Enhanced T cell activation and cytotoxicity against AML via targeted anti-CD99 nanoparticle treatment.

CD99 is a transmembrane protein overexpressed in Acute Myeloid Leukemia (AML), presenting a potential novel therapeutic target. Our group has previously developed anti-CD99-A192 (α-CD99-A192), comprising of single chain variable fragment (scFv) and elastin-like polypeptides (ELPs), and reported promising anti-leukemic activity in AML preclinical models. Treatment with α-CD99-A192 induced apoptosis in AML cell lines and prolonged survival in AML xenograft models. Considering CD99's expression and role in T cell activation, in the current study, we propose that α-CD99-A192 plays a dual function, i.e., targeting leukemic cells and activating T cells. This manuscript reports the effects of α-CD99-A192 on T cells in the context of AML. α-CD99-A192 treatment enhances T cell proliferation and activation and increases the release of pro-inflammatory cytokines along with increased aggregation of T cells, which culminates in heightened cytotoxicity against leukemic cells. Altogether, these findings suggest α-CD99-A192 enhances T cell activation and cytotoxic potential consistent with dual mechanisms of action for α-CD99-A192.

FLT3/CD99 Bispecific Antibody-Based Nanoparticles for Acute Myeloid Leukemia.

Cluster of differentiation 99 (CD99) is a receptor that is significantly upregulated in acute myeloid leukemia (AML). FMS-like tyrosine kinase 3 internal tandem duplication mutation in AML (FLT3-ITD AML) exhibits even higher levels of CD99 expression. Our group previously employed a novel peptide platform technology called elastin-like polypeptides and fused it with single-chain antibodies capable of binding to FLT3 (FLT3-A192) or CD99 (CD99-A192). Targeting either FLT3 or CD99 using FLT3-A192 or CD99-A192 led to AML cell death and reduced leukemia burden in AML mouse models. Here, we report on the development of a novel Co-Assembled construct that is capable of binding to both CD99 and FLT3 and the antileukemia activity of the bispecific construct in FLT3-ITD AML preclinical models. This dual-targeting Co-Assembled formulation exhibits cytotoxic effects on AML cells (AML cell lines and primary blasts) and reduced leukemia burden and prolonged survival in FLT3-ITD AML mouse models. Altogether, this study demonstrates the potential of an innovative therapeutic strategy that targets both FLT3 and CD99 in FLT3-ITD AML. SIGNIFICANCE: This study investigates a dual-targeting strategy in acute myeloid leukemia (AML), focusing on FLT3 and CD99. The approach demonstrates enhanced therapeutic potential, presenting a novel option for AML treatment.

Differences in the mutational landscape of clonal hematopoiesis of indeterminate potential among races and between male and female patients with cancer.

Clonal hematopoiesis of indeterminate potential (CHIP) has emerged as a significant precursor to hematological malignancies and is associated with several age-related diseases. We leveraged public data to explore differences in the mutational landscape of CHIP between males (Ms) and females (Fs) and across diverse racial populations. DNA (cytosine-5) methyltransferase 3 alpha (DNMT3A) mutations were substantially more prevalent in Fs than in Ms (38.94% vs. 31.37%, p-value: < 0.001, q-value: < 0.001). Additional sex combs-like 1 (ASXL1) mutations were more frequent in Ms than Fs (5.82% vs. 2.69%, p-value < 0.001, q-value < 0.001). In the racial cohorts with sufficient sample sizes, STAT5B and CSF1R mutations were most frequent in Asian populations (1.40% and 0.84%), followed by Black populations (0.98% and 0.24%) and White populations (0.29% and 0.09%) (p-value: < 0.001 , q-value: 0.023 for both genes). Several other CHIP mutations were enriched in Black: RARA, SMAD2, CDKN1B, CENPA, CTLA4, EIF1AX, ELF3, MSI1, MYC, SOX17, and AURKA. On the other hand, H3C1, H3C4, and MYCL were enriched in the Asian cohort. Our analysis highlights sex and racial differences in CHIP mutations among patients with cancer. As CHIP continues to gain recognition as a critical precursor to malignancies and other diseases, understanding how these differences contribute to CHIP's underlying mechanisms and clinical implications is critical.

