JCI Insight Research Articles

EV-idence uncovered: kidney-on-chip study links circulating EVs of cardiorenal syndrome to renal pathology

The intertwined nature of cardiac and renal failure, where dysfunction in one organ predicts a poor outcome in the other, has long driven the interest in uncovering the exact molecular links between the two. Elucidating the mechanisms driving Cardiorenal Syndrome (CRS) will enable the development of targeted therapies that disrupt this detrimental cycle, potentially improving outcomes for patients. A recent study by Chatterjee et al . (JCI insight 2023) demonstrated the feasibility of utilizing a humanized microfluidic kidney-on-chip model to elucidate the role of circulating extracellular vesicles (EVs) in the development of CRS (type 1 and type 2) in heart failure (HF) patients. The study also identified and validated EV miRNAs that correlated with kidney function by targeting several genes involved in kidney damage pathways, including transforming growth factor- β (TGF-β) signaling. These findings suggest that plasma EVs from CRS patients induce harmful responses in renal cells by regulating key pathways, highlighting their role in both type 1 and type 2 CRS.

A phase II trial of nivolumab plus axitinib in patients with anti-PD1 refractory advanced melanoma.

TPS9600 Background: A significant portion of patients (pts) with advanced melanoma (mel) either do not respond or become refractory to immune checkpoint inhibitor (ICI) therapy, with no established second line treatment regimen. A key ICI resistance mechanism includes formation of an immunosuppressive tumor microenvironment (TME). We have shown that high oxidative metabolism (OXPHOS) of mel tumor cells is associated with a hostile TME (1). Furthermore, this is associated with high intra-tumoral hypoxia (ITH). In mel tumor cells with high OXPHOS, CD8+ tumor infiltrating lymphocytes (TIL) display increased exhaustion and decreased functionality (decreased IFN-γ and TNF-α production). Pts with metastatic mel who progressed on ICI had tumor cells with high OXPHOS and ITH. Axitinib is a VEGFR small molecule tyrosine kinase inhibitor with high inhibitory activity for VEGFR 1-3. We previously showed that low dose axitinib reduces ITH in B16 murine mel, and our pre-clinical data show that axitinib synergizes with ICI to produce an improved tumor response (2). We hypothesize that axitinib will re-sensitize mel to ICI by modulating angiogenesis and reducing ITH and resultant T cell dysfunction. Methods: This is an investigator-initiated, single arm phase II trial of nivolumab plus axitinib for pts with unresectable stage III or IV cutaneous, acral, or mucosal mel who have progressed on prior anti-PD1 based therapy in the adjuvant or metastatic setting (NCT04493203). Pts previously treated with concomitant anti-CTLA4, anti-LAG3, BRAF/MEK inhibitors, and/or pts with brain metastases (asymptomatic or stable treated disease x 2 weeks) are eligible. Pts will receive nivolumab 480 mg IV every 4 wks with axitinib PO 5 mg twice daily for up to 2 years, or until progression or unacceptable toxicity. Pts will undergo biopsy at baseline and at 12 wks, with optional biopsy at progression. Pts will receive an oral dose of pimonidazole 0.5 mg/m2 prior to each biopsy to permit in-vivo evaluation of ITH. Staging scans (full body PET-CT or CT and MRI brain) will be obtained at baseline, with restaging scans at 12 week intervals. Primary endpoint: overall response rate (ORR) by RECIST 1.1. Responses are determined by Blinded Independent Central Review (BICR). Secondary endpoints: safety analysis, progression-free survival (PFS) and overall survival (OS). Extensive correlative translational analyses will focus on immunophenotypic changes in the TME, ITH, profiling of tumor cell metabolism, and TIL function. The null hypothesis that the true ORR is 10% will be tested versus a one-sided alternative hypothesis of ≥25%, using Simon’s minimax two-stage design. This design has a type I error rate 0.08 and power 0.81 when the true response rate is 0.25. PFS and OS will be estimated via Kaplan-Meier method. 31 of planned 31 pts have been enrolled in January 2024, with no data analysis yet available. 1. Najjar et al., JCI Insight 2019. 2. Scharping et al., Nat Immunol 2021. Clinical trial information: NCT04493203 .

Humanized Diets Define Optimal Pressure Lowering Response to Dietary Potassium in Mice

High potassium (K) diets are known to lower blood pressure (BP) in rodents and humans but can also raise BP when consumed in extreme. Interestingly, this response also appears to be influenced by dietary sodium (Na) such that when Na intake is high, high K lowers BP, whereas when sodium intake is lower, high K increases BP (Little et al, Hypertension, 2023). We recently reported (Little et al, JCI Insight, 2023) that the K-mediated BP lowering response, driven by the inactivation of the thiazide-sensitive sodium chloride cotransporter (NCC), is offset by an increase in ENaC (epithelial sodium channel) when an extremely high K diet is chronically consumed. Here, we define the optimal dietary K level needed to inactivate NCC without activating ENaC in the high Na setting and show this K level approximates the recommended level for humans by the DASH (Dietary Approaches to Stop Hypertension) trials. Mice were fed diets containing 6 different K contents, all with high Na (1.7%), so the effects of the control diet (1% K, 1.7% Na) could be compared to traditional extremes (0.1% and 5% K) and others (0.35%, 0.7%, 1.7%) that mimic typical variation in human diets (30-120 mEq/d) when mouse consumption is normalized for caloric intake relative to body mass. Metabolic cage and renal clearance studies were used to assess electrolyte and aldosterone (aldo) excretion as well as NCC- (hydrochlorothiazide (HCTZ) sensitive) and ENaC- (benzamil sensitive) dependent sodium handling. Blood potassium (PK) was measured using iSTAT. BP was measured hourly for 10 days using DSI telemetry probes in male mice (C57Bl6J). The BP response to the diuretics was also measured. PK levels changed significantly with each diet relative to the control diet from 3.2mM to 4.8mM. Systolic blood pressure (SBP; 12-hour active period average) also changed significantly with the different K diets. Extreme high or low K diets (0.1% and 5%) increased SBP by 7mmHg, 0.35% and 0.7% K diets increased SBP by 6 and 3mmHg, while the 1.7% K diet lowered SBP by 4mmHg relative to control (1% K, 132 mmHg). Thus, the relationship between SBP and PK is ‘U-shaped’. Diuretic studies revealed that HCTZ reduces SBP in the 0.1%, 0.35%, 0.7% K groups but had minimal effect in the 1.7% and 5% K groups. Conversely, inhibiting ENaC activity with benzamil normalized SBP in the 5% K group but had a minimal effect in the 1.7% group and no BP effect in the other groups. The BP-lowering effect of each diuretic correlated with increased natriuresis suggesting dietary K influences blood pressure by affecting distal nephron sodium transport. Lastly, urinary aldo levels increased with increasing dietary K, but compared to the control group, aldo levels were significantly lower in the 0.1%, 0.35%, and 0.7% groups, significantly higher in the 5% group, and not different in the 1.7% group. As previously reported, our results demonstrate that extremes in dietary intake can elevate blood pressure by stimulating distal nephron sodium transport proteins with low K inducing NCC activation and high K stimulating ENaC with elevated aldosterone. An optimal dietary K level (1.7%) that maximally reduces BP in mice was identified, suppressing NCC activity while limiting aldosterone production and ENaC activity. Remarkably, this diet mimics the human DASH diet in K content and further supports its effcacy as a blood pressure-lowering modality. DE (DK054231, DK51496); PW (DK054231, DK093501, DK110375): Leducq Foundation U54 DK137331-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 6212: A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden that leads to the generation of few neo-epitopes derived from mutated genes that can be recognized by cytotoxic T cells. The immunosuppressive microenvironment of PDAC comprises suppressive cell populations that include myeloid cells, cancer associated fibroblast subpopulations, and regulatory T cells that reduce T cell infiltration and cytotoxic function. We have developed a murine personalized cancer vaccine to enhance cytotoxic T cell function against PDAC, PancVAX (Kinkead et al, JCI Insight 2018). Survival benefit with PancVAX was dependent on the addition of immune checkpoint inhibitor (ICI) therapy to increase the number of cytotoxic CD8 T cells infiltrating tumors and maintain their cytotoxic function (Huff et al, JCI Insight 2023). Yet, the impact of cell-cell communication on effector T cell function arising from the immunosuppressive microenvironment of PDAC remains unknown. We previously developed a ligand-receptor inference method called Domino that infers inter- and intra-cellular signaling networks in single-cell RNA-seq data (Cherry et al, Nat Biomed Eng 2021). However, this technique is limited to data from single conditions. Understanding how this combination immunotherapy alters cellular signaling networks requires new computational tools that identify changes in regulatory networks between treatment conditions. We developed capabilities for assessment of differential signaling with Domino founded on a basis of differential signal receipt. Active receptors are identified by receptor expression in recipient cells that is correlated with downstream transcription factors in the intracellular signaling cascade. Identification of causative ligands for receptor activation is assessed by a probability of interaction for ligand-receptor interactions between cell types. This probability metric considers mean expression of ligand and receptor genes as well as the number of sender cells for comparison across treatment groups. Comparing communication networks in PancVAX alone to PancVAX with ICI, PancVAX with ICI enhances cell-cell adhesion and interferon gamma signaling in cytotoxic CD8 T cells towards exhausted CD8 T cells. Macrophages in PancVAX with ICI enhance expression of immune checkpoint ligands towards CD8 T cells, despite the presence of blocking ICI antibodies. Intracellular signaling downstream of PD-1 in exhausted T cells shows diminished numbers of transcription factors linked to PD-1 signaling. This software informs prioritization of inhibitory signals for modulation in future immunotherapy regimens and can be applied generally to other tumor subtypes and treatment contexts. Citation Format: Jacob T. Mitchell, Atul Deshpande, Amanda L. Huff, Chris Cherry, Sushma Nagaraj, Kavita Krishnan, Dmitrijs Lvovs, Jennifer H. Elisseeff, Elizabeth M. Jaffee, Neeha Zaidi, Elana J. Fertig. A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma [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 6212.

NIAAA/NIDA animal study: Diet drug may help treat AUD

Researchers at the National Institute on Alcohol Abuse and Alcoholism (NIAAA) and the National Institute on Drug Abuse (NIDA), and collaborators from The Scripps Research Institute, have found that semaglutide (brand name for the injection drug is Wegovy) — a gut hormone that promotes a feeling of fullness and thus promotes weight loss — can help reduce alcohol consumption and binge‐like drinking. The research is based on a rodent model. Published in the June 2023 issue of the journal JCI Insight, the study adds to growing preclinical evidence that the glucagon‐like peptide‐1 (GLP‐1) system plays a role in alcohol and other substance use disorders, and that GLP‐1 receptor agonists show potential for treating people with alcohol use disorder (AUD).

Abstract PR-006: A novel bispecific NK cell engager targeting FSHR and Siglec-7 displays potent anti-tumor immunity against ovarian cancer

Abstract Ovarian cancer (OC) is the deadliest gynecologic malignancy, with the highest mortality among cancers of the female reproductive system. Because of the close interaction between the OC cells and tumor microenvironment (TME), development of approaches which not only target tumor cells but also can maintain their anti-tumor function in this TME is of high importance. Uniquely, Follicle stimulating hormone receptor (FSHR) represents such an important target with specificity for OC and ovarian granulosa cells. We have recently developed monoclonal antibodies (mAbs) that specifically recognize human FSHR on the surface of multiple OC. We characterized them, selected potent anti-FSHR mAb: D2AP11 for detailed investigation and immune engineering in the form of a bispecific T cell engager (D2AP11-TCE) (Bordoloi et al., JCI Insight, 2022). Since most OCs are extremely aggressive and have restricted treatment choices, engaging additional immune effector components may further help. We have developed mAbs against PAN NK marker; Siglec 7 in humanized mice. We identified several Siglec-7 mAbs through high throughput screening and down-selected the pertinent clones. By using a highly potent anti-Siglec-7 clone DB7.2, we have developed a novel NK cell engager (NKCE): DB7.2xD2AP11 NKCE, by linking two antibody binding fragments (scFVs) arranged to bind FSHR, the OC antigen and hman Siglec-7, the NK marker. The NKCE was efficiently expressed in vitro and showed potent binding to both FSHR overexpressing K562 cells as well as Siglec-7 overexpressed HEK293T cells. This novel NKCE resulted in robust and specific killing of multiple OC targets in vitro in the presence of human PBMCs or NK cells, from multiple donors. The OC targets evaluated for killing by the NKCE include high grade serous adenocarcinoma, ovarian clear cell adenocarcinoma and ovarian cystadenocarcinoma lines which carry different mutations; BRCA1&2, AKT, TP53, PIK3CA, BRAF etc. and exhibit resistance against a wide array of cancer drug targets; PARP, HSP90, HDAC, MTORC, DNA alkylating agents, EGFR, PI3K, and WEE1, among others. DB7.2xD2AP11 NKCE displayed killing with EC50-142.87 pM and 236.6 pM against target OVISE-FSHR and OVCAR3 cells respectively, indicating its potency. Further, DNA encoded DB7.2xD2AP11 NKCE significantly attenuated the tumor burden in OVCAR3-FSHR and OVISE challenged humanized mice model. Additionally, this novel NKCE significantly enhanced the median survival of OVCAR3-FSHR challenged mice, suggesting in vivo potency. These data support further research on this novel NKCE as crucial tools for treating OC as well as targeted therapies for other malignancies. Of note, it is the first study of the development of Siglec-7 or any Siglec family member as a bispecific immune engager for targeted immunotherapy. Citation Format: Devivasha Bordoloi, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, Pratik S. Bhojnagarwala, Shushu Zhao, Candice Ionescu, Mohamed bdel-Mohsen, David B. Weiner. A novel bispecific NK cell engager targeting FSHR and Siglec-7 displays potent anti-tumor immunity against ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr PR-006.

Glucose-dependent insulinotropic polypeptide receptor systems in the hypothalamus and the brainstem regulate feeding and weight through distinct pathways.

The report by Adriaenssens et al. in JCI Insight 22 May 2023 explored the role and property of the neurons that express glucose-dependent insulinotropic polypeptide receptor (GIPR) in the brainstem and hypothalamus. The chemogenetic activation of the brainstem GIPR neurons and that of the hypothalamic GIPR neurons showed different feeding and behavior responses. The brainstem GIPR neurons projected to the paraventricular hypothalamus and lateral parabrachial nucleus. Fluorescent-labeled, stabilized peptide GIPR agonist (GIPRA), peripherally injected, localized to the area postrema, nucleus tractus solitarius, median eminence and arcuate hypothalamus. This report showed the role of brainstem GIPR neurons in receiving GIPRA to drive the neural circuit to reduce feeding and bodyweight. In this commentary, distinct and possible cooperative roles of the hypothalamic and the brainstem GIPR pathways will also be discussed.

Single Cell Transcriptome Comparisons of Diagnosis-Relapse Pairs Uncover Programs Associated with Chemoresistance in Pediatric AML

Background: Relapse rates for pediatric AML remain unacceptably high at nearly 50% after frontline therapy. Once relapse occurs, the disease is often refractory to further chemotherapy. Prior studies have shown that acquired mutations conferring chemoresistance are uncommon, and most relapses are thought to arise from the selection of clones already present at diagnosis. Therefore, characterizing chemoresistant disease at the gene expression level could lead to new insights and novel therapy targets. Profiling cases by bulk RNA sequencing has revealed important disease features such as novel gene fusions. However, identifying resistant subpopulations by bulk sequencing is challenging, particularly if they constitute small fractions of the leukemia cells at diagnosis. We hypothesize that by applying single-cell transcriptome profiling (scRNA-seq) to diagnosis-relapse pairs we can identify and characterize the subclones selected by treatment. Additionally, we will discover pathways and gene-expression programs that are associated with chemoresistance. Methods: We profiled thirteen diagnosis-relapse pairs from pediatric AML patients who were treated on or similarly to the Children's Oncology Group (COG) study AAML1031 and consented to banking of cells for future research. Six pairs were banked at Texas Children's Cancer Center and 7 were obtained from the COG biobank. The COG cases were flow-sorted to enrich for blasts before undergoing scRNA-seq with 10X-Chromium and Illumina NovaSeq systems. In addition, two local normal bone marrow cases were profiled, one with prior sorting and one with and without prior sorting. We included scRNA-seq profiles of 8 pediatric AML patients and 4 non-cancer bone marrow samples from Bailur et al. (JCI insight, 2020) as additional controls. Sequencing data were analyzed using CellRanger and Seurat. Four of the COG cases were profiled at the protein level by mass cytometry (CyTOF) and analyzed in Cytobank. Results: In total, for the 13 diagnosis-relapse pairs, 17,233 to 34,246 cells were profiled per patient, and 22,800 transcripts were detected. Unsupervised clustering of our samples and controls yielded 43 clusters (Figure 1) that could be confidently identified as either malignant or non-malignant (normal). We further classified all clusters into inferred hematopoietic developmental stages based on comparisons to publicly available expression reference data (Figure 2). We distinguished between patients with cell types at relapse that were also identified at diagnosis (Group 1), albeit often at a lower proportion, and patients whose cell types at relapse were not detected at diagnosis (Group 2). We also characterized 4 of the diagnosis-relapse pairs by CyTOF with an antibody panel that quantified surface lineage markers and intracellular signaling proteins. The protein expression data validated the RNA data in both the degree of differentiation of AML cells and the degree of similarity between diagnosis and relapse. We identified a Relapse-Up signature composed of 139 differentially expressed genes that were upregulated in AML clusters that expanded between diagnosis and relapse, compared to clusters that diminished between diagnosis and relapse. The Relapse-Up gene signature is enriched in genes that are co-expressed with FLT3, CDK6, and ILK kinase genes. With publicly available TARGET gene expression data, we confirmed that this gene signature is upregulated in relapsed AML samples when compared to diagnosis samples. We further used the profiles of the cell types that expanded between diagnosis and relapse to construct a risk-predictive function that predicted patient outcomes based on bulk RNA-Seq profiles of their diagnostic samples with significant accuracy. Conclusion: Single-cell RNA-seq of diagnosis-relapse pairs from pediatric patients with AML revealed distinct patterns of relapse that were also apparent at the protein level. The Relapse-Up signature was enriched in pediatric AML relapse samples in a large bulk RNA-seq data set, confirming its broader applicability. The kinase co-expression programs present in the signature are likely to be functionally associated with chemotherapy resistance and represent targets for future preclinical therapeutic studies.

Sinusoidal Obstruction Syndrome (SOS) Biomarkers-Derived Predictive Model to Individualize SOS Preemption in Patients Undergoing Allogeneic Stem Cell Transplant

Background: We have previously reported that the predictive model based on 3 biomarkers [L-ficolin, hyaluronic acid (HA), and stimulation 2 (ST2)] can accurately stratify the risk of sinusoidal obstruction syndrome (SOS) in high (>30%) vs low (<5%) risk groups [ JCI Insight. 2023; 8(10):e168221]. However, the improved risk stratification for SOS does not necessarily help guide the administration of the risk-adapted preemptive therapy. Here we set out to assess the clinical utility of the SOS model by integrating its prediction with the consequences (benefits and harms) of preemptive treatment with defibrotide. Methods: We used the data set from the prospective evaluation of the accuracy of the SOS predictive model. We have reformulated the SOS predictive model as a simple fast-and-frugal (FFT) decision tree. FFT consisted of sequentially ordered cues of 3 biomarkers [L-ficolin, HA, and ST2] via a series of binary (yes/no) classification decisions according to the optimal cutpoints chosen by Youden's index. After assessing the model's performance (discrimination, calibration) we applied a generalized decision curve analysis (gDCA) to integrate the effects of treatments with the predictive accuracy of the model. This, in turn, allowed us to develop individualized recommendations for the use of preemptive treatment of SOS with defibrotide. We calculated net benefits (NB) to compare 3 management strategies: don't provide any treatment vs. administer prophylaxis to all patients undergoing allo-transplant vs. use the SOS model to guide the preemption treatment with defibrotide. According to gDCA the best treatment is the one with the highest net benefits. We informed gDCA analysis using data from the published randomized trial comparing defibrotide vs. placebo treatment effects [ Lancet. 2012; 379 :1301]. Adverse events (AE) were seen in 2.5% more patients compared with placebo, and defibrotide reduced the risk of SOS by 40% (=relative risk reduction [RRR]). Results: The model had a good performance characteristic. Harell's C discrimination statistics was 0.77 with good calibration properties (with intercept and the slope not statistically significantly different from 0 and 1, respectively). Assuming AE of 2.5%, offering prophylaxis to all patients with defibrotide represents the best management strategy for very large RRR (>65%). Similarly, for RRR<2.5%, no prophylaxis should be offered. For all other values of treatment effects (ranging from RRR 2.5% to 65%), relying on the SOS model represents the best strategy to guide the use of defibrotide preemptively (Figure 1A). Assuming the baseline treatment values [AE=2.5%, RRR=40%], the SOS model almost perfectly individualizes the use of prophylaxis: all patients with a risk of SOS>6.25% should be given prophylaxis, otherwise not (Figure 1B). However, the risk threshold was sensitive to the assumptions about defibrotide treatment effects. Conclusion: The SOS biomarker-based model offers a new method to guide risk-adapted prophylactic or preemptive therapy for SOS.

