms of neuroprotection.ConclusionSpatial transcriptomics from human brain tissue is a powerful approach to reveal cellular mechanisms of pathogenic lesions and enables us to identify neuroprotective mechanisms that may prevent the development of tauopathy and ultimately neuronal loss. The combination of single cell and spatial transcriptomics together with protein‐protein interaction networks provides a promising approach for the identification of novel therapeutic targets for AD.

Signalling pathways associated with impaired angiogenesis in Alzheimer’s Disease

AbstractBackgroundBrain perfusion and blood brain barrier (BBB) integrity are reduced in Alzheimer’s disease (AD). We hypothesized that b‐amyloid (Aβ) induces dysfunction of pathways in vascular cells that regulate angiogenesis.MethodWe performed single nucleus RNA sequencing (snRNAseq) of vascular cells isolated from post mortem samples from multiple cortical regions from AD patients (Braak V‐VI) and non‐diseased controls (NDC, Braak 0‐II). We developed a meta‐analytic approach to integrate these data with related publicly available datasets. We performed differential gene expression (DGE) analyses as a categorical comparison of AD vs NDC and as a confound‐controlled regression of transcriptomic alterations by regional total Aβ. We also performed cell‐specific gene co‐expression analyses identify modules differentially expressed in AD relative to NDC.ResultOur results describe differential expression of genes central to pathways sustaining angiogenesis, proliferation and migration of endothelial cells and the integrity of the BBB with either AD or greater pathological protein load. HIF1A and FGF2, proangiogenic molecules which both induce VEGF, are upregulated in EC, consistent the parenchymal hypoxic response shown repeatedly in prior studies. However, components of signalling pathways downstream of VEGFR2 are downregulated, suggesting a pathological block to angiogenesis with greater Aβ load. While the proangiogenic stimuli promote the expression of genes such as PPP3CA and ITPR1, coding for calcineurin, which contribute to VEGF signalling through the PLCγ‐Ca2+ signalling pathway are upregulated in AD, expression of RAC1 and RHOJ, which enable cellular migration and adhesion, is downregulated. While ANGPT2 expression is also upregulated, the angiopoietin receptor (TIE2, coded by TEK) is downregulated. Downregulation of ADAM10 in AD and upregulation of other NOTCH pathway genes with greater Aβ load suggests altered NOTCH signalling.ConclusionOverall, a hypoxic tissue environment in AD is accompanied by dysfunctional angiogenesis related to Aβ load. Evidence for multiple sites of dysfunction were identified, involving impaired angiopoietin responses, vascular cell migration and NOTCH signaling.

Abstract C005: Inferring global ancestry using scRNA sequencing reads of the fallopian tube and ovarian cancer

Abstract Introduction Single cell RNA sequencing (scRNA-seq) is a powerful technique to profile the transcriptome of each single cell in the sample to characterize its gene expression profile. Final 10x scRNA-seq libraries are comprised of standard Illumina paired end constructs. Read 2 encodes the sequence of the actual cDNA fragment used in library preparation to amplify the RNA sequences, so read 2 is a result of transcription from the DNA encoded in a particular gene. We hypothesize that single nucleotide polymorphisms (SNPs) and global ancestry can be inferred from read 2. Methods Using the read 2, FASTQ files were mapped to the reference genome using the STAR algorithm (2-pass), subsequent steps were performed according to GATK best practices. For reference populations, we selected 2142 unrelated individuals from the 1000 genomes project with admixture >80% in a single ancestral population using k=5 ancestral populations. SNPs quality control for the FT samples and reference included call rates of < 95% or minor allele frequency (MAF) < 1% which were filtered out. Genotypes of fallopian tube (FT) cases were merged with pruned reference genotypes (markers) and intersecting SNPs were used for further analysis. Data was visualized using PCA and ADMIXTURE (k=5). Clinical and demographic variables available in our dataset included age at time of surgery, race, ethnicity, country of birth and specific germline mutation. Whole genome sequencing on the matched germline DNA of the same samples was performed as a validation step. Results A total of 34 cases underwent scRNA-seq. FT with germline mutations included seven BRCA1, seven BRCA2, five PALB2, two RAD51, two MLH1 and one ATM. Additionally, we sequenced six FT with no germline mutations and four ovarian cancer tumors. Self-identified race included 2 Asian, 9 Black, 16 White Hispanic and 6 White non-hispanic. A total of 730 382 SNPs intersected between all samples and pruned references. PCA visualization showed correlation between self-identified race and inferred global ancestry using five ancestral populations. Furthermore, there was high correlation between country of birth, position of cases in the PCA and ADMIXTURE with K=5. Conclusion Inferring global ancestry from scRNA seq reads will help estimate the genetic ancestry from data in which this variable is missing or was not captured. Genetic ancestry is a powerful tool that should be considered when generalizing gene expression results to populations. Citation Format: Alex P. Sanchez-Covarrubias, Ashlee Sealy, Dylan Thompson, David Samuel, Ayodele Omotoso, Destiny Burnett, Matthew Schlumbrecht, Sophia George. Inferring global ancestry using scRNA sequencing reads of the fallopian tube and ovarian cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C005.