The DNA methylation landscape across the TCR loci in patients with acute myeloid leukemia.

The capacity of T cells to initiate anti-leukemia immune responses is determined by the ability of their receptors (TCRs) to recognize leukemia neoantigens. Epigenetic mechanisms including DNA methylation contribute to shaping the TCR repertoire composition and diversity. The DNA hypomethylating agents (HMAs) have been widely used in the treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Whether DNA HMAs directly influence TCR gene loci methylation patterns remains unknown. By analyzing public datasets, we compared methylation patterns across TCR loci in AML patients and healthy controls. We also explored how HMAs influence TCR loci DNA methylation in patients with AML. While methylation patterns are largely conserved across the TCR loci, certain V genes exhibit high interindividual variability. Although overall methylation levels within the TCR loci did not show significant differences, specific sites, including 32 TRAV and 12 TRBV sites exhibited distinct methylation patterns when comparing T cells from healthy donors to those from patients with AML. In leukemic cells, decitabine treatment demethylates sites across the TRAV and TRBV genes. While not as significant, a similar pattern of demethylation is observed in T cells. Pretreatment AML samples exhibit higher methylation beta values in differentially methylated positions (DMPs) compared with non-DMPs. Methylation levels of certain TRAV and TRBV genes in leukemic cells are associated with patients' risk status. The presence of disease specific TCR loci methylated signatures that are associated with clinical outcome presents an opportunity for therapeutic intervention. HMAs can modulate the TCR loci methylation patterns, yet whether they could reprogram the TCR repertoire composition remains to be explored.

A rigorous benchmarking of alignment-based HLA typing algorithms for RNA-seq data.

Accurate identification of human leukocyte antigen (HLA) alleles is essential for various clinical and research applications, such as transplant matching and drug sensitivities. Recent advances in RNA-seq technology have made it possible to impute HLA types from sequencing data, spurring the development of a large number of computational HLA typing tools. However, the relative performance of these tools is unknown, limiting the ability for clinical and biomedical research to make informed choices regarding which tools to use. Here we report the study design of a comprehensive benchmarking of the performance of 12 HLA callers across 682 RNA-seq samples from 8 datasets with molecularly defined gold standard at 5 loci, HLA-A, -B, -C, -DRB1, and -DQB1. For each HLA typing tool, we will comprehensively assess their accuracy, compare default with optimized parameters, and examine for discrepancies in accuracy at the allele and loci levels. We will also evaluate the computational expense of each HLA caller measured in terms of CPU time and RAM. We also plan to evaluate the influence of read length over the HLA region on accuracy for each tool. Most notably, we will examine the performance of HLA callers across European and African groups, to determine discrepancies in accuracy associated with ancestry. We hypothesize that RNA-Seq HLA callers are capable of returning high-quality results, but the tools that offer a good balance between accuracy and computational expensiveness for all ancestry groups are yet to be developed. We believe that our study will provide clinicians and researchers with clear guidance to inform their selection of an appropriate HLA caller.

Features of the TCR repertoire associate with patients' clinical and molecular characteristics in acute myeloid leukemia.

