Through alternative splicing, the TP53 gene can generate multiple protein isoforms with distinct biochemical properties. The p53psi isoform has been identified as a shorter variant than full-length p53 as it lacks nuclear localization, oligomerization, and part of the DNA binding domains due to the use of an alternative 3' splice site in intron 6. Several TP53-truncating mutations, including those producing p53psi, have been detected in a significant proportion of human tumors. However, the mechanistic roles of these truncated p53 proteins remain poorly understood. Here, we describe the generation and analysis of a genetically engineered mouse model that expresses the p53psi protein in place of the full-length p53 protein. In the C57/BL6J genetic background, mice heterozygous for the targeted p53psi allele (p53KI/+) appear phenotypically normal, survive to adulthood, and reproduce. However, heterozygote matings fail to yield viable p53psi homozygote knock-in (p53KI/KI) pups, indicating that forced p53psi expression disrupts embryogenesis. Timed matings revealed that homozygous p53psi expression is embryonically lethal on day E16.5. E14.5-16.5 embryos were pale, reduced in size, and exhibited exencephaly, a defect typically associated with neural tube closure failure. Mouse embryonic fibroblasts (MEFs) derived from p53psi embryos and transformed with the E1A and H-RasV12 oncogenes formed tumors with a decreased growth rate compared to their p53null counterparts, suggesting that p53psi retains at least some tumor-suppressive functions. Our mechanistic studies suggest that p53psi modulates tumorigenesis by triggering senescence. These findings provide insights into the role of the p53psi variant, paving the way for a better interpretation of TP53 mutational patterns in human cancers.

In human acute myeloid leukemia (AML), mutations of isocitrate dehydrogenase-1 (IDH1) often co-occur with NPM1 mutations, and less frequently with FLT3 mutations. To investigate whether the effects of IDH1 mutation differ according to the specific co-occurring mutation, we generated two strains of double knock-in mutant mice. Idh1R132H combined with Npm1c induced overt AML, whereas Idh1R132H plus Flt3ITD resulted in Flt3ITD-driven myelo- or lymphoproliferation that was minimally affected by Idh1R132H and rarely generated AML. Gene expression profiling revealed differences between Idh1R132H;Npm1c cells and Idh1R132H;Flt3ITD cells and suggested altered heme metabolism and immune responses in the former. The profile of Idh1R132H;Npm1c cells corresponded to that of human IDH-mutated AML cells, particularly those resistant to inhibitors of mutant IDH. Compared to treatment with a menin inhibitor, IDH1-targeted therapy of Idh1R132H;Npm1c AML-bearing mice was less efficacious in improving cell differentiation and extending survival. The differential cooperation of Idh1R132H with Npm1c vs. Flt3ITD may have implications for the devising of subtype-specific treatments for human AML.

PirB is an inhibitory cell surface receptor particularly prominent on myeloid cells. PirB curtails the phenotypes of activated macrophages during inflammation or tumorigenesis, but its functions in macrophage homeostasis are obscure. To elucidate PirB-related functions in macrophages at steady-state, we generated and compared single-cell RNA-sequencing (scRNAseq) datasets obtained from myeloid cell subsets of wild type (WT) and PirB-deficient knockout (PirB KO) mice. To facilitate this analysis, we developed a novel approach to clustering parameter optimization called “Cluster Similarity Scoring and Distinction Index” (CaSSiDI). We demonstrate that CaSSiDI is an adaptable computational framework that facilitates tandem analysis of two scRNAseq datasets by optimizing clustering parameters. We further show that CaSSiDI offers more advantages than a standard Seurat analysis because it allows direct comparison of two or more independently clustered datasets, thereby alleviating the need for batch-correction while identifying the most similar and different clusters. Using CaSSiDI, we found that PirB is a novel regulator of Cebpb expression that controls the generation of Ly6Clo patrolling monocytes and the expansion properties of peritoneal macrophages. PirB’s effect on Cebpb is tissue-specific since it was not observed in splenic red pulp macrophages (RPMs). However, CaSSiDI revealed a segregation of the WT RPM population into a CD68loIrf8+ “neuronal-primed” subset and an CD68hiFtl1+ “iron-loaded” subset. Our results establish the utility of CaSSiDI for single-cell assay analyses and the determination of optimal clustering parameters. Our application of CaSSiDI in this study has revealed previously unknown roles for PirB in myeloid cell populations. In particular, we have discovered homeostatic functions for PirB that are related to Cebpb expression in distinct macrophage subsets.