Abstract A004: Immune response to sub-ablative radiation before surgery in pleural mesothelioma – Results from the SMARTER trial

Abstract Introduction. We developed a new treatment paradigm in pleural mesothelioma using a short course of sub-ablative hemithoracic radiation followed by surgery and observed encouraging results in epithelioid subtypes. We therefore performed a phase I trial to determine the safety and immunological benefit of 3 fractions of radiation before surgery. Methods. The trial, Surgery for Mesothelioma After Radiation Therapy using Extensive pleural Resection (SMARTER), was designed as a 3+3 phase I trial with incremental hemithoracic radiation dose up to 18 Gy in 3 fractions (background dose) combined with a boost of at least 39 Gy to the gross disease (boost dose). Patients with histologically confirmed stage I-III pleural mesothelioma with tumor size of at least 2 cm in diameter on CT scan were eligible for the study. Surgery was performed 7-14 days after the last fraction of radiation. CyTOF and Luminex were performed on peripheral blood at baseline pre-radiation, post-radiation at the time of surgery, and during follow-up within 3 months from surgery. Tumor was collected at the time of surgery from the sites of low dose radiation (background, T1) and high dose radiation (boost, T2) and compared to pre-radiation (baseline, T0) using single cell RNA sequencing (scRNA seq). CD8 tumor infiltrating lymphocytes (TILs) was determined by immunostaining. The results were validated in a second cohort of patients treated off trial. Results. A total of 12 patients were included in the trial with all patients safely proceeding to surgery after a background radiation dose of 0 Gy, 6 Gy, 12 Gy and 18 Gy. Total radiation boost dose ranged from 39 Gy to 54 Gy. The results were validated in a second cohort of 12 patients. Circulating IL-15, FLT-3L, and CXCL10 significantly increased at the time of surgery compared to baseline. CXCL10 continued to rise during follow-up and was associated with better survival at 1 year (86% vs 17% in the absence of CXCL10 upregulation, p=0.005). The peak of circulating IL-15 correlated with CD8 TILs in the resected tumor specimen. scRNA seq on tumor samples demonstrated that monocytes expressed IL-15 and IL-15Ra, and increased after radiation. Pseudotime trajectory analysis showed an evolution from classical to non-classical monocytes with a shift from M2-like to M1-like phenotype. This was associated with reinvigoration of cytotoxic CD8 TILs and expansion of GITR negative NK cells. CyTOF analysis on peripheral blood confirmed the upregulation of circulating monocytes followed by upregulation of CD8 effector memory T cells, which progressively decreased before tumor recurrence. Tumors with larger populations of IFN-TAM and CD8 ISG were characterized by high CXCL10 expression at baseline and a lack of CXCL10 upregulation after radiation associated with an absence of CD8 T cell reinvigoration and poor survival. Conclusions. These results demonstrate the immunogenic benefit and clinical safety of sub-ablative radiation before surgery in pleural mesothelioma. The lack of benefit was observed in tumors expressing high CXCL10 at baseline. Citation Format: Marc de Perrot, Amin Zia, Licun Wu, Giselle Boukhaled, Ramy Gadalla, Ben Wang, David Brooks, Jeffrey Man, Trevor Pugh, Prodipto Pal, Fatemeh Zaeimi, Hebe Chu, Ming-Sound Tsao, Penelope Bradbury, John Cho. Immune response to sub-ablative radiation before surgery in pleural mesothelioma – Results from the SMARTER trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A004.