Background: Allogeneic hematopoietic stem cell transplant remains the most effective strategy for patients with high-risk acute myeloid leukemia (AML). Leukemia-specific neoantigens presented by the major histocompatibility complexes (MHCs) are recognized by the T cell receptors (TCR) triggering the graft-versus-leukemia effect. A unique TCR signature is generated by a complex V(D)J rearrangement process to form TCR capable of binding to the peptide-MHC. The generated TCR repertoire undergoes dynamic changes with disease progression and treatment. Method: Here we applied two different computational tools (TRUST4 and MIXCR) to extract the TCR sequences from RNA-seq data from The Cancer Genome Atlas (TCGA) and examine the association between features of the TCR repertoire in adult patients with AML and their clinical and molecular characteristics. Results: We found that only ~30% of identified TCR CDR3s were shared by the two computational tools. Yet, patterns of TCR associations with patients' clinical and molecular characteristics based on data obtained from either tool were similar. The numbers of unique TCR clones were highly correlated with patients' white blood cell counts, bone marrow blast percentage, and peripheral blood blast percentage. Multivariable regressions of TCRA and TCRB median normalized number of unique clones with mutational status of AML patients using TRUST4 showed significant association of TCRA or TCRB with WT1 mutations, WBC count, %BM blast, and sex (adjusted in TCRB model). We observed a correlation between TCRA/B number of unique clones and the expression of T cells inhibitory signal genes (TIGIT, LAG3, CTLA-4) and foxp3, but not IL2RA, CD69 and TNFRSF9 suggestive of exhausted T cell phenotypes in AML. Conclusion: Benchmarking of computational tools is needed to increase the accuracy of the identified clones. The utilization of RNA-seq data enables identification of highly abundant TCRs and correlating these clones with patients' clinical and molecular characteristics. This study further supports the value of high-resolution TCR-Seq analyses to characterize the TCR repertoire in patients.

Genomics and transcriptomic alterations of the glutamate receptors in acute myeloid leukemia.

Glutamine and glutamate have been widely explored as potential therapeutic targets in acute myeloid leukemia (AML). In addition to its bioenergetic role in leukemia cell proliferation, L-glutamate is a neurotransmitter that acts on glutamate receptors. However, the role of glutamate receptors in AML is largely understudied. Here, we comprehensively analyze the genomic and transcriptomic alterations of glutamate receptor genes in AML using publicly available data. We investigated the frequency of mutations in the glutamate receptor genes and whether an association exist between the presence of these mutations and clinical and molecular characteristics or patient's clinical outcome. We also assessed the dysregulation of glutamate receptor gene expression in AML with and without mutations and whether gene dysregulation is associated with clinical outcomes. We found that 29 (14.5%) of 200 patients with AML had a mutation in at least one glutamate receptor gene. The DNMT3A mutations were significantly more frequent in patients with mutations in at least one glutamate receptor gene compared with patients without mutations (13 of 29 [44.8%] vs. 41 of 171 [23.9%], p value: 0.02). Notably, patients with mutations in at least one glutamate receptor gene survived shorter than patients without mutations; however, the results did not reach statistical significance (overall survival: 15.5 vs. 19.0 months; p value: 0.10). Mutations in the glutamate receptor genes were not associated with changes in gene expression and the transcriptomic levels of glutamate receptor genes were not associated with clinical outcome.

Asparaginase toxicity in Hispanic adult and pediatric patients with acute lymphoblastic leukemia: current understanding.

INTRODUCTION: Asparaginase is essential to chemotherapy regimens for acute lymphoblastic leukemia (ALL). Survival of patients with ALL has improved since incorporating asparaginase into chemotherapy backbones. Hispanic patients have a higher incidence of ALL than other ethnicities and suffer inferior outcomes. The inferior outcome of Hispanics is due to several factors, including the increased incidence of high-risk genetic subtypes and susceptibility to treatment-related toxicity. AREAS COVERED: We summarize the current knowledge of asparaginase-related toxicity by comparing their incidence between Hispanic and non-Hispanic patients. These toxicities include hypersensitivity, hepatotoxicity, pancreatitis, thrombosis, and hypertriglyceridemia. The PubMed database and Google Scholar were used to search for this review from October 2022 to June 2023. EXPERT OPINION: Except for hepatotoxicity and hypertriglyceridemia secondary to asparaginase-based treatments, which may develop more frequently among Hispanic patients with ALL, other toxicities were comparable between Hispanic and non-Hispanic patients. Nevertheless, studies with larger cohorts and more accurate capturing of Hispanic ethnicity should be conducted to fill the gaps in the current knowledge.

