IL-9 Secreted by Leukemia Stem Cells Induces Th1-Skewed CD4+ T-Cells, which Promote Their Expansion.

In acute myeloid leukemia (AML), leukemia stem and progenitor cells (LSCs and LPCs) interact with various cell types in the bone marrow (BM) microenvironment, regulating their expansion and differentiation. To study the interaction of CD4+ and CD8+ T-cells in the BM with LSCs and LPCs, we analyzed their transcriptome and predicted cell-cell interactions by unbiased high-throughput correlation network analysis. We found that CD4+ T-cells in the BM of AML patients were activated and skewed towards Th1-polarization whereas IL-9 producing (Th9) CD4+ T-cells were absent. In contrast to normal hematopoietic stem cells (HSCs), LSCs produced IL-9 and the correlation modelling predicted IL9 in LSCs as a main hub-gene that activates CD4+ T-cells in AML. Functional validation revealed that IL-9R signaling in CD4+ T-cells leads to activation of the JAK-STAT pathway that induces the upregulation of KMT2A, KMT2C genes resulting in methylation on histone H3 at lysine 4 (H3K4) to promote genome accessibility and transcriptional activation. This induced Th1-skewing, proliferation and effector cytokine secretion, including interferon (IFN)-É£ and tumor necrosis factor (TNF)-α. IFN-É£ and to a lesser extend TNF-α produced by activated CD4+ T-cells, induced the expansion of LSCs. In accordance with our findings, high IL9 expression in LSCs and high IL9R, TNF and IFNG expression in BM-infiltrating CD4+ T-cells correlated with worse overall survival in AML. Thus, IL-9 secreted by AML LSCs shapes a Th1-skewed immune environment that promotes their expansion by secreting IFN-É£ and TNF-α.

Reverse Phase Proteomic Array Profiling of Asparagine Synthetase Expression in Newly Diagnosed Acute Myeloid Leukemia.

Asparaginase-based therapy is a cornerstone in acute lymphoblastic leukemia (ALL) treatment, capitalizing on the methylation status of the asparagine synthetase (ASNS) gene, which renders ALL cells reliant on extracellular asparagine. Contrastingly, ASNS expression in acute myeloid leukemia (AML) has not been thoroughly investigated, despite studies suggesting that AML with chromosome 7/7q deletions might have reduced ASNS levels. Here, we leverage reverse phase protein arrays to measure ASNS expression in 810 AML patients and assess its impact on outcomes. We find that AML with inv(16) has the lowest overall ASNS expression. While AML with deletion 7/7q had ASNS levels slightly lower than those of AML without deletion 7/7q, this observation was not significant. Low ASNS expression correlated with improved overall survival (46 versus 54 weeks, respectively, p = 0.011), whereas higher ASNS levels were associated with better response to venetoclax-based therapy. Protein correlation analysis demonstrated association between ASNS and proteins involved in methylation and DNA repair. In conclusion, while ASNS expression was not lower in patients with deletion 7/7q as initially predicted, ASNS levels were highly variable across AML patients. Further studies are needed to assess whether patients with low ASNS expression are susceptible to asparaginase-based therapy due to their inability to augment compensatory ASNS expression upon asparagine depletion.

Time-Dependent and Coating Modulation of Tomato Response upon Sulfur Nanoparticle Internalization and Assimilation: An Orthogonal Mechanistic Investigation.

Nanoenabled strategies have recently attracted attention as a sustainable platform for agricultural applications. Here, we present a mechanistic understanding of nanobiointeraction through an orthogonal investigation. Pristine (nS) and stearic acid surface-modified (cS) sulfur nanoparticles (NPs) as a multifunctional nanofertilizer were applied to tomato (Solanum lycopersicumL.) through soil. Both nS and cS increased root mass by 73% and 81% and increased shoot weight by 35% and 50%, respectively, compared to the untreated controls. Bulk sulfur (bS) and ionic sulfate (iS) had no such stimulatory effect. Notably, surface modification of S NPs had a positive impact, as cS yielded 38% and 51% greater shoot weight compared to nS at 100 and 200 mg/L, respectively. Moreover, nS and cS significantly improved leaf photosynthesis by promoting the linear electron flow, quantum yield of photosystem II, and relative chlorophyll content. The time-dependent gene expression related to two S bioassimilation and signaling pathways showed a specific role of NP surface physicochemical properties. Additionally, a time-dependent Global Test and machine learning strategy applied to understand the NP surface modification domain metabolomic profiling showed that cS increased the contents of IA, tryptophan, tomatidine, and scopoletin in plant leaves compared to the other treatments. These findings provide critical mechanistic insights into the use of nanoscale sulfur as a multifunctional soil amendment to enhance plant performance as part of nanoenabled agriculture.