Establishing glial responses to anti‐Aβ immunotherapy through single‐cell RNAseq to gain insights into mechanisms of ARIA

AbstractBackgroundWhile anti‐Aβ immunotherapies effectively clear Aβ plaques, mouse and human data show consistent and significant cerebrovascular adverse events of this treatment approach. Anti‐Aβ immunotherapies have been plagued in clinical trials by the occurrence of amyloid‐related imaging abnormalities (ARIA), signifying cerebrovascular disruptions of the edema (ARIA‐E) or hemorrhagic (ARIA‐H) type in the brain. Despite these cerebrovascular adverse effects, the FDA granted accelerated approval to aducanumab and lecanemab. However, our mechanistic understanding underlying ARIA remains minimal. Elucidating the mechanism(s) by which anti‐Aβ antibodies cause ARIA is critical towards improving safety of these therapies for AD. MethodIn the current study, we aimed to determine the glial responses to anti‐Ab antibodies with the hypothesis that astrocytes and microglia have temporal cellular shifts that contribute to cerebrovascular dysfunction after acute anti‐Aβ antibody administration. To identify changes in glial gene expression in response to acute anti‐Aβ antibody we performed a single cell sequencing and digital spatial profiling (DSP) on 14mo Tg2576 APP mice (Fig. 1). We observed differences in size of clusters between anti‐Aβ (6E10) and IgG control (IgG1) intracranial injections after 24hr and 3days, suggesting some clusters of cells respond to anti‐Aβ in a time‐dependent or antibody manner (Fig. 2).ResultPreliminary data identified some of the clusters as microglia (Cx3cr1) and astrocytes (Aldoc), as well as oligodendrocytes (Plp). Apoe was increased in multiple cell types. DSP on these mice is ongoing, however we aim to further characterize the transcriptional profile of glia at the vasculature, and in the vicinity of Aβ plaques and cerebral amyloid angiopathy (CAA). Contralateral hemispheres are utilized for downstream validation studies.ConclusionOur work will elucidate the mechanisms of anti‐Aβ antibody‐induced glial response and establish the role of astrocytes and microglia in cerebrovascular dysfunction and ARIA. This work is critical to improve safety and increase generalizability of these therapies.

Abstract A047: Tumor specific γδ T cells expand and respond to PD-1 blockade

Abstract With the breakthrough of checkpoint blockade, immunotherapies targeting PD-1/PD-L1 have had remarkable success in the clinic and are now widely used to treat a variety of malignancies. While the majority of research on T cell exhaustion and PD-1 blockade has been focused on conventional αβ T cells, the contribution of innate-like T cells such as γδ T cells to PD-1 blockade is not clear. γδ T cells are generally not restricted to conventional MHC-peptide molecules and can recognize a diverse range of ligands including phosphoantigens presented on butyrophilins and MHC class I-like family members MR1, and CD1 isoforms. Using flow cytometry, we evaluated the γδ T cell responses in tumor biopsies and peripheral blood from six MCC patients treated with pembrolizumab. Furthermore, bulk and single cell γδ TCR sequencing was used to measure diversity and clonal expansion in the blood upon PD-1 blockade. TCRs were cloned and expressed in Jurkat cells to screen for reactivity against tumor cells lines. Finally, we used CRISPR-Cas9 to knockout known γδ T cell ligands to identify potential tumor antigen recognized by γδ T cells. We identified a MCC patient who experienced a complete response to pembrolizumab treatment and had a ten-fold expansion of γδ T cells in their tumor biopsies. Tumor-infiltrating γδ T cells expressed high levels of inhibitory receptors PD-1 and TIGIT. Furthermore, γδ T cells characterized in peripheral blood were predominantly Vδ1 cells and had increased Ki-67 expression upon pembrolizumab treatment. From TCR sequencing of peripheral blood, we observed the emergence of a dominant γδ T cell clonotype that was also found in the tumor. Upon TCR gene transfer into Jurkat 76 cells, we identified a γδ TCR that was able to recognize multiple Merkel cancer cell lines. CRISPR knockout of B2M in tumor cells still retained γδ TCR reactivity, suggesting that γδ TCR recognition is independent of B2M expression. Together, these results show innate-like T cells such as γδ T cells also have the capacity to respond to checkpoint blockade. As mutations in antigen presentation can be a resistance mechanism to PD-1 blockade, our study provides the rationale to utilize γδ T cells for treating cancer variants that have escaped the immune system and warrants further investigation of the interplay between γδ T cells and checkpoint blockade. Citation Format: Scott C. Lien, Dalam Ly, S.Y. Cindy Yang, Ben X. Wang, Michael St. Paul, Ramy Gadalla, Babak Noamani, Sarah Boross-Harmer, Trevor J. Pugh, Anna Spreafico, Naoto Hirano, Albiruni R.A. Razak, Pamela S. Ohashi. Tumor specific γδ T cells expand and respond to PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A047.