Current vaccine efforts to combat SARS-CoV-2 are focused on the whole spike protein administered as mRNA, viral vector, or protein subunit. However, the SARS-CoV-2 receptor-binding domain (RBD) is the immunodominant portion of the spike protein, accounting for 90% of serum neutralizing activity. In this study, we constructed several versions of RBD and together with aluminum hydroxide or DDA (dimethyldioctadecylammonium bromide)/TDB (d-(+)-trehalose 6,6'-dibehenate) adjuvant evaluated immunogenicity in mice. We generated human angiotensin-converting enzyme 2 knock-in mice to evaluate vaccine efficacy in vivo following viral challenge. We found that 1) subdomain (SD)1 was essential for the RBD to elicit maximal immunogenicity; 2) RBDSD1 produced in mammalian HEK cells elicited better immunogenicity than did protein produced in insect or yeast cells; 3) RBDSD1 combined with the CD4 Th1 adjuvant DDA/TDB produced higher neutralizing Ab responses and stronger CD4 T cell responses than did aluminum hydroxide; 4) addition of monomeric human Fc receptor to RBDSD1 (RBDSD1Fc) significantly enhanced immunogenicity and neutralizing Ab titers; 5) the Beta version of RBDSD1Fc provided a broad range of cross-neutralization to multiple antigenic variants of concern, including Omicron; and 6) the Beta version of RBDSD1Fc with DDA/TDB provided complete protection against virus challenge in the knock-in mouse model. Thus, we have identified an optimized RBD-based subunit vaccine suitable for clinical trials.

Introduction: Immune checkpoint inhibitors can elicit remarkable tumor regressions in non-small cell lung cancer (NSCLC), but not all patients are eligible for these drugs and only a small fraction of those who are respond. Thus, additional immunotherapies (IO) for NSCLC patients are needed. CD47 is an immunosuppressive protein frequently overexpressed in NSCLC. When bound to its receptor, SIRPα, the phagocytic function of antigen presenting cells (APCs) is impaired which dampens the innate immune response and anti-tumor immunity. CD47-SIRPα-targeted IOs are under investigation in clinical trials but little efficacy has been seen in solid tumours, suggesting further preclinical knowledge is needed to guide effective use in NSCLC. To address this need, we aim to decipher immune-mediated and cell-intrinsic mechanisms governing NSCLC response to CD47 inhibition. Methods: CRISPR/Cas9 was used to generate mixed and clonal Cd47 knockout (KO) populations in two murine, syngeneic NSCLC models (LLC and CMT167). The effect of Cd47 inactivation on tumor cell fitness was assessed by multicolour competition assays conducted in vitro and in orthotopic tumors grown in immunocompetent C57BL/6 mice. Survival studies were also done in immune competent (C57BL/6) and deficient (NCG) mice to provide insights into cell-intrinsic versus immune-mediated effects of Cd47 KO on tumor growth. Immunophenotyping of tumours grown in syngeneic hosts was done using flow cytometry to compare immune cell infiltration in wildtype (WT) versus Cd47 KO tumors. Results: Multicolour competition assays revealed that Cd47 LOF reduced the fitness of LLC and CMT167 cells grown in mice but not in cells grown in vitro, suggesting Cd47 KO does not compromise cell proliferation. Consistent with these results, survival studies conducted in immune competent hosts showed prolonged survival of mice with Cd47 KO compared to WT tumors in both models. An increase in activated Cd8+ cytotoxic T cells and M1-polarized macrophages was observed in Cd47 KO tumors relative to WT controls. Interestingly, the same survival studies conducted in immune compromised mice also showed a significant survival benefit for mice with Cd47 KO tumors, suggesting immune- and proliferation-independent, cell-intrinsic functions of Cd47 may also regulate NSCLC growth and progression. Conclusions: Our results confirm the therapeutic potential of Cd47-targeted IO in NSCLC. The increased infiltration of anti-tumor lymphocytes and myeloid cells in Cd47 KO tumours supports a role for the immune system in mediating enhanced survival. However, because immune deficient mice with Cd47 KO tumors also exhibited a survival benefit, additional studies are required to deduce how Cd47-regulated cell-intrinsic mechanisms promote NSCLC biology. Our findings warrant further preclinical research to define effective anti-CD47 strategies for NSCLC. Citation Format: Asa P. Lau, Shawn P. Kubli, Andrew Wakeham, Tak W. Mak, Kelsie L. Thu. CD47 is a promising therapeutic target in non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5182.