Rigorous benchmarking of T-cell receptor repertoire profiling methods for cancer RNA sequencing.

The ability to identify and track T-cell receptor (TCR) sequences from patient samples is becoming central to the field of cancer research and immunotherapy. Tracking genetically engineered T cells expressing TCRs that target specific tumor antigens is important to determine the persistence of these cells and quantify tumor responses. The available high-throughput method to profile TCR repertoires is generally referred to as TCR sequencing (TCR-Seq). However, the available TCR-Seq data are limited compared with RNA sequencing (RNA-Seq). In this paper, we have benchmarked the ability of RNA-Seq-based methods to profile TCR repertoires by examining 19 bulk RNA-Seq samples across 4 cancer cohorts including both T-cell-rich and T-cell-poor tissue types. We have performed a comprehensive evaluation of the existing RNA-Seq-based repertoire profiling methods using targeted TCR-Seq as the gold standard. We also highlighted scenarios under which the RNA-Seq approach is suitable and can provide comparable accuracy to the TCR-Seq approach. Our results show that RNA-Seq-based methods are able to effectively capture the clonotypes and estimate the diversity of TCR repertoires, as well as provide relative frequencies of clonotypes in T-cell-rich tissues and low-diversity repertoires. However, RNA-Seq-based TCR profiling methods have limited power in T-cell-poor tissues, especially in highly diverse repertoires of T-cell-poor tissues. The results of our benchmarking provide an additional appealing argument to incorporate RNA-Seq into the immune repertoire screening of cancer patients as it offers broader knowledge into the transcriptomic changes that exceed the limited information provided by TCR-Seq.

Deletion of CD36 exhibits limited impact on normal hematopoiesis and the leukemia microenvironment.

BACKGROUND: CD36 has been identified as a potential therapeutic target both in leukemic cells and in the tumor immune microenvironment. In acute myeloid leukemia (AML), we found that APOC2 acts with CD36 to promote leukemia growth by activating the LYN-ERK signaling. CD36 also plays a role in lipid metabolism of cancer associated T-cells leading to impaired cytotoxic CD8+ T-cell and enhanced Treg cell function. To establish CD36 as a viable therapeutic target in AML, we investigated whether targeting CD36 has any detrimental impact on normal hematopoietic cells. METHODS: Differential expression data of CD36 during human and mouse normal hematopoiesis were examined and compared. Cd36 knockout (Cd36-KO) mice were evaluated for blood analysis, hematopoietic stem cells and progenitors (HSPCs) function and phenotype analyses, and T cells in vitro expansion and phenotypes in comparison with wild type (WT) mice. In addition, MLL-PTD/FLT3-ITD leukemic cells were engrafted into Cd36-KO and WT mice, and leukemia burden was compared between groups. RESULTS: RNA-Seq data showed that Cd36 expression was low in HSPCs and increased as cells matured. Phenotypic analysis revealed limited changes in blood count except for a slight yet significantly lower red blood cell count and hemoglobin and hematocrit levels in Cd36-KO mice compared with WT mice (P < 0.05). In vitro cell proliferation assays of splenocytes and HSPCs from Cd36-KO mice showed a similar pattern of expansion to that of cells from WT mice. Characterization of HSPCs showed similar percentages of the different progenitor cell populations between Cd36-KO with WT mice. However, Cd36-KO mice exhibited ~ 40% reduction of the number of colonies developed from HSPCs cells compared with WT mice (P < 0.001). Cd36-KO and WT mice presented comparably healthy BM transplant in non-competitive models and developed similar leukemia burden. CONCLUSIONS: Although the loss of Cd36 affects the hematopoietic stem cell and erythropoiesis, limited detrimental overall impact was observed on normal Hematopoietic and leukemic microenvironments. Altogether, considering the limited impact on normal hematopoiesis, therapeutic approaches to target CD36 in cancer are unlikely to result in toxicity to normal blood cells.