Modulating amyloid‐beta proteostasis in the brain of 5xFAD mice by selective expression of angiotensin converting enzyme in microglia

AbstractBackgroundAlzheimer’s Disease (AD) is a common and progressive neurodegenerative disease characterized by abnormalities in amyloid‐beta (Ab) proteostasis in the brain. Abnormal cleavage of Ab and aggregation into extracellular amyloid‐containing plaques is widely believed to contribute to neuroinflammation, synapse loss and neurotoxicity in AD. Microglia are brain‐resident myeloid‐lineage cells that mediate synapse homeostasis and the immune response to Ab deposition in the brain, and many Genome‐Wide Association Studies (GWAS) have identified late‐onset AD (LOAD) risk‐associated genes that are primarily expressed in microglia, suggesting their important role in disease pathogenesis (Kunkle et al., 2019, Wightman et al. 2021). Identifying signaling pathways in microglia that modulate Ab proteostasis may provide important targets for disease therapy.MethodThe gene encoding for Angiotensin Converting Enzyme (ACE) has been identified as an AD risk‐associated gene (Kunkle et al., 2019, Wightman et al, 2021), and ACE modulates the function of myeloid‐lineage cells, such as neutrophils and macrophages. ACE expression in macrophages appears to modulate their ability to decrease Ab plaque load in the brain and reverse cognitive abnormalities in AD model mice. Here, we developed a novel mouse model to express ACE specifically in microglia (RACE). We mated RACE mice to 5xFAD mice and examined the role of ACE expression in microglia using molecular, physiologic and behavioral techniques. Single nucleus RNA sequencing (10x Genomics) and digital spatial profiling on tissues sections (Nanostring GeoMx) was used to identify gene expression modulated by ACE in microglia.ResultWe find that ACE expression in microglia in 5xFAD mice increases amyloid phagocytosis, reduces plaque burden, rescues synapse and neuron loss, enhances microglial recruitment to plaques, rescues learning and memory deficits, and modulates gene expression associated with injury and autophagy in plaque‐associated microglia. Single‐nucleus RNA sequencing, proteomic profiling, synaptic plasticity, and pharmacological studies are in process to interrogate the molecular basis of ACE function in microglia.ConclusionACE expression in microglia has the remarkable capacity to restore Ab proteostasis, rescue synapse and neuron loss, and learning and memory abnormalities in 5xFAD mice. Signaling pathways and gene expression modulated by ACE in microglia may be targets for novel disease therapy.

Abstract C057: Fibrolamellar carcinoma single-nucleus RNA sequencing reveals alteration of mitochondrial energetic pathways

Abstract Background: Fibrolamellar carcinoma (FLC) is a primary liver cancer, which arises in a background of normal liver. The DNAJB1-PRKACA (DP) fusion gene is present in the majority of FLC and is an oncogenic driver in animal models. The oncogenic mechanism of DP is unclear. To date, transcriptomic analyses of FLC have been based on bulk RNA sequencing. Given the lack of cell-specific data in bulk transcriptomic analysis, we hypothesize that using single nucleus RNA sequencing (snRNA-Seq) will allow for identification of significantly dysregulated signaling pathways specific to tumor cells. Methods: Single nuclei were isolated from 7 FLC tumors and 4 normal liver (NL). The Chromium Next GEM Single Cell 3’ Reagent Kit was used to generate sequencing libraries. All samples passing QC were submitted for sequencing by GeneWiz with a read depth of 20,000 reads/cell. An R-based pipeline for the single-cell profiling was built using Seurat R toolkit. Cells exhibiting extremely low or high library sizes, gene features outside the 95% confidence interval, or displaying high mitochondrial content were filtered out. Clusters from snRNA-Seq analysis were annotated using singleR, enrichR, and publicly available datasets. Results: FLC and NL nuclei counts were 98664 and 56644, respectively. We generated thirty one clusters and annotated 21 cell types. The FLC tumor group was annotated based on previously published FLC bulk transcriptomic data. We identified increased expression of SLC16A14, TMEM163, PDE10A, OAT, and GALNTL6 in FLC tumor cells. The predominant cells types and percentage of cells (%NL/%FLC) per group were: hepatocytes (43/57), endothelial cells (18/82), FLC tumor cells (15/85), stellate cells (28/72), Kupffer cells (50/50), cholangiocytes (62/38), Other 1 (22/78), T cells (36/64), and fibroblasts (69/31). FLC tissue contained only 3% of the liver sinusoidal endothelial cells. Comparison of FLC tumor cells demonstrated differential upregulation of 1879 and downregulation of 1579 genes in tumor vs. NL. Gene ontology (GO) enrichment identified the following top processes in FLC tumor cells: ATP metabolic process, cellular respiration, and response to peptide hormone. GO analysis of hepatocytes identified: ribonucleotide metabolic process, energy derivation by oxidation of organic compounds, and response to peptide hormone as significant pathways. Conclusions: FLC tumor cells demonstrate alterations in mitochondrial processes and energetics. FLC cells are known to contain abundant mitochondria, which is not observed in other primary liver epithelial cancers. The PRKACA component of DP fusion protein is the catalytic subunit of protein kinase A (PKA). Since PKA is known to regulate mitochondrial function, these data support the possibility that DP may directly alter mitochondrial function. Another interesting finding is the presence of the FLC tumor cell type in NL group (15%), which may suggest its dedifferentiation status or its cell-of-origin as a subpopulation of liver cells distinct from mature hepatocytes or cholangiocytes. Citation Format: Nihal Bharath, Biju Isaac, Marc S. Sherman, Colton J. Smith, Michael Karski, Liang Sun, Brian J. Pepe-Mooney, Ina Kycia, Michael Rogers, Wolfram Goessling, Khashayar Vakili. Fibrolamellar carcinoma single-nucleus RNA sequencing reveals alteration of mitochondrial energetic pathways [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 C057.

Aging drives chronic exacerbated interactions of disease associated microglia and CD8+ T cells following traumatic brain injury

AbstractBackgroundAged individuals are highly susceptible to adverse outcomes following traumatic brain injury (TBI). Despite worsened correlates related to functional outcomes following injury, as well as increased likelihood to acquire a TBI, our understanding of the cellular mechanisms governing the poorer outcomes associated with aged individuals post‐TBI are not well‐characterized.MethodIn the current study, we examined microglial heterogeneity following TBI, in our mouse model of focal TBI across both acute (3d) and chronic (28d) intervals in both young (4m) and aged (18m) male C57B6J mice. At the appropriate interval, brains were collected and processed for single cell RNAseq (scRNAseq), spatial transcriptomics, and histology. A follow up study examining the interaction of TBI and aging upon T cell receptor (TCR) sequences was utilized to examine clonal expansion and lymphocyte heterogeneity in young and aged mice chronically after TBI.ResultOur findings demonstrate that TBI drives diverse transcriptional heterogeneity of microglia in both the young and aged condition. Acutely at 3 days post injury an inflammatory response is seen both in the young and aged mice. However, chronically at 28 days post injury, the microglia from young animals more closely resemble their pre‐injury (i.e. Sham) phenotypes, in comparison to the aged brain where there is a persistence of Apoe‐linked ‘DAM‐like’ microglial response. Inferential analyses point toward these aging‐related chronic populations of microglia as having distinct transcription factor utilization and metabolic pathway enrichment, including Hif1α upregulation and interferon gamma (INFγ) signaling. Furthermore, linkage with chronically accumulated CD8+ T cells and interferon signaling may underlie these phenotypes. Using single cell TCR sequencing, we demonstrate stark contrasts in the responses due to aging and TBI in the clonal expansion of infiltrated CD8+ T cells chronically following TBI.ConclusionOur studies demonstrate that aging is a centrally associated with the persistence of chronically reactive microglia in the brain and that these responses are linked, in part, with the protracted accumulation of CD8+ T cells following TBI.