Opioid Prescribing and Outcomes in Patients With Sickle Cell Disease Post-2016 CDC Guideline.

Importance: Although the intention of the 2016 US Centers for Disease Control and Prevention (CDC) Guideline for Prescribing Opioids for Chronic Pain was not to limit pain treatment for patients with sickle cell disease (SCD), clinicians and patients have recognized the possibility that the guideline may have altered outcomes for this population. However, the outcomes of the 2016 guideline for this patient population are unknown. Objective: To examine changes in opioid prescribing patterns and health outcomes among patients with SCD before and after the release of the 2016 CDC guideline. Design, Setting, and Participants: This retrospective cohort study conducted interrupted time series analysis of claims data from the Merative MarketScan Commercial Database from January 1, 2011, to December 31, 2019. In this population-based study in the US, individuals with SCD who were at least 1 year of age, had no cancer diagnosis, and had pharmacy coverage for the month of measurement were included. The data were analyzed from January 2021 to November 2023. Exposure: The CDC Guideline for Prescribing Opioids for Chronic Pain released in March 2016. Main Outcomes and Measures: The main variables measured in this study included the practice of opioid prescribing among patients with SCD (ie, rate of opioid prescriptions dispensed, mean number of days supplied, mean total morphine milligram equivalents [MME] per patient, and mean daily MME per opioid prescription) and pain-related health outcomes (rates of emergency department visits related to vaso-occlusive crises [VOC] and hospitalizations related to VOC). Results: The cohort included 14 979 patients with SCD (mean [SD] age, 25.9 [16.9] years; 8520 [56.9%] female). Compared with the preguideline trends, the following changes were observed after the guideline was released: significant decreases in the coefficient for change in slope of the opioid dispensing rate (-0.29 [95% CI, -0.39 to -0.20] prescriptions per 100 person-month; P < .001), the number of days supplied per prescription (-0.05 [95% CI, -0.06 to -0.04] days per prescription-month; P < .001), and opioid dosage (-141.0 [95% CI, -219.5 to -62.5] MME per person-month; P = .001; -10.1 [95% CI, -14.6 to -5.6] MME/prescription-month; P < .001). Conversely, a significant increase in VOC-related hospitalizations occurred after the guideline release (0.16 [95% CI, 0.07-0.25] hospitalizations per 100 person-month; P = .001). These changes were observed to a greater extent among adult patients, but pediatric patients experienced similar changes in several measures, even though the guideline focused exclusively on adult patients. Conclusions and Relevance: This retrospective cohort study showed that the 2016 CDC guideline may have had unintended negative outcomes on the patient population living with SCD.

Paired single-B-cell transcriptomics and receptor sequencing reveal activation states and clonal signatures that characterize B cells in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is associated with a dismal prognosis. Immune checkpoint blockade (ICB) to induce antitumor activity in AML patients has yielded mixed results. Despite the pivotal role of B cells in antitumor immunity, a comprehensive assessment of B lymphocytes within AML's immunological microenvironment along with their interaction with ICB remains rather constrained. METHODS: We performed an extensive analysis that involved paired single-cell RNA and B-cell receptor (BCR) sequencing on 52 bone marrow aspirate samples. These samples included 6 from healthy bone marrow donors (normal), 24 from newly diagnosed AML patients (NewlyDx), and 22 from 8 relapsed or refractory AML patients (RelRef), who underwent assessment both before and after azacitidine/nivolumab treatment. RESULTS: We delineated nine distinct subtypes of B cell lineage in the bone marrow. AML patients exhibited reduced nascent B cell subgroups but increased differentiated B cells compared with healthy controls. The limited diversity of BCR profiles and extensive somatic hypermutation indicated antigen-driven affinity maturation within the tumor microenvironment of RelRef patients. We established a strong connection between the activation or stress status of naïve and memory B cells, as indicated by AP-1 activity, and their differentiation state. Remarkably, atypical memory B cells functioned as specialized antigen-presenting cells closely interacting with AML malignant cells, correlating with AML stemness and worse clinical outcomes. In the AML microenvironment, plasma cells demonstrated advanced differentiation and heightened activity. Notably, the clinical response to ICB was associated with B cell clonal expansion and plasma cell function. CONCLUSIONS: Our findings establish a comprehensive framework for profiling the phenotypic diversity of the B cell lineage in AML patients, while also assessing the implications of immunotherapy. This will serve as a valuable guide for future inquiries into AML treatment strategies.

