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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (©2024 The Authors; Published by the American Association for Cancer Research.)

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., Competing Interests: Conflict of Interest Disclosure The authors do not have any conflicts of interest to declare in relation to this work., (Copyright © 2024 International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pospiech, Tamizharasan, Wei, Kumar, Lou, Milstein and Alachkar.)

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., (© 2023 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Background: Detailed demographic data on people with hereditary angioedema (HAE) and acquired C1 inhibitor deficiency in the United Kingdom are relatively limited. Better demographic data would be beneficial in planning service provision, identifying areas of improvement, and improving care., Objective: To obtain more accurate data on the demographics of HAE and acquired C1 inhibitor deficiency in the United Kingdom, including treatment modalities and services available to patients., Methods: A survey was distributed to all centers in the United Kingdom that look after patients with HAE and acquired C1 inhibitor deficiency to collect these data., Results: The survey identified 1152 patients with HAE-1/2 (58% female and 92% type 1), 22 patients with HAE with normal C1 inhibitor, and 91 patients with acquired C1 inhibitor deficiency. Data were provided by 37 centers across the United Kingdom. This gives a minimum prevalence of 1:59,000 for HAE-1/2 and 1:734,000 for acquired C1 inhibitor deficiency in the United Kingdom. A total of 45% of patients with HAE were on long-term prophylaxis (LTP) with the most used medication being danazol (55% of all patients on LTP). Eighty-two percent of patients with HAE had a home supply of acute treatment with C1 inhibitor or icatibant. A total of 45% of patients had a supply of icatibant and 56% had a supply of C1 inhibitor at home., Conclusions: Data obtained from the survey provide useful information about the demographics and treatment modalities used in HAE and acquired C1 inhibitor deficiency in the United Kingdom. These data are useful for planning service provision and improving services for these patients., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Published by Oxford University Press 2023.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Peng, Moore, Vahed, Brito, Kao, Burkhardt, Alachkar and Mangul.)

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., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion and differentiation arrest of the myeloid progenitor cells, which leads to the accumulation of immature cells called blasts in the bone marrow and peripheral blood. Mutations in the receptor tyrosine kinase FLT3 occur in 30% of normal karyotype patients with AML and are associated with a higher incidence of relapse and worse survival. Targeted therapies against FLT3 mutations using small-molecule FLT3 tyrosine kinase inhibitors (TKIs) have long been investigated, with some showing favorable clinical outcomes. However, major setbacks such as limited clinical efficacy and the high risk of acquired resistance remain unresolved. FLT3 signaling, mutations, and FLT3 inhibitors are topics that have been extensively reviewed in recent years. Strategies to target FLT3 beyond the small molecule kinase inhibitors are expanding, nevertheless they are not receiving enough attention. These modalities include antibody-based FLT3 targeted therapies, immune cells mediated targeting strategies, and approaches targeting downstream signaling pathways and FLT3 translation. Here, we review the most recent advances and the challenges associated with the development of therapeutic modalities targeting FLT3 beyond the kinase inhibitors., (Copyright © 2021. Published by Elsevier Inc.)

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a public health emergency. The most common symptoms of COVID-19 are fever, cough, and fatigue. While most patients with COVID-19 present with mild illness, some patients develop pneumonia, an important risk factor for mortality, at early stage of viral infection, putting these patients at increased risk of death. So far, little has been known about differences in the T cell repertoires between COVID-19 patients with and without pneumonia during SARS-CoV-2 infection. Herein, we aimed to investigate T cell receptor (TCR) repertoire profiles and patient-specific SARS-CoV-2-associated TCR clusters between COVID-19 patients with mild disease (no sign of pneumonia) and pneumonia. The TCR sequencing was conducted to characterize the peripheral TCR repertoire profile and diversity. The TCR clustering and CDR3 annotation were exploited to further discover groups of patient-specific TCR clonotypes with potential SARS-CoV-2 antigen specificities. Our study indicated a slight decrease in the TCR repertoire diversity and a skewed CDR3 length usage in patients with pneumonia compared to those with mild disease. The SARS-CoV-2-associated TCR clusters enriched in patients with mild disease exhibited significantly higher TCR generation probabilities and most of which were highly shared among patients, compared with those from pneumonia patients. Importantly, using similarity network-based clustering followed by the sequence conservation analysis, we found different patterns of CDR3 sequence motifs between mild disease- and pneumonia-specific SARS-CoV-2-associated public TCR clusters. Our results showed that characteristics of overall TCR repertoire and SARS-CoV-2-associated TCR clusters/clonotypes were divergent between COVID-19 patients with mild disease and patients with pneumonia. These findings provide important insights into the correlation between the TCR repertoire and disease severity in COVID-19 patients., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.