Abstract B051: GSTT2 modulates the bladder tumor environment and response to BCG Immunotherapy

Abstract The purpose of this study was to elucidate the impact of Glutathione -S-transferase Theta 2 (GSTT2) loss on the response of bladder tumors to Mycobacterium Bovis, Bacillus Calmette Guerin (BCG) immunotherapy. GSTT2 gene expression is controlled by a promoter deletion that occurs frequently in 25-29% of Asians and 41% of Caucasians. The GSTT2 promoter deletion correlated with better response to BCG therapy in a retrospective analysis of patients who received less BCG therapy. To understand this response, we generated GSTT2 knockout mice to elucidate the role of GSTT2 in BCG immunotherapy. Female C57BL/6J mice from GSTT2 wildtype (WT) and KO backgrounds were orthotopically implanted with MB49-PSA cells (murine MB49 cells secreting human PSA). To view the impact of GSTT2 loss on BCG immunotherapy, mice were treated with only 4 instillations of BCG Tokyo. This therapeutic schedule would not result in a high cure rate and but was chosen as we expected it to provide insights into the anti-tumor response. Mice were treated weekly with intravesical BCG Tokyo instillations and tumor growth was monitored by urinary PSA secretion. Final tumor presence was confirmed by real-time PCR analysis of PSA gene expression. One day after the last instillation the bladders (n=7) were harvested for single cell RNA sequencing (Parse Biosciences). Cell clusters were identified by their gene expression profiles. The response to BCG in terms of the cure rate was similar in KO and WT mice (cure rate of 18-20%) but several genes were differentially expressed. Genes with a Log2FC value of +1.5 in KO mice (Peak1, Rpph1, Gphn and Gm19551) and -1.5 in KO (Rora, Tshz2, Sh3gl2, Ankfn1, Rbms3, Naaladl2, Prkn, Mecom, Fmo5, Saa3, Tmprss2, C3, Gm10800 and Gm10801) relative to WT mice were examined. Based on the literature, these genes either block tumor progression (Rora, Tshz2, Sh3gl2 and Gphn); promote tumor progression (Ankfn1, Rbms3, Naaladl2, Prkn, Mecom, Fmo5, Tmprss2, Saa3, C3, Peak1, Rpph1) or modulate immune cells (C3, Saa3 and Rpph1). The majority of these genes were not previously associated with bladder cancer. Interrogation of the TCGA database showed that high expression of Gphn, Peak1 and GSTT2 correlated with poorer outcomes in bladder cancer patients in terms of disease-free survival (HR=1.9 log rank p=0.00038). While expression of the genes downregulated in KO mice and GSTT2 resulted in better outcomes with lower expression of all the genes (HR=1.4, log rank p=0.042). This data indicates that the loss of GSTT2 expression impacts the response of tumors to BCG immunotherapy, primarily by modulating the expression of genes associated with cancer progression and the expression of genes in multiple cell types. Given the common loss of GSTT2 expression in man this gene may be a parameter that should be evaluated in further detail in patients receiving BCG immunotherapy. Citation Format: Ratha Mahendran, Mugdha Vijay Patwardhan, Qin Kane Toh, Edmund Chiong. GSTT2 modulates the bladder tumor environment and response to BCG Immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B051.

PSEN2 poison exon inclusion is common across brain regions in late‐onset Alzheimer’s disease