Prediction of Peptide and TCR CDR3 Loops in Formation of Class I MHC-Peptide-TCR Complexes Using Molecular Models with Solvation.

Formation of major histocompatibility (MHC)-peptide-T cell receptor (TCR) complexes is central to initiation of an adaptive immune response. These complexes form through initial stabilization of the MHC fold via binding of a short peptide, and subsequent interaction of the TCR to form a ternary complex, with contacts made predominantly through the complementarity-determining region (CDR) loops of the TCR. Stimulation of an immune response is central to cancer immunotherapy. This approach depends on identification of the appropriate combinations of MHC molecules, peptides, and TCRs to elicit an antitumor immune response. This prediction is a current challenge in computational biochemistry. In this chapter, we introduce a predictive method that involves generation of multiple peptides and TCR CDR 3 loop conformations, solvation of these conformers in the context of the MHC-peptide-TCR ternary complex, extraction of parameters from the generated complexes, and use of an AI model to evaluate the potential for the assembled ternary complex to support an immune response.

pyTCR: A comprehensive and scalable solution for TCR-Seq data analysis to facilitate reproducibility and rigor of immunogenomics research.

T cell receptor (TCR) studies have grown substantially with the advancement in the sequencing techniques of T cell receptor repertoire sequencing (TCR-Seq). The analysis of the TCR-Seq data requires computational skills to run the computational analysis of TCR repertoire tools. However biomedical researchers with limited computational backgrounds face numerous obstacles to properly and efficiently utilizing bioinformatics tools for analyzing TCR-Seq data. Here we report pyTCR, a computational notebook-based solution for comprehensive and scalable TCR-Seq data analysis. Computational notebooks, which combine code, calculations, and visualization, are able to provide users with a high level of flexibility and transparency for the analysis. Additionally, computational notebooks are demonstrated to be user-friendly and suitable for researchers with limited computational skills. Our tool has a rich set of functionalities including various TCR metrics, statistical analysis, and customizable visualizations. The application of pyTCR on large and diverse TCR-Seq datasets will enable the effective analysis of large-scale TCR-Seq data with flexibility, and eventually facilitate new discoveries.

Hispanic ethnicity and the rs4880 variant in SOD2 are associated with elevated liver enzymes and bilirubin levels in children receiving asparaginase-containing chemotherapy for acute lymphoblastic leukemia.

Asparaginase is an integral component of acute lymphoblastic leukemia (ALL)3 treatment. Hepatotoxicity related to asparaginase is one of the most common treatment-related toxicities in ALL therapy. Hispanic children are at higher risk of developing ALL, and toxicities from ALL therapy. The rs4880 variant in the superoxide dismutase 2 (SOD2)4 gene, a critical mitochondrial enzyme that protects cells against oxidative stress, was found to be associated with increased incidence of asparaginase-related hepatotoxicity in adult cohort of largely White non-Hispanics patients with ALL. The risk genotype (rs4880-CC) is more frequent among adult Hispanic patients with ALL. To assess the prevalence of hepatotoxicity and risk genotype among pediatric patients with ALL, particularly of Hispanic ethnicity, we conducted a prospective study of 143 pediatric patients with ALL (62.2% Hispanic). Bilirubin and hepatic transaminase levels were collected at different times during multiagent therapy including asparaginase treatment. Germline DNA blood samples were genotyped for the SOD2 rs4880. We found that the frequency of hepatotoxicity and the rs4880-CC risk genotype are higher in Hispanic patients than non-Hispanic. Patients with the CC genotype exhibit higher bilirubin and hepatic transaminase levels compared with patients with the TT and CT genotypes. In a multivariate Cox analysis, Hispanic ethnicity was identified as a strong predictor of hepatotoxicity (hazard ratio [HR] = 1.9, 95% confidence interval [95% CI] 1.0-3.5, p = 0.05). Altogether, these findings demonstrate that hepatotoxicity is highly prevalent among Hispanic pediatric patients with ALL, and those with rs4880-CC genotype.