The DNA Sensor AIM2 Determines B Cell Fate after BCR Activation in Chronic Graft-Vs-Host Disease

Prevention and treatment of chronic graft-vs-host disease (cGVHD), a debilitating late immune toxicity in patients after allogeneic hematopoietic stem cell transplantation (allo-HCT), who are otherwise cured of hematological malignancies, remains a major challenge. T cells incite and promote cGVHD through coordinated T-B cell responses. Alloantibody production ( Srinivasan et al, Blood 2012, Jin et al, Blood 2016) and aberrant B Cell Receptor (BCR) signaling ( Allen et al, Blood 2014, Flynn et al, Blood 2015) play fundamental roles in the pathology of cGVHD. B cell-intrinsic pathways in cGVHD remain incompletely understood, limiting our therapeutic capacity for patients. In a single cell RNA sequencing study, we found that transcript levels of the DNA sensor Absent in Melanoma 2 (AIM2) were significantly increased in CD27+ BCR-activated B cell clusters in circulating as well as in tissue-resident cGVHD B cells ( Poe et al, JCI Insight 2023). After confirming AIM2 expression is restricted to the nucleus, we also previously showed that AIM2-expressing B cells were augmented after BCR and interferon-gamma (IFNγ) and/or Toll-like receptor (TLR)-7 or 9 stimulation ( Visentin J, Blood 2022;140 Supp 1), suggesting a context-dependent role for AIM2 after allo-HCT. Our new findings now afford the hypothesis that an AIM2-BCR signaling axis determines B cell fate after allo-HCT. To address this hypothesis, we first examined whether AIM2 modulates the promotion of antibody-secreting cells (ASCs) after BCR activation using a hapten-carrier immunization model. We compared numbers of hapten-specific ASCs from immunized WT and AIM2-KO mouse splenocytes using enzyme-linked immunosorbent spot (ELISPOT). We compared numbers of ASCs on day 3 post-immunization, when early germinal center (GC) formation is known to occur. We found that hapten-specific IgM ASCs were significantly increased in WT splenocytes compared to AIM2-KO (p=0.002). By contrast, when we looked at day 8, when mature GC formation and rapid ASC proliferation is known to occur, we found that AIM2-KO immunized mice had significantly lower numbers of hapten-specific IgM (p=0.005) and IgG (p=0.004) ASCs in AIM2-expressing mice (Figure 1a). Our results suggest a role for AIM2 in regulating the survival of short-lived plasmablast-like ASCs after high affinity antigen encounter and maturation of germinal centers. To further investigate whether AIM2 affects expansion of GC-derived (GL7+CD95+) ASCs, we stimulated B cells purified from wildtype (WT) or global AIM2-deficient (AIM2-KO) mice in vitro with lipopolysaccharide (LPS) and IL-4 +/- BCR stimulation. We found that AIM2-deficient B cells, especially GC-like cell populations, expanded significantly in an anti-IgM concentration-dependent manner up to 12-fold (p=0.03) after 72 hr (Figure 1b). By contrast, B cell expansion was constrained in AIM2-sufficient B cells. This expansion was unlikely to be due to differences in proliferation alone, as we did not detect differential Ki67 expression in stimulated B cells with or without AIM2. Together, these data suggest a role for AIM2 in modulating BCR signaling. Finally, we examined the role of AIM2 in BCR-mediated cell death after chronic antigen exposure using our previously described bone marrow transplant (BMT) model, where recipients of bone marrow and splenocytes (BM+Spl) exhibit cGVHD manifestations ( Poe et al, JCI Insight 2018, Jia et al, Blood 2021). We have previously generated C57BL/6J mice with conditional deficiency of AIM2 only in B cells (AIM2 fl/flMb1 Cre/+, CKO), and used these in our BMT model as donors. At day 58 post BMT, we performed ex vivo stimulation of splenic B cells from BMT recipients. While B cells from CKO BM+Spl recipients expanded ex vivo in an anti-IgM concentration-dependent manner, B cells from AIM2-sufficient BM+Spl recipients demonstrated limited responsiveness ex vivo to BCR stimulation. This extends our in vitro observations by suggesting a role for AIM2 in B cell tolerance after chronic antigen exposure. Ongoing experiments will determine if B cell-intrinsic AIM2 contributes to cGVHD pathogenesis. Thus far, our data reveal a novel role for AIM2 as a B cell survival checkpoint after BCR activation. Further delineation of context-dependent and synergistic AIM2-BCR signaling after allo-HCT will lead to strategies that block the development of cGVHD without adversely affecting anti-tumor activity.

Development and Characterization of a Novel Murine Peripheral T-Cell Lymphoma Cell Line Which Closely Mimics Human Disease

Peripheral T-cell lymphoma (PTCL) represents a heterogenous group of aggressive non-Hodgkin lymphomas with poor prognostic outcomes and limited treatment options. Despite the advances in genomic sequencing and molecular characterization, the complex and elusive nature of PTCL's underlying biology has proven difficult to fully understand and manage effectively. The development and refinement of therapeutic strategies for PTCL are impeded by a paucity of reliable preclinical models that accurately mimic the disease's pathophysiology. There is a dire need for more physiologically relevant models for PTCL. Improved models allow for a better understanding of the disease's molecular and cellular mechanisms, facilitate the development of innovative therapeutic strategies, and ultimately improve patient outcomes. Here we described a new T-cell lymphoma cell line (LM-23) derived from a 12-week-old female Balb/cJ mouse which developed diffuse adenopathy and splenomegaly. Tumors from this mouse were cultured in cRPMI + 10% fetal bovine serum for 4 weeks without supplementation of additional cytokines and passaged 3-4 times per week where we found the cells to be non-adherent. Immunophenotyping of LM-23 demonstrated positivity for CD90, CD3, TCR-beta, CD4(partial/subset), CD8, PD1 (positive), ICOS (positive) and negative for B-cell and myeloid lineage markers (Fig. 1B). This immunophenotype most closely resembles PTCL-NOS in humans at an immunophenotypic characterization level. Whole exome sequencing is in progress and will be presented. Intravenous administration of this cell line to syngeneic Balb/cJ mice resulted in rapid establishment of diffuse adenopathy, splenomegaly, and liver metastases. Bulky adenopathy was observable within 14 days of administration. Histologically, these tumors consisted of sheets of medium to large sized lymphocytes with clumped chromatin and prominent nucleoli, similar to human T-cell lymphomas (Fig. 1A). Subcutaneous administration demonstrated rapid tumor growth with mice reaching terminal endpoints (>2,000mm 3) within 21 days following administration. As CHOP-based (cyclophosphamide, doxorubicin, vincristine, and prednisone) therapies are the primary backbone for treatment of PTCL, we tested whether CHOP therapy alone had anti-tumor activity to LM-23. Tumor bearing mice (~150mm 3) were treated with CHOP and monitored for tumor growth. All mice treated were non-responsive to CHOP therapy with no difference in tumor growth compared to untreated mice. Additionally, LM-23 bearing mice treated with anti-PD1 showed signs of hyperprogression, which has been observed in human patients in clinical trials (Gao Y et al. JCI Insight 2023, Bennani NN et al. Blood Supplement 2019). Additional therapeutic target testing is underway and will be presented. In conclusion, the newly established LM-23 T-cell lymphoma cell line provides a novel, physiologically relevant preclinical model for the study of PTCL. Its phenotypic characteristics, rapid growth, and metastatic behavior in syngeneic mice, along with its resistance to CHOP therapy and hyperprogression upon anti-PD1 treatment, mimicking the clinical scenarios observed in some human PTCL patients. These features highlight LM-23's potential as a valuable tool for understanding the biological mechanisms underlying PTCL and for the development and testing of innovative therapeutic strategies. We hope that the LM-23 cell line will contribute significantly to these efforts, ultimately improving outcomes for patients afflicted with PTCL.