AbstractBackgroundPathogenic variants in APP, PSEN1 and PSEN2 can cause early‐onset familial Alzheimer’s disease (AD). The molecular events leading to AD in individuals with late‐onset sporadic AD are still unclear even though they share the same clinical and pathological features of AD.MethodUsing targeted PacBio long read isoform sequencing (Iso‐Seq), we recently reported the characterization of tens of thousands of complete PSEN1 and PSEN2 transcripts in the prefrontal cortex of individuals with sporadic AD, familial AD (carrying PSEN1 and PSEN2 pathogenic variants), and controls.ResultWe found several alternative splicing differences in PSEN2 that are significantly elevated in sporadic AD including inclusion of a human‐specific cryptic exon present in intron 9 of PSEN2 (termed exon 9B) present in 11.8% of full‐length reads in sporadic AD samples relative to 1.9% of controls (p = 0.002) and 3.4% of PSEN variant carriers (p = 0.005) as well as a 77bp intron retention product prior to exon 6. These PSEN2 splice products are predicted to generate a prematurely truncated PSEN2 protein and were confirmed in an independent RNA‐seq dataset of ∼80 AD cases and ∼80 controls from cerebellum tissue (Course et al., Brain 2022). To extend these findings we analyzed PSEN2 exon 9B isoform levels in parietal and temporal lobe tissue samples. We found exon 9B was significantly elevated in sporadic AD relative to controls in the parietal lobe (p = 0.02) as well as the temporal lobe (p = <0.001). However, levels of exon 9B were independent of TDP‐43 pathology or MSUT2 protein levels, which is intriguing given their roles as RNA binding proteins implicated in AD. We are working to establish the consequence of exon 9B inclusion in APP processing, Aβ and PSEN2 protein products in addition to possible cell autonomous roles for PSEN2 splice variants in microglia by leveraging single cell sequencing data.ConclusionCollectively, our findings expand our understanding of PSEN2 splice variants in the development of late‐onset sporadic AD, which can point to novel therapeutic strategies for AD.

Abstract A176: Loss of SMAD4 unleashes mTOR and increases dependency on cap-dependent translation in esophageal tumorigenesis

Abstract Application of molecular targeted therapies for esophageal adenocarcinoma (EAC) has been limited by a lack of druggable oncogenic drivers. We propose that synthetic lethal interactions may provide new opportunities for targeted therapies in EAC. We have taken an integrated multi-omics approach incorporating Perturb-Seq (CRISPR editing combined with single cell RNA sequencing) and in vivo tumorigenesis assays to perform high-throughput characterisation of >70 high-confidence EAC driver genes, and genome-wide CRISPR-Cas9 knockout screens in isogenic models of EAC tumorigenesis to identify disease relevant synthetic lethal genetic interactions. MS-based proteomics, reverse phase protein arrays, polysome profiling and bulk RNA-sequencing of isogenic models were utilised to interrogate the biology of bona fide EAC drivers and associated driver specific gene dependencies. The overall goals were to (i) enhance our understanding of EAC tumorigenesis, (ii) identify potential opportunities for therapeutic interventions targeting EAC drivers via synthetic lethal-like approaches, and (iii) reduce the complexity of genetic heterogeneity by categorising EAC drivers with similar phenotypic outcomes. Through our approach we have identified complex crosstalk between the tumor suppressor SMAD4 and regulation of mTOR signaling, with specific downstream effects on translational reprogramming in EAC. Mutation or loss of SMAD4 occurs in up to 20% of EAC, but not pre-malignant tissue (Barrett’s esophagus), and is sufficient to promote transformation of pre-malignant cells in our in vivo tumorigenesis model. In this model, xenotransplanted SMAD4-deficient (via CRISPR-Cas9 knockout or shRNA knockdown) Barrett’s metaplasia cells formed invasive, metastatic tumors after a period of latency. SMAD4 deficient cells had downregulated expression of 4E-BP1, which inhibits EIF4E, the cap-dependent translation initiation factor. This was accompanied by increased mTOR activity, including phosphorylation and inactivation of 4EBP1. Moreover, we found that SMAD4-deficient cells preferentially upregulate cap-dependent translation and become addicted to translation of oncogenic mRNAs. Furthermore, perturbation of additional negative regulators of mTOR signaling in combination with SMAD4 knockout exacerbated these effects and accelerated tumorigenesis in vivo. We have extended these findings to a model of Barrett’s esophagus patient-derived organoids (PDOs) and observed increased proliferative potential of our genetically modified PDOs. Finally, analysing gene ontologies for differentially expressed genes from Perturb-seq revealed that driver-dependent transcriptional changes can be categorized into a smaller number of functional pathways allowing us to potentially consider groups of drivers as functional units. This work advances our understanding of EAC tumorigenesis, provides new mechanistic insights into SMAD4-driven transformation as well as novel potential therapeutic avenues for SMAD4-deficient EAC. Citation Format: Julia V Milne, Ebtihal Mustafa, Kenji Fujihara, Eric Kusnadi, Niko Thio, Maree Pechlivanis, Carlos Cabalag, Twishi Gulati, Kaylene Simpson, Cuong Duong, Luc Furic, Wayne Phillips, Nicholas J Clemons. Loss of SMAD4 unleashes mTOR and increases dependency on cap-dependent translation in esophageal tumorigenesis [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 A176.