Single-Cell CD4 and CD8 T-Cell Secretome Profiling Reveals Temporal and Niche Differences in Acute Myeloid Leukemia Following Immune Checkpoint Blockade Therapy.

Acute myeloid leukemia (AML) is a heterogeneous malignancy of the blood primarily treated with intensive chemotherapy. The allogeneic T-cell antileukemic activity via donor lymphocyte infusions and stem cell transplantation suggests a potential role for checkpoint blockade therapy in AML. While clinical trials employing these treatments have fallen short of expected results, a deeper exploration into the functional states of T cells in AML could bridge this knowledge gap. In this study, we analyzed the polyfunctional activity of T cells in a cohort of patients with relapsed/refractory (RelRef) AML treated on the clinical trial (ClinicalTrials.gov identifier: NCT02397720) of combination therapy using azacitidine and nivolumab (Aza/Nivo). We utilized the single-cell polyfunctional multiplexed immune assay IsoPlexis to evaluate the CD4 and CD8 T cells in peripheral blood and bone marrow samples collected before and after immunotherapy. This revealed at a pseudobulk level that the CD4 T cells exhibited higher functional activity post-immunotherapy (post-IO), suggesting that CD4-directed therapies may play a role in RelRef AML. Additional single-cell analysis revealed significant differences in baseline polyfunctionality in bone marrows of responders as compared with nonresponders for both CD4 and CD8 T cells. Overall, this study highlights the impact of polyfunctional assessment in understanding CD4 and CD8 dynamics in contexts of therapy in AML. SIGNIFICANCE: We found T-cell polyfunctionality differs between local and systemic microenvironments. Enhanced variability in proteomic profiles of bone marrow CD4 T cells post-IO suggests their pivotal role in AML treatment response. Single-cell analysis identified novel CD4 and CD8 T-cell functional groups linked to immunotherapy response within the bone marrow.

Comprehensive characterization of IFNγ signaling in acute myeloid leukemia reveals prognostic and therapeutic strategies.

Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation, inflammation, and tumor surveillance. However, while IFNγ levels were elevated in sera of most newly diagnosed acute myeloid leukemia (AML) patients, its complex interplay in AML remains insufficiently understood. We aim to characterize these complex interactions through comprehensive bulk and single-cell approaches in bone marrow of newly diagnosed AML patients. We identify monocytic AML as having a unique microenvironment characterized by IFNγ producing T and NK cells, high IFNγ signaling, and immunosuppressive features. IFNγ signaling score strongly correlates with venetoclax resistance in primary AML patient cells. Additionally, IFNγ treatment of primary AML patient cells increased venetoclax resistance. Lastly, a parsimonious 47-gene IFNγ score demonstrates robust prognostic value. In summary, our findings suggest that inhibiting IFNγ is a potential treatment strategy to overcoming venetoclax resistance and immune evasion in AML patients.

Association of elevated autoantibody to high expression of GNAS in hepatocellular carcinoma.

Purpose: This study was based on hepatocellular carcinoma (HCC) patients of early-stage to explore the diagnostic capability and possible production causes of anti-GNAS autoantibody. Methods: We evaluated the frequency of anti-GNAS autoantibody in sera from patients with early-stage HCC by enzyme-linked immunosorbent assay (ELISA) and the expression of GNAS protein in early-stage HCC tissues by immunohistochemistry. Western blotting (WB) and real-time polymerase chain reaction (RT-PCR) were utilized to examine the expressions of GNAS protein and mRNA in cell lines. GEO and International Cancer Genome Consortium (ICGC) databases were inquired to explore mRNA expression and mutation of GNAS in HCC tissues. Results: The positive rates of anti-GNAS autoantibody in HCC patients at clinical stage I (78.1 %) and clinical stage II (57.1 %) were all significantly higher than that in healthy control (20 %). There was also a significant difference in GNAS protein expression between HCC and its adjacent normal liver tissues. The results from WB and RT-PCR showed a significant difference at the mRNA level but no statistical difference at the protein level between HCC and normal liver cell lines. The difference in mRNA level between HCC and adjacent normal liver tissues was verified to be significant. Furthermore, the ICGC database demonstrated a 10.6 % mutation frequency for GNAS in HCC patients. Conclusion: The coordination of elevated anti-GNAS autoantibody, high expression of GNAS in the mRNA and protein levels in HCC, and high frequency of GNAS mutation indicates that anti-GNAS autoantibody may be used as an early indicator of HCC.