TL-895, a Highly Selective, Covalent Inhibitor of Bruton's Tyrosine Kinase (BTK), Sensitizes Myeloproliferative Neoplasm (MPN)-Blast Phase Stem Cells to Navtemadlin By Targeting Intrinsic Dysregulated MDM2/p53 and NF-Κb Pathways and Disrupting the Protective Tumor Microenvironment (TME)

We recently reported that navtemadlin (nvtm), a potent, orally available, selective inhibitor of MDM2 (MDM2i), can deplete/eliminate TP53WT MPN-blast phase (BP) stem cells (SC) in an MPN-BP patient (pt)-derived xenograft (PDX) model (Wang EHA 2023). Single agent nvtm has demonstrated significant clinical activity in pts with advanced myelofibrosis (MF) (Vachhani EHA 2023, Verstovsek Future Oncology 2022). We hypothesized that effects of nvtm on MPN-BP, which is characterized by multiple SC subclones, could be enhanced by combination with agents that target additional MPN-BP SC vulnerabilities, such as hyperactivation of p65 subunit of NF-κB (Fisher Leukemia 2017; our unpublished data) and dependence on TME support. BTK and bone marrow (BM) kinase X-linked (BMX) are key Tec family members expressed in cells of myeloid origin. Here, we show BTK is constitutively activated in MPN-BP SC subpopulations relative to normal donor (ND) hematopoietic SCs (HSC) and progenitors [phospho (p)-BTK Y233 mean fluorescence intensity fold change (FC)]: CD34 +CD38 - cells: 1.34±0.18, HSC/multipotent progenitor (MPP): 1.50±0.23, multi-lymphoid progenitor (MLP): 1.59±0.59, megakaryocyte-erythroid progenitor (MEP): 1.56±0.49; p <0.05]. TL-895 is a highly selective, potent, orally available, second-generation covalent inhibitor of BTK and BMX, currently under clinical evaluation for treatment of pts with CLL (NCT02825836) and MF (NCT04655118). We used multi-modal single-cell analysis to study mechanistic effects of combination TL-895 + nvtm on dysregulated intracellular signaling pathways in primary MPN-BP SC, and investigated the combination in a PDX model of MPN-BP. In vitro treatment with TL-895 alone (100 nM-5 µM) did not affect growth of ND CD34 + cells [FC (TL-895 vs. vehicle, veh): 2.5 µM: 1.27±0.11, 5 µM: 0.97±0.14, n=3)] but modestly decreased growth of primary TP53WT MPN-BP CD34 + cells, in a dose dependent fashion [FC (TL-895 vs. veh): 2.5 µM: 0.88±0.11, 5 µM: 0.62±0.12, n=4. p<0.05). Addition of TL-895 (5 µM) to nvtm (400 nM) resulted in much greater reductions in MPN-BP CD34 + and CD34 +CD38 - cell numbers, along with increased apoptosis (n=6, p<0.05, TL-895 + nvtm vs. veh, TL-895, or nvtm alone). By contrast, at these doses, combination TL-895 + nvtm did not affect ND HSC subpopulations (n=3). We previously demonstrated that, multiple MPN-BP SC subclones contribute to the development of MPN-BP irrespective of clone size, and minor subclones may be responsible for clonal evolution and disease progression in most cases (Wang JCI insight 2022). Strikingly, single cell mutational profiling and epitope analysis of primary MPN-BP CD34 + cells treated with TL-895 + nvtm showed depletion of all 8 distinct MPN-BP SC subclones with differing variant allele frequencies, each containing 2-3 oncogenic mutations in WT1, ETV6 and/or KRAS. Combination TL-895 + nvtm treatment depleted 7 of 8 subclones to a greater degree than either agent alone (Fig. 1). Bulk RNA-seq and gene set enrichment analysis showed that combination TL-895 + nvtm synergistically upregulated p53 pathway gene transcription ( CDKN1 HDM2, DDB2, BTG2, GADD45A, FAS, FUCA1, S100A10, ATF3) in primary MPN-BP CD34 + cells. Mass cytometry analysis and intracellular flow cytometry of MPN-BP CD34 + cells treated with combination TL-895 + nvtm further revealed significant (p<0.05) upregulation of p53 and BAX proteins and decreases in the anti-apoptotic factors BCL-xL and activated NF-κB (p-p65), as well as significant reductions in DNA synthesis and protein markers associated with glycolysis (p-AMPK, MCT1). Downregulation of NF-κB activation might result from decreases in the upstream modulators p-S6 and p-4EBP1 (p<0.05, TL-895 + nvtm vs. veh). Treatment in vivo withTL-895 promoted dose-dependent egress of hCD45 +CD34 + cells from BM into the spleen and peripheral blood (PB) of MPN-BP PDX mice (Fig. 2). Furthermore, a limited course of combination TL-895 + nvtm treatment for 3 cycles led to clearance of hCD45 +CD34 + cells from the BM, spleen and PB of MPN-BP PDX mice (Fig. 2); neither treatment had this effect when administered alone. Our results indicate that BTKi therapy might increase susceptibility of MPN-BP SC to MDM2i therapy, by upregulating p53 activity and dampening NF-κB signaling, and also by disrupting protective TME interactions that sustain MPN-BP SC. This novel combination merits further clinical investigation in advanced phase MPN.

Reduced Polycomb Repressor Complex 2 (PRC2) Activity and Increased TP53 Activity Mediate Hematopoietic Stem Cell Dysfunction in RPS19-Mutated Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia caused by heterozygous loss-of-function mutations in one of 23 ribosomal protein (RP) genes, with RPS19 being mutated in approximately 25% of patients. While DBA typically manifests in infancy as isolated anemia with reticulocytopenia, older patients can develop multilineage cytopenias and bone marrow (BM) hypocellularity suggesting impaired hematopoietic stem cell (HSC) function. Defects in HSCs have been observed in Rps19-depleted mice but studies on human DBA HSCs have been inconclusive, largely because patient BM cells are difficult to obtain for research. To address this gap, we developed a new, robust, and scalable cellular model using CRISPR/Cas9 to create heterozygous RPS19 loss-of-function mutations in healthy donor CD34 + hematopoietic stem and progenitor cells (HSPCs). Transplantation of RPS19+/- HSPCs into NBSGW immunodeficient mice revealed a defect in BM repopulation that was partially rescued by co-disruption of the TP53 gene (Bhoopalan et al, JCI Insight, 2023). Here we provide further mechanistic insight into this HSC defect by showing that RP imbalance caused by RPS19 deficiency leads to the activation of TP53 and inhibition of Polycomb Repressor Complex 2 (PRC2), which has been previously shown to be required for HSC maintenance. Healthy donor CD34 + HSPC were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 safe harbor locus as a negative control. RPS19 disruption was verified by next-generation sequencing of the targeted region and by Western blot analysis showing reduction of RPS19 protein. RNA-Seq at 3 days after electroporation showed enrichment of TP53 pathway genes in RPS19-edited HSPCs compared to control cells, confirming our previous observations. Additionally, we noted an enrichment of HSPC genes that were previously shown to harbor the repressive histone mark H3K27me3 in normal HSPCs. Methylation of H3K27 is catalyzed by EZH2, the catalytic component of PRC2, which maintains HSCs by inhibiting transcription of genes that promote differentiation. Gene set enrichment analysis (GSEA) revealed significant induction of PRC2 target genes in RPS19-disrupted HSPCs and in induced pluripotent stem cells (iPSCs) generated from two different DBA patients with heterozygous RPS19 mutations. The “Cleavage under targets and release using nuclease (CUT&RUN)” assay indicated globally reduced EZH2 binding to chromatin in RPS19-disrupted HSPCs. These findings suggest that PRC2 activity is inhibited by RPS19 haploinsufficiency. Previous studies have shown that RPS19 haploinsufficiency can cause accumulation of large RP subunits, RPL5 and RPL11, which bind 5S ribosomal RNA to form 5S RNP. This complex can sequester the ubiquitin ligase MDM2 to inhibit its function. MDM2 ubiquitinates TP53, targeting it for proteasomal degradation. Additionally, MDM2 binds PRC2 to augment its activity. Thus, we theorized that RPS19 haploinsufficiency leads to the accumulation of 5S RNP complex which sequesters MDM2, causing increased TP53 activity and suppression of PRC2 activity, both of which can inhibit HSC maintenance. To test this, we inhibited formation of 5S RNP complex by co-disrupting RPL5 or RPL11 along with RPS19 in CD34 + HSPCs, followed by RNA-Seq analysis or transplantation into NBSGW mice. Remarkably, haploinsufficiency of either large RP gene rescued the BM repopulation defect of RPS19 +/- HSPC (p<0.05) ( Fig. 1A), reduced TP53 activity and improved PRC2 activity (p<0.001 and FDR q<0.01) ( Fig. 1B). In summary, our data support the model that RPS19 haploinsufficiency leads to increased TP53 activity and reduced PRC2 activity through 5S RNP-mediated sequestration of MDM2, causing impairment of HSC function. These studies provide insights into the molecular mechanisms of DBA and could potentially inform new therapies.

Editorial Comment.

No AccessJournal of UrologyTranslational Research Article26 May 2023Editorial CommentThis article comments on the following:Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance Udit Singhal and Simpa S. Salami Udit SinghalUdit Singhal Department of Urology, Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan Department of Urology, Mayo Clinic, Rochester, Minnesota More articles by this author and Simpa S. SalamiSimpa S. Salami Department of Urology, Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003507.01AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookLinked InTwitterEmail "Editorial Comment." The Journal of Urology, Publish Ahead of Print, pp. REFERENCES 1. Molecular biomarkers in localized prostate cancer: ASCO guideline. J Clin Oncol. 2020; 38:1474-1494. Crossref, Medline, Google Scholar 2. Transcriptomic heterogeneity in multifocal prostate cancer. JCI Insight. 2018; 3(21):e123468. Crossref, Medline, Google Scholar 3. Multimodal biomarkers that predict the presence of Gleason pattern 4: potential impact for active surveillance. J Urol. 2023; 210(2):000. Google Scholar 4. . EDRN Prostate MRI Biomarker Study (P-MRI); 2023. https://ClinicalTrials.gov/show/NCT03784924. Google Scholar Submitted April 19, 2022; accepted April 20, 2023; published 000. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetailsRelated articlesJournal of Urology1 May 2023Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Udit Singhal Department of Urology, Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan Department of Urology, Mayo Clinic, Rochester, Minnesota More articles by this author Simpa S. Salami Department of Urology, Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan More articles by this author Expand All Submitted April 19, 2022; accepted April 20, 2023; published 000. Advertisement PDF downloadLoading ...

Abstract 663: Resistin mediates the immunoediting activity exerted by primary human macrophages towards mesothelioma cells

Abstract Tumor-associated macrophages (TAM) are particularly abundant (i.e. 27% ± 9%) in the microenvironment of malignant pleural mesothelioma (MPM). These macrophages do not correspond to classical (M1) and alternatively-activated (M2) phenotypes but, instead, are shaped by local inflammatory mediators released in the pleura. We previously showed that, besides classical functions exerted by macrophages (i.e. phagocytosis, cytokine expression and antigen presentation), murine RAW264.7 cells are directly cytotoxic to MPM tumors (Hamaidia et al, JCI Insight 4:e128474). Upon direct cell-to-cell contact, RAW264.7 macrophages kill mesothelioma cells by a mechanism of oxeiptosis involving the enhancer of zeste homolog 2 (EZH2) methyltransferase. We have now investigated this immune-editing activity in human mesothelioma. We show that M1-activated primary macrophages are more cytotoxic for M14K cells than M2. The killing activity of M1 macrophages is dependent on NADPH oxidase activity and peroxynitrite levels. Mesothelioma cells and M2 macrophages interact through an inhibitory synapse characterized by engagement of the PD-1 receptor. Consistently, the immune-editing activity of M2 macrophages is partially restored in presence of neutralizing anti-PD1 antibody. Primary human macrophages cultured in presence of pleural effusions of MPM patients are less cytotoxic than M1 and are unable to inhibit tumor growth in mice. Macrophages differentiated in pleural effusions display a broad spectrum of cytotoxic activities (i.e. from complete inactivity to rates similar to those of M2). Luminex profiling of pleural fluids has identified a series of key mediators associated with the cytotoxic phenotype. The best correlation is obtained with resistin (RETN, FIZZ3, ADSF). Recombinant human resistin improves the killing activity of pleural effusion macrophages in cell culture. Gene transduction of RETN in RAW264.7 impairs growth of AB12 tumors in BALB/c mice. In conclusion, we have shown that resistin is the main factor that mediates immunoediting activity exerted by primary human macrophages towards mesothelioma cells, opening new prospects for therapeutic intervention. Citation Format: Malik Hamaidia, Majeed Jamakhani, Jean-Rock Jacques, Alexis Fontaine, Arnaud Scherpereel, Eric Wasielewski, Louis Renaud, Vincent Heinen, Bernard Duysinx, Luc Willems. Resistin mediates the immunoediting activity exerted by primary human macrophages towards mesothelioma cells [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 663.

Abstract 5076: Combination PancVAX neo-epitope vaccine with anti-CTLA-4 and anti-PD-1 antibodies enhances infiltration of cytotoxic T cells and mitigates T cell exhaustion in a murine model of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden and therefore few neoantigen targets that can be recognized by cytotoxic T cells. Most PDACs are thus insensitive to either single or dual immune checkpoint inhibitor (ICI) therapy. Personalized neoantigen vaccines can expand the number and repertoire of anti-tumor T cells that infiltrate the tumor and mediate cytotoxicity. To model a personalized neoantigen vaccine treatment strategy in PDAC, we previously developed PancVAX, a peptide-based vaccine targeting 12 neoantigens expressed in the murine pancreatic cell line Panc02 (Kinkead et al, JCI Insight 2018). Although we observed increased T cell infiltration present in the tumor post-vaccination, these cells expressed high levels of exhaustion markers. We therefore hypothesized that sequential administration of anti-CTLA-4 and anti-PD-1 would enhance the pool of T cells primed by the neoantigen vaccine and maintain activation of antigen-experienced T cells, respectively, to yield optimal and durable neoantigen-specific anti-tumor immunity in PDAC. To address this, mice bearing subcutaneous Panc02 tumors were vaccinated with two rounds of the PancVAX neoantigen vaccine followed by anti-CTLA-4 and anti-PD-1 3 days later. Anti-PD-1 maintenance was given twice weekly beginning at the first vaccine dose. Twelve days after the last peptide vaccine dose, tumors were harvested and dissociated into single-cell suspensions for paired single-cell RNA-sequencing and TCR-sequencing. Mice that were untreated or given ICIs without PancVAX had the highest proportions of CD8+ T cells expressing exhaustion markers. PancVAX-treated mice had more intratumoral cycling CD8 T cells and effector CD8+ T cells with high cytotoxic gene expression. Among mice treated with PancVAX, tumors from mice treated with PancVAX + anti-PD1 or PancVAX + anti-PD1 + anti-CTLA-4 had the highest proportions of effector CD8+ T cells. Ongoing analyses include differential gene expression and pathway analysis between treatment conditions in the T cell compartment in mice treated with combination ICI and PancVAX. Additionally, we will assess changes in T cell clonality and diversity within the tumors when mice are treated with single or combination therapy. These results will define a transcriptional signature associated with the generation of a productive anti-tumor immune response when neoantigen vaccines and ICI are used in combination. This work demonstrates how the addition of ICIs to personalized neo-epitope vaccines for PDAC can further enhance the quality of vaccine-induced T cell effector function in an otherwise immunologically cold tumor type and supports their inclusion in neoantigen vaccination strategies for patients with PDAC. Citation Format: Jacob T. Mitchell, Amanda Huff, Emily Davis-Marcisak, Fangluo Chen, Todd D. Armstrong, Luciane T. Kagohara, James Leatherman, Rulin Wang, Srinivasan Yegnasubramanian, Elizabeth M. Jaffee, Elana J. Fertig, Neeha Zaidi. Combination PancVAX neo-epitope vaccine with anti-CTLA-4 and anti-PD-1 antibodies enhances infiltration of cytotoxic T cells and mitigates T cell exhaustion in a murine model of pancreatic ductal adenocarcinoma. [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 5076.

Abstract 608: Low avidity neoepitope-specific CD8+ tumor infiltrating lymphocytes are less exhausted and more effective than their high avidity counterparts in a BALB/c murine fibrosarcoma

Abstract The frequency of CD8+ tumor infiltrating lymphocytes (TILs) has been positively associated with overall survival in several cancers. The interaction of T cell receptors (TCRs) on T cells with Major Histocompatibility Complex (MHC)-peptide complexes on cancer cells elicits the cytotoxic activity of CD8+ T cells. Here we set out to understand how the strength of this interaction, or T cell avidity, shapes the phenotype of CD8+ TILs and how that dictates anti-tumor immunity. We developed a novel tetramer decay assay to isolate T cells based on their TCR avidities. We then used this method to isolate from tumors of Meth A tumor-bearing mice (28 days after tumor challenge), Low (K off < 15 min), Medium (75 min > K off > 45 min), and High (K off > 105 min) avidity CD8+ TILs responding to the tumor rejection neoepitope, PDPRMUT of this fibrosarcoma (Ebrahimi-Nik et al. JCI Insight 2019). Unbiased clustering of single cell RNA sequencing, TCR sequencing, and CITE-seq of these PDPRMUT -specific CD8+ TILs revealed five clusters: Stem-Like (LY108+Tcf7+), Effector-Exhausted (Eff-Exh PD-1Low,CX3CR1+) Terminally-Exhausted-1 (Term-Exh1 PD-1+TIM3High), Terminally-Exhausted-2 (Term-Exh2 PD-1HighToxHigh), and NK-Like (PD-1Low, KlrHigh). The low avidity CD8+ TILs have more than twice the proportion of Eff-Exh (60.0%) when compared to high avidity CD8+ TILs (28.39%). The high avidity CD8+ TILs have more Stem-Like (12.37% vs 6.31%), Term-Exh1 (27.38% vs 21.88%), and Term-Exh2 (24.03% vs 5.33%) cells than low avidity CD8+ TILs. Furthermore, trajectory inference of these clusters, by scVelo, predicts that Stem-Like cells differentiate into Eff-Exh cells that can differentiate into either Term-Exh1 or Term-Exh2. Our data demonstrates that low avidity Eff-Exh cells preferentially differentiate into Term-Exh1, while high avidity Eff-Exh cells differentiate into both Term-Exh1 and Term-Exh2. To validate these results, we isolated CD8+ TILs responding to PDPRMUT with low or high avidity from tumors of 21 Meth A bearing mice 28 days after tumor challenge and analyzed their exhaustion phenotype by flow cytometry. There were significantly more TIM3+PD-1+ cells (Paired t-test; P < 0.0001) and TOX+PD-1+ cells (Paired t-test; P < 0.05) in the high avidity CD8+ TILs than the low avidity CD8+ TILs. We conclude that CD8+ TILs with high avidity are more exhausted and less effector like than low avidity CD8+ TILs. This finding has major implications for T cell-based cancer immunotherapy. Citation Format: Summit Singhaviranon, Joseph P. Dempsey, Adam T. Hagymasi, Pramod K. Srivastava. Low avidity neoepitope-specific CD8+ tumor infiltrating lymphocytes are less exhausted and more effective than their high avidity counterparts in a BALB/c murine fibrosarcoma [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 608.