Abstract C064: Combination of D3L-002, an anti-TIGIT/PVRIG bispecific antibody, with D3S-001, a KRAS G12C inhibitor, transformed tumor microenvironment from “cold” to “hot” and achieved durable tumor remission in preclinical models

Abstract KRAS G12C inhibitors have demonstrated encouraging clinical benefit in patients with solid tumors harboring KRAS G12C mutation. However, the overall response rate and duration of response remain to be improved. Though combining KRAS G12C inhibitors with anti-PD-1 antibodies showed enhanced preclinical efficacy, clinical benefit for this regimen was compromised by treatment-related toxicities. While the mechanisms underlining these toxicities are being actively investigated, combination strategies incorporating other immunotherapies into KRAS G12C inhibition may provide an alternative to anti-PD-1 combination. We have discovered a new bispecific antibody (BsAb), D3L-002, targeting the TIGIT and PVRIG immune checkpoint molecules that are expressed on activated T and NK cells and mediate inhibitory signaling to counteract the immune stimulatory DNAM-1 pathway. To assess the combination effect of D3L-002 with KRAS G12C inhibitors, the anti-tumor efficacies of KRAS G12C inhibitor D3S-001, a murine surrogate anti-TIGITxPVRIG BsAb (mTxP BsAb), a murine anti-PD-1 antibody (mPD-1 Ab), the combination of D3S-001 with mTxP BsAb and with mPD-1 Ab were evaluated in CT-26 KRAS G12C syngeneic mouse tumor models. Tumor microenvironment (TME) profiling was analyzed by flow cytometry, immunohistology and single cell sequencing. In this study, D3S-001 monotherapy inhibited tumor growth with a 50% complete remission (CR) rate. While mTxP BsAb alone was not effective in this immune “cold” model, combination of D3S-001 with mTxP BsAb resulted in a 70% CR rate, comparable to that of the combination of D3S-001 with the mPD-1 Ab. TME profiling indicated that D3S-001 remodeled the TME from “cold” to “hot” by reducing M2 macrophages and MDSCs while increasing T cells in the tumor. Interestingly, D3S-001 treatment led to increased expression of DNAM-1 on T cells, which has been implicated in augmenting T cell sensitivity towards environmental stimulation. The expression of TIGIT and PD-1 was also upregulated on T cells after D3S-001 treatment, correlating with enhanced T cell activation and anti-tumor efficacy in the combination group of mTxP BsAb and D3S-001 or mPD-1 Ab and D3S-001, respectively. In single cell analysis, mTxP BsAb demonstrated a distinctive effect in regulating immune suppressive Treg population from that of the mPD-1 Ab. While Treg population was increased in D3S-001 or the combination of D3S-001 with mPD-1 Ab group, it was decreased in mTxP BsAb monotherapy or D3S-001 and mTxP BsAb combination group. Collectively, these data showed that D3S-L002’s surrogate mTxP BsAb synergized with D3S-001 in achieving enhanced and durable anti-tumor effect in KRAS G12C-mutant tumors. The combination treatment transformed TME from “cold” to “hot” and inhibited Treg population. These results provide a strong rationale for combining D3L-002 with D3S-001 in clinic trials, which may lead to an alternative treatment option, with differentiated mechanisms and potentially better tolerability, from current strategy in combining anti-PD-1 antibodies with KRAS G12C inhibitors. Citation Format: George Chen, Jing Zhang, Haopeng Rui, Xiaofeng Yang, Tienan Wang. Combination of D3L-002, an anti-TIGIT/PVRIG bispecific antibody, with D3S-001, a KRAS G12C inhibitor, transformed tumor microenvironment from “cold” to “hot” and achieved durable tumor remission in preclinical models [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 C064.