Citations

CD19-targeted chimeric antigen receptor T (CART19) cell therapy has shown outstanding clinical success. However, patients can develop CART-induced toxicities, including cytokine release syndrome (CRS) and neurotoxicity. Studies have found that activation of myeloid cells by granulocyte monocyte-colony stimulating factor (GM-CSF) can contribute to toxicity development. GM-CSF depletion leads to reduced myeloid activation, prevention of CART-induced toxicities, and improved CART efficacy. CART cells upregulate cytokine receptors upon activation, and our results indicate that CART19 cells significantly upregulate GMCSFR upon antigen-specific stimulation (<10% vs >30%, p<0.01). This led us to hypothesize that GMCSFR signaling contributes to CART cell activity. To understand the role of GM-CSFR upregulation on CART cell functions, we compared the impacts of neutralizing GM-CSF or blocking GM-CSF receptor alpha (GM-CSFRα) on CART19. First, 4-1BB-costimulated CART19 cells generated from healthy donors were stimulated with CD3/CD28 beads at a 1:3 ratio for 6 days. CART cells were maintained at 1 x 106/mL throughout the culture and supplemented with IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade at 10 μg/mL, 20 μg/mL, or 100 μg/mL. On day 6, cells were debeaded and rested for 2 days. Then, CART cells were cocultured with NALM6, a CD19+ acute lymphoblastic leukemia cell line, at a ratio of 1:1 for 3 days. The cocultures were supplemented with the same doses of antibodies listed above. Antigen-specific proliferation of CART cells was measured by flow cytometry. Our data suggested that CART19 proliferated is not impacted by GM-CSF neutralization but is inhibited by GM-CSFRα blockade (IgG isotype vs 10 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 20 μg/mL GM-CSFRα blockade, p<0.0001; IgG isotype vs 100 μg/mL GM-CSFRα blockade, p=0.009). CART cells were assessed in a cytotoxicity assay with NALM6 luciferase+ cells as well, which did not show significant impacts from GM-CSF neutralization or GM-CSFRα blockade. To evaluate the impacts of GM-CSF neutralization and GM-CSFRα blockade in a mouse model, we engrafted NSG mice with 1 million NALM6 luciferase+ cells intravenously. Mice were then treated with 1.4 million CART19 plus 10 mg/kg IgG isotype control, GM-CSF neutralizing antibody, or GM-CSFRα blockade intraperitoneally. GM-CSFRα blockade drastically suppressed antitumor activities of CART (CART19+GM-CSFRα blockade vs CART19+IgG isotype control, p<0.0001; CART19+GM-CSFRα blockade vs CART19+GM-CSF neutralizing antibody, p<0.0001; CART19+ GM-CSFRα blockade vs untransduced T cell, n.s.). Serum cytokines were analyzed 2 days after CART treatment, showing that mice with GM-CSFRα blockade had significantly lower levels of human IFNγ (p=0.0189) and IL-2 (p=0.0121) compared to IgG isotype control group, while GM-CSF neutralization did not show significant impacts. Mice treated with CART plus IgG isotype control or GM-CSF neutralizing antibody showed prolonged survival compared to mice treated with GM-CSFRα blockade or untransduced T cells (p=0.0285). To unravel the mechanisms behind functional differences between GM-CSFRα blockade and GM-CSF neutralization, CART cells (n = 3 biological replicates) were cocultured with NALM6 at a 1:1 ratio supplemented with 10 μg/mL GM-CSF neutralizing antibody, GM-CSFRα blockade, or IgG isotype control overnight, followed by T cell isolation, bulk RNA isolation, and RNA sequencing (RNAseq) analysis. Our RNAseq data indicated significant upregulation (Log2FC>0.5, padj. <0.05) of T cell exhaustion- and dysfunction-associated genes (RGS16, PDCD1, CTLA4, NR4A3, CD101, LAG3) as well as negative regulators of T cell functions (G0S2, PIK3IP1, LGALS3) in the GM-CSFRα blockade group compared to GM-CSF neutralizing antibody or IgG isotype control groups. T cell activation-associated pathways were enriched in the GM-CSFRα blockade group. Overall, our data show that GM-CSFRα blockade impacts CART functions differently from GM-CSF neutralization. Specifically, GM-CSFRα blockade inhibits CART proliferation in vitro and antitumor activity in vivo. Interestingly, GM-CSFRα blockade leads to enrichment of T cell exhaustion- and dysfunction-associated genes in CART19, which is worth further investigation. These findings may be applicable to other cytokine receptors upregulated on activated CART cells.

Introduction: The incidence of cardiovascular events in the first 100 days post-HSCT was found to be 19%, with arrhythmia having the highest incidence of 12% (p<0.0001). The incidence of cardiovascular events is 6.7% in patients with multiple myeloma treated with HSCT (HSCT-MM) [1]. However, the literature on arrhythmia in HSCT-MM patients is limited. Given the high risk of cardiac events in HSCT patients, we aimed to determine the impact of arrhythmia in patients with HSCT-MM. Methods: We conducted a retrospective analysis using the National Inpatient Sample Database (2018-2021). Adult patients with HSCT-MM who were primarily admitted for life-threatening arrhythmias were identified using ICD-10 codes [2]. We performed propensity matching and multivariate regression, considering a p-value of ≤0.05 as significant. Results: In 14,232 HSCT-MM patients, 168 (1.2%) had arrhythmia. The prevalence of arrhythmia in HSCT-MM patients was higher than in the remaining HSCT recipients (1.2% vs 0.9%, p<0.01). In HSCT-MM, atrial fibrillation (69.6%) and atrial flutter (19%) were the common arrhythmias. Arrhythmia in HSCT-MM patients was more prevalent in those over the age of 60 years (84.5% vs 68.5%, p<0.001), males (72.6% vs 58.2%, p<0.001), and in the White race (84.8% vs 67.3%, p<0.001) compared to those without arrhythmia. Arrhythmia was less common in those with bone marrow dysfunction compared to those with normocellular marrow (anemia 1% vs 1.5%, p<0.01; thrombocytopenia 1% vs 1.2%, p>0.05; neutropenia 0.7% vs 1.3%, p<0.05; pancytopenia 0.9% vs 1.4%, p<0.01). Among the HSCT-MM patients with arrhythmia, long-term anticoagulation was less frequently given than those without arrhythmia (44.6% vs 55.4%, p<0.001). The arrhythmia cohort had less frequent acute kidney injury (19.1% vs 27.5%, p=0.01). The arrhythmia cohort required more vasopressors (2.4% vs 2.1%, p>0.05) but had lower mortality (1.2% vs 4.5%, p<0.05). Patients with arrhythmia had shorter hospital stays (Coefficient -3 days; 95% CI -3.8 to -2.3 days; p<0.001) and lesser hospital charges (Coefficient $-41,200; CI $-55,700 to $-26,600; p<0.001) compared to those without. However, the arrhythmia cohort had higher rates of acute heart failure (17.9% vs 9.1%, p<0.001) and the risk of acute heart failure was also higher in those with arrhythmia (adjusted odds ratio 2.2; 95% CI 1.3 to 3.6; p<0.01). In patients with acute heart failure and arrhythmia, atrial fibrillation was present in 76.7% and atrial flutter in 20%. In patients with acute heart failure and arrhythmia, bone marrow dysfunction was more common than normocellular bone marrow (anemia 22.2% vs 12.8%, p>0.05; thrombocytopenia 38.7% vs 13.1%, p=0.001; neutropenia 18.2% vs 14.3%, p>0.05; pancytopenia 8.7% vs 21.3%, p>0.05). In the arrhythmia cohort, long-term anticoagulation was commonly given in those with acute heart failure compared to those without (53.3% vs 46.7%, p>0.05), and acute kidney injury also occurred more frequently in those with heart failure (26.7% vs 17.4%, p>0.05). Conclusion: Acute heart failure is a serious risk associated with arrhythmia in multiple myeloma patients treated with HSCT, with atrial fibrillation emerging as the most common arrhythmia. It is essential to prevent heart failure in those with arrhythmia, especially in patients with thrombocytopenia. Preventive measures could include more frequent follow-ups and having a multidisciplinary team to manage renal failure, monitor anticoagulation, and ensure rhythm control in these patients. References: Aghel N, Lui M, Wang V, et al.: Cardiovascular events among recipients of hematopoietic stem cell transplantation-A systematic review and meta-analysis. Bone Marrow Transplant. 2023, 58:478-90. 10.1038/s41409-023-01928-22021 ICD-10-CM. Accessed: July 14, 2024. https://www.cms.gov/medicare/coding-billing/icd-10-codes/2021-icd-10-cm.

Abstract Senescent cells drive tissue dysfunction through the senescence-associated secretory phenotype (SASP). We uncovered a central role for mitochondria in the epigenetic regulation of the SASP, where mitochondrial-derived metabolites, specifically citrate and acetyl-CoA, fuel histone acetylation at SASP gene loci, promoting their expression. We identified the mitochondrial citrate carrier (SLC25A1) and ATP-citrate lyase (ACLY) as critical for this process. Inhibiting these pathways selectively suppresses SASP without affecting cell cycle arrest, highlighting their potential as therapeutic targets for age-related inflammation. Notably, SLC25A1 inhibition reduces systemic inflammation and extends healthspan in aged mice, establishing mitochondrial metabolism as pivotal to the epigenetic control of aging.

Abstract DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematologic neoplasms. Although individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here, we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity (acute respiratory distress syndrome [ARDS]) and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant (DNMT3Amt) CH on COVID-19–related ARDS and mortality. Using single-cell proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid lineage cells. Using single-cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of interleukin-32 (IL-32), a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single-cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Hence, we demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19–related ARDS and mortality. DNMT3Amt CH in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can thus serve as a potential biomarker for adverse outcomes in COVID-19.