Abstract 1105: Carbon ion radiotherapy eradicates glioblastoma via tumor immune environment reprograming

Abstract Background and purpose: Glioblastoma multiforme (GBM) is a stroma rich immune cold tumor with still dismal prognosis of despite multimodal therapy consisting of postoperative surgery and radiochemotherapy. Consequently, all randomized Phase III clinical GBM trials attempting to integrate immune checkpoint blockade (ICB) have failed to demonstrate efficacy. We have recently shown that tumor immune microenvironment (TIME) reprograming via Bintrafusp alpha (Anti-PD-L1/TGF-beta bispecific molecule) combined with conventional photon radiotherapy elicits beneficial effects and prolong survival in experimental models (Lan, Knoll, Moustafa et al. Cancer Cell 2021). Moreover, eradication of radioresistant i.e., stem cell like, invasive and hypoxic tumor subpopulations by high linear energy transfer (LET) carbon ion radiotherapy (CIRT) was shown to generate an immune permissive niche in experimental GBM models (Chiblak et al. JCI Insight 2019, Tang et al. IJRBP 2022). Therefore, we aimed to systematically study the effect of CIRT vs. conventional photon radiotherapy (RT) in two (GL261 and SB28) orhotopic syngeneic mouse GBM models. Methods: Tumor growth monitoring was conducted longitudinally by means of bioluminescence and small animal MR-imaging. Mice were irradiated in five fractions (Fx) with a total dose of 15Gy RT vs. 5, 10 and 15Gy CIRT. Results: Intriguingly, all GL261 harbouring mice were cured after 5 × 3Gy Fx CIRT with no sign of tumor recurrence up to 80 days post irradiation. In contrast, no tumor control was found after 5 × 3Gy Fx RT led despite a significant prolongation of OS. The tumor control probability 50% (TCP50) for CIRT in GL261 was estimated to be 10Gy in 5 Fx. To asses if the curative effect was related to tumor eradication or reconstitution of immune surveillance. GL261 tumor cells were implanted in the contralateral hemisphere of cured mice. Single cell and Bulk RNAseq as well as immunostaining were conducted to deconvolute TIME effects post CIRT. Conclusions: In line with mechanistic studies, lack of tumor growth indicating persistent immunity against GL261 was found in CIRT cured mice. Similar experiments are conducted in SB28 model and will be presented. Together, these encouraging data indicate potential benefits for high-LET CIRT in immune modulation of GBM in experimental models that warrants for study and optimization towards clinical translation. Citation Format: Federica Ciamarone, Sarah Meister, Jennifer Furkel, Maximilian Knoll, Christian Schwager, Jovana Bojcevski, Yannik Streibel, Christel Herold-Mende, Michael Breckwoldt, Juergen Debus, Ivana Dokic, Amir Abdollahi, Katharina Schregel. Carbon ion radiotherapy eradicates glioblastoma via tumor immune environment reprograming [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 1105.

The importance of a skilled clinical assessment in the management and healing of complex wounds

British Journal of Community NursingVol. 28, No. Sup3 Society UpdateThe importance of a skilled clinical assessment in the management and healing of complex woundsSylvie HamptonSylvie HamptonE-mail Address: [email protected]Tissue Viability Consultant Nurse; Director of Wound Care Consultancy LtdSearch for more papers by this authorSylvie HamptonPublished Online:21 Feb 2023https://doi.org/10.12968/bjcn.2023.28.Sup3.S40AboutSectionsView articleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareShare onFacebookTwitterLinked InEmail View article References Foster DS, Jones RE, Ransom RC, Longaker MT, Norton JA. The evolving relationship of wound healing and tumor stroma. JCI Insight. 2018;3(18):e99911. https://doi.org/10.1172/jci.insight.99911 Crossref, Medline, Google ScholarNational Institute for Health. Fungi may impair wound healing in Crohn’s disease. 2021. https://tinyurl.com/hwh8228k (accessed 08 February 2023) Google ScholarWound Care Solutions. Hypertension and Wound Healing: How Does Hypertension Affect Pressure Ulcers? Wound Care Solutions. 2017. https://tinyurl.com/mv5rzjms (accessed 08 February 2023) Google Scholar FiguresReferencesRelatedDetails 2 March 2023Volume 28Issue Sup3ISSN (print): 1462-4753ISSN (online): 2052-2215 Metrics History Published online 21 February 2023 Published in print 2 March 2023 Information© MA Healthcare LimitedPDF download

Redox Signaling in Doxorubicin-Induced Ferroptosis

Doxorubicin is among the most widely used anticancer drugs; however, its clinical use is associated with cardiomyopathy and heart failure. Studies show ferroptosis as a pivotal form of cell death underlying doxorubicin cardiomyopathy. Recently, multiple redox signaling pathways have been discovered to underly doxorubicin-induced ferroptosis. This Cutting-Edge Research Highlights discusses these latest advances, focusing on pathways involving Nrf2/HO-1, GPx4, and Alas1/heme synthesis. (First online: February 19, 2023) REFERENCES Zhu H, Sarkar S, Scott L, Danelisen I, Trush MA, Jia Z, et al. Doxorubicin Redox biology: redox cycling, topoisomerase inhibition, and oxidative stress. React Oxyg Species (Apex) 2016; 1(3):189–98. doi: https://dx.doi.org/10.20455/ros.2016.835 Higgins AY, O'Halloran TD, Chang JD. Chemotherapy-induced cardiomyopathy. Heart Fail Rev 2015; 20(6):721–30. doi: https://dx.doi.org/10.1007/s10741-015-9502-y Page RL, 2nd, O'Bryant CL, Cheng D, Dow TJ, Ky B, Stein CM, et al. Drugs that may cause or exacerbate heart failure: a scientific statement from the American Heart Association. Circulation 2016; 134(6):e32–69. doi: https://dx.doi.org/10.1161/CIR.0000000000000426 Fang X, Wang H, Han D, Xie E, Yang X, Wei J, et al. Ferroptosis as a target for protection against cardiomyopathy. Proc Natl Acad Sci USA 2019; 116(7):2672–80. doi: https://dx.doi.org/10.1073/pnas.1821022116 Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 2012; 149(5):1060–72. doi: https://dx.doi.org/10.1016/j.cell.2012.03.042 Yang WS, Kim KJ, Gaschler MM, Patel M, Shchepinov MS, Stockwell BR. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc Natl Acad Sci USA 2016; 113(34):E4966–75. doi: https://dx.doi.org/10.1073/pnas.1603244113 Kagan VE, Mao G, Qu F, Angeli JP, Doll S, Croix CS, et al. Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat Chem Biol 2017; 13(1):81–90. doi: https://dx.doi.org/10.1038/nchembio.2238 Ichikawa Y, Ghanefar M, Bayeva M, Wu R, Khechaduri A, Naga Prasad SV, et al. Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation. J Clin Invest 2014; 124(2):617–30. doi: https://dx.doi.org/10.1172/JCI72931 Tadokoro T, Ikeda M, Ide T, Deguchi H, Ikeda S, Okabe K, et al. Mitochondria-dependent ferroptosis plays a pivotal role in doxorubicin cardiotoxicity. JCI Insight 2020; 5(9). doi: https://dx.doi.org/10.1172/jci.insight.132747 Abe K, Ikeda M, Ide T, Tadokoro T, Miyamoto HD, Furusawa S, et al. Doxorubicin causes ferroptosis and cardiotoxicity by intercalating into mitochondrial DNA and disrupting Alas1-dependent heme synthesis. Sci Signal 2022; 15(758):eabn8017. doi: https://dx.doi.org/10.1126/scisignal.abn8017