Asbestos is the main cause of malignant mesothelioma. Previous studies have linked asbestos-induced mesothelioma to the release of HMGB1 from the nucleus to the cytoplasm, and from the cytoplasm to the extracellular space. In the cytoplasm, HMGB1 induces autophagy impairing asbestos-induced cell death. Extracellularly, HMGB1 stimulates the secretion of TNFα. Jointly, these two cytokines kick-start a chronic inflammatory process that over time promotes mesothelioma development. Whether the main source of extracellular HMGB1 were the mesothelial cells, the inflammatory cells, or both was unsolved. This information is critical to identify the targets and design preventive/therapeutic strategies to interfere with asbestos-induced mesothelioma. To address this issue, we developed the conditional mesothelial HMGB1-knockout (Hmgb1ΔpMeso) and the conditional myelomonocytic-lineage HMGB1-knockout (Hmgb1ΔMylc) mouse models. We establish here that HMGB1 is mainly produced and released by the mesothelial cells during the early phases of inflammation following asbestos exposure. The release of HMGB1 from mesothelial cells leads to atypical mesothelial hyperplasia, and in some animals, this evolves over the years into mesothelioma. We found that Hmgb1ΔpMeso, whose mesothelial cells cannot produce HMGB1, show a greatly reduced inflammatory response to asbestos, and their mesothelial cells express and secrete significantly reduced levels of TNFα. Moreover, the tissue microenvironment in areas of asbestos deposits displays an increased fraction of M1-polarized macrophages compared to M2 macrophages. Supporting the biological significance of these findings, Hmgb1ΔpMeso mice showed a delayed and reduced incidence of mesothelioma and an increased mesothelioma-specific survival. Altogether, our study provides a biological explanation for HMGB1 as a driver of asbestos-induced mesothelioma.

Cholinergic nerves are involved in tumor progression and dissemination. In contrast to other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected. It remains unclear whether there is any form of cholinergic regulation of liver cancer. Here, we show that cholinergic T cells curtail the development of liver cancer by supporting antitumor immune responses. In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response and induction of two populations of CD4 + T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1 + T cells. Tumor antigens drove the clonal expansion of these cholinergic T cells in HCC. Genetic ablation of Chat in T cells led to an increased prevalence of preneoplastic cells and exacerbated liver cancer due to compromised antitumor immunity. Mechanistically, the cholinergic activity intrinsic in T cells constrained Ca 2+ –NFAT signaling induced by T cell antigen receptor engagement. Without this cholinergic modulation, hyperactivated CD25 + T regulatory cells and dysregulated PD-1 + T cells impaired HCC immunosurveillance. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology.

The success of PD-1 and PD-L1-targeted immunotherapies in lung cancer patients demonstrates the power or harnessing the immune system to fight cancer. Thus, the next generation of immunotherapies holds great promise to achieve similar improvements in patient outcomes. CD47 is an immunosuppressive, cell surface protein that is overexpressed and associated with poor prognosis in lung and other cancers. Since tumours exploit CD47 to escape destruction by the immune system, therapeutic antibodies targeting CD47 are being evaluated as a new type of immunotherapy in ongoing clinical trials. Given its prognostic significance in lung cancer, we aimed to evaluate the consequence of CD47 inhibition on lung tumour growth in syngeneic models.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS) in which Th17 cells have a crucial but unclear function. Here we show that choline acetyltransferase (ChAT), which synthesizes acetylcholine (ACh), is a critical driver of pathogenicity in EAE. Mice with ChAT-deficient Th17 cells resist disease progression and show reduced brain-infiltrating immune cells. ChAT expression in Th17 cells is linked to strong TCR signaling, expression of the transcription factor Bhlhe40, and increased Il2, Il17, Il22, and Il23r mRNA levels. ChAT expression in Th17 cells is independent of IL21r signaling but dampened by TGFβ, implicating ChAT in controlling the dichotomous nature of Th17 cells. Our study establishes a cholinergic program in which ACh signaling primes chronic activation of Th17 cells, and thereby constitutes a pathogenic determinant of EAE. Our work may point to novel targets for therapeutic immunomodulation in MS.

Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the tran-scription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.

Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts. In erythroid cells of these mice, D-2-hydroxyglutarate, an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase activity and diminishes succinyl-coenzyme A (CoA) production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells, while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species that induce the cell death of IDH1-mutant erythroid cells. Our results clearly show the essential role of IDH1 in normal erythropoiesis and describe how its mutation leads to myeloid disorders. These data thus have important implications for the devising of new treatments for IDH-mutant tumors.

Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram-negative bacteria, enriched in the microbiota of inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We address the role of typhoid toxin in modulation of the host-microbial interaction in health and disease. Infection with a genotoxigenic Salmonella protects mice from intestinal inflammation. We show that the presence of an active genotoxin promotes DNA fragmentation and senescence in vivo, which is uncoupled from an inflammatory response and unexpectedly associated with induction of an anti-inflammatory environment. The anti-inflammatory response is lost when infection occurs in mice with acute colitis. These data highlight a complex context-dependent crosstalk between bacterial genotoxin-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.

In order to sustain proficient life-long hematopoiesis, hematopoietic stem cells (HSCs) must possess robust mechanisms to preserve their quiescence and genome integrity. DNA-damaging stress can perturb HSC homeostasis by affecting their survival, self-renewal, and differentiation. Ablation of the kinase ataxia telangiectasia mutated (ATM), a master regulator of the DNA damage response, impairs HSC fitness. Paradoxically, we show here that loss of a single allele of Atm enhances HSC functionality in mice. To explain this observation, we explored a possible link between ATM and the tumor suppressor phosphatase and tensin homolog (PTEN), which also regulates HSC function. We generated and analyzed a knocicin mouse line (Pten(S398A)/S398S()), in which PTEN cannot be phosphorylated by ATM. Similar to Atm(+/-), Pten(S398A/S398S) HSCs have enhanced hematopoietic reconstitution ability, accompanied by resistance to apoptosis induced by genotoxic stress, Single-cell transcriptomic analyses and functional assays revealed that dormant Pten(S398A/S398S) HSCs aberrantly tolerate elevated mitochondria! activity and the accumulation of reactive oxygen species, which are normally associated with HSC priming for self-renewal or differentiation. Our results unveil a molecular connection between ATM and PTEN, which couples the response to genotoxic stress and dormancy in HSCs.

The tumor suppressor PTEN is disrupted in a large proportion of cancers, including in HER2-positive breast cancer, where its loss is associated with resistance to therapy. Upon genotoxic stress, ataxia telangiectasia mutated (ATM) is activated and phosphorylates PTEN on residue 398. To elucidate the physiological role of this molecular event, we generated and analyzed knock-in mice expressing a mutant form of PTEN that cannot be phosphorylated by ATM (PTEN-398A). This mutation accelerated tumorigenesis in a model of HER2-positive breast cancer. Mammary tumors in bi-transgenic mice carrying MMTV-neu and Pten398A were characterized by DNA damage accumulation but reduced apoptosis. Mechanistically, phosphorylation of PTEN at position 398 is essential for the proper activation of the S phase checkpoint controlled by the PI3K-p27Kip1-CDK2 axis. Moreover, we linked these defects to the impaired ability of the PTEN-398A protein to relocalize to the plasma membrane in response to genotoxic stress. Altogether, our results uncover a novel role for ATM-dependent PTEN phosphorylation in the control of genomic stability, cell cycle progression, and tumorigenesis.

Primary tumor-derived factors (TDFs) act upon normal cells to generate a pre-metastatic niche, which promotes colonization of target organs by disseminated malignant cells. Here we report that TDFs-induced activation of the p38α kinase in lung fibroblasts plays a critical role in the formation of a pre-metastatic niche in the lungs and subsequent pulmonary metastases. Activation of p38α led to inactivation of type I interferon signaling and stimulation of expression of fibroblast activation protein (FAP). FAP played a key role in remodeling of the extracellular matrix as well as inducing the expression of chemokines that enable lung infiltration by neutrophils. Increased activity of p38 in normal cells was associated with metastatic disease and poor prognosis in human melanoma patients whereas inactivation of p38 suppressed lung metastases. We discuss the p38α-driven mechanisms stimulating the metastatic processes and potential use of p38 inhibitors in adjuvant therapy of metastatic cancers.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3bK27M and Pik3caH1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.