CD99 is a transmembrane glycoprotein shown to be upregulated in various malignancies. We have previously reported CD99 to be highly upregulated and present a viable therapeutic target in acute myeloid leukemia (AML). Currently, no therapy against CD99 is under clinical investigation. As a surface molecule, CD99 can be targeted with an antibody-based approach. Here, we have developed a new modality to target CD99 by engineering a fusion protein composed of a single-chain variable fragment antibody (anti-CD99 scFv) conjugated with a high molecular weight elastin-like polypeptide (ELP), A192: α-CD99-A192. This fusion protein assembles into multi-valent nanoworm with optimal physicochemical properties and favorable pharmacokinetic parameters (half-life: 16 h). α-CD99-A192 nanoworms demonstrated excellent in vitro and in vivo anti-leukemic effects. α-CD99-A192 induced apoptotic cell death in AML cell lines and primary blasts and prolonged overall survival of AML xenograft mouse model., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) is a devastating hematologic malignancy that affects the hematopoietic stem cells. The 5-year overall survival (OS) of patients with AML is less than 30%, highlighting the urgent need to identify new therapeutic targets. Here, we analyze gene expression datasets for genes that are differentially overexpressed in AML cells compared with healthy hematopoietic cells. We report that apolipoprotein C2 ( APOC2 ) mRNA is significantly overexpressed in AML, particularly in patients with mixed-lineage leukemia rearrangements. By multivariate analysis, high APOC2 expression in leukemia blasts is significantly associated with decreased OS (HR: 2.51; 95% CI, 1.03-6.07; P = 0.04). APOC2 is a small secreted apolipoprotein that constitutes chylomicrons, very-low-density lipoproteins, and high-density lipoproteins with other apolipoproteins. APOC2 activates lipoprotein lipase and contributes to lipid metabolism. By gain and loss of function approaches in cultured AML cells, we demonstrate that APOC2 promotes leukemia growth via CD36-mediated LYN-ERK signaling activation. Knockdown or pharmacological inhibition of either APOC2 or CD36 reduces cell proliferation, induces apoptosis in vitro, and delays leukemia progression in mice. Altogether, this study establishes APOC2-CD36 axis as a potential therapeutic target in AML., Competing Interests: Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

FLT3 receptor is an important therapeutic target in acute myeloid leukemia due to high incidence of mutations associated with poor clinical outcome. Targeted therapies against the FLT3 receptor, including small-molecule FLT3 tyrosine kinase inhibitors (TKIs) and anti-FLT3 antibodies, have demonstrated promising preclinical and even clinical efficacy. Yet, even with the current FDA approval for two FLT3 inhibitors, these modalities were unable to cure AML or significantly extend the lives of patients with a common mutation called FLT3-ITD. While FLT3 is a viable target, the approaches to inhibit its activity were inadequate. To develop a new modality for targeting FLT3, our team engineered an α-FLT3-A192 fusion protein composed of a single chain variable fragment antibody conjugated with an elastin-like polypeptide. These fusion proteins assemble into multi-valent nanoparticles with excellent stability and pharmacokinetic properties as well as in vitro and in vivo pharmacological activity in cellular and xenograft murine models of AML. In conclusion, α-FLT3-A192 fusions appear to be a viable new modality for targeting FLT3 in AML and warrant further preclinical development to bring it into the clinic., Competing Interests: Declaration of Competing Interest JAM, MP, VPV, and HA are inventors on pending provisional patent application related to this work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected several millions and killed more than quarter of a million worldwide to date. Important questions have remained unanswered: why some patients develop severe disease, while others do not; and what roles do genetic variabilities play in the individual immune response to this viral infection. Here, we discuss the critical role T cells play in the orchestration of the antiviral response underlying the pathogenesis of the disease, COVID-19. We highlight the scientific rationale for comprehensive and longitudinal TCR analyses in COVID-19 and reason that analyzing TCR repertoire in COVID-19 patients would reveal important findings that may explain the outcome disparity observed in these patients. Finally, we provide a framework describing the different strategies, the advantages, and the challenges involved in obtaining useful TCR repertoire data to advance our fight against COVID-19., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is crucial in the immune system as it stimulates survival, proliferation, differentiation, and functional activation of myeloid hematopoietic cells. hGMCSF is integral to approved therapies, including monoclonal antibodies against checkpoint inhibitors, chimeric antigen receptors, and prevention of chemotherapy-induced neutropenia. Recombinant hGMCSF can be purified from Escherichia. coli ; however, it forms inclusion bodies that require solubilization and refolding. Alternatively, this manuscript describes its fusion with an elastin-like polypeptide (ELP). Previously reported as purification tags and solubility enhancers, ELPs are recombinant polypeptides that undergo reversible temperature-dependent phase separation. This report is the first to show that fusion to an ELP enables direct purification of hGMCSF fusions from the soluble fraction of bacterial lysate. Surprisingly, these ELP-fusions assemble stable, small, spherical nanoparticles that maintain pro-mitotic activity of hGMCSF. These nanoparticles exhibit ELP-mediated phase separation; however, nanoparticle assembly significantly increases the entropic and enthalpic cost of phase separation compared to ELP alone. The attachment of a high molecular weight ELP to a difficult-to-express protein, like hGMCSF, appears to be a useful strategy to stabilize bioactive, protein-based nanoparticles, which may have broad applications in medicine and biology.

In an effort to identify target genes in acute myeloid leukemia (AML), we compared gene expression profiles between normal and AML cells from various publicly available datasets. We identified CD99 , a gene that is up-regulated in AML patients. In 186 patients from The Cancer Genome Atlas AML dataset, CD99 was over-expressed in patients with FLT3 -ITD and was down-regulated in patients with TP53 mutations. CD99 is a trans-membrane protein expressed on leukocytes and plays a role in cell adhesion, trans-endothelial migration, and T-cell differentiation. The CD99 gene encodes two isoforms with distinct expression and functional profiles in both normal and malignant tissues. Here we report that, although the CD99 long isoform initially induces an increase in cell proliferation, it also induces higher levels of reactive oxygen species, DNA damage, apoptosis and a subsequent decrease in cell viability. In several leukemia murine models, the CD99 long isoform delayed disease progression and resulted in lower leukemia engraftment in the bone marrow. Furthermore, the CD99 monoclonal antibody reduced cell viability, colony formation, and cell migration, and induced cell differentiation and apoptosis in leukemia cell lines and primary blasts. Mechanistically, CD99 long isoform resulted in transient induction followed by a dramatic decrease in both ERK and SRC phosphorylation. Altogether, our study provides new insights into the role of CD99 isoforms in AML that could potentially be relevant for the preclinical development of CD99 targeted therapy., (Copyright© 2020 Ferrata Storti Foundation.)

The role of adhesion G protein-coupled receptors (aGPCRs) in cancer has become increasingly evident in recent years. Yet, data supporting the contribution of this family of genes to hematological malignancies, particularly acute myeloid leukemia (AML) are limited. Here, we use publicly available genomic data to characterize the expression of the 33 aGPCRs in patients with AML and examine whether upregulation of these genes is associated with the clinical and molecular characteristics of patients. Upregulation in one or more of eight aGPCR genes (ADGRB1, ADGRC2, ADGRD1, ADGRE1, ADGRE2, ADGRE5, ADGRG1, and/or ADGRG3) was significantly associated with shorter overall survival (OS) (median OS: 11.8 vs 55.4 months; P < 0.0001). This was also significant in multivariate survival analysis (hazard ratio: 1.73; 95% confidence interval 1.11-2.69; P = 0.015) after adjusting for age, molecular risk status, and transplant status. High expression of the eight aGPCRs was significantly associated with older age (≥60; P = 0.011). Patients with high aGPCRs expression were more frequently classified in the poor molecular risk status group and less in the good risk status group compared with patients with low aGPCRs expression (31% vs 17% P = 0.049 and 14% vs 28% P = 0.027, respectively). Via Ingenuity Pathway Analysis, we identified the interleukin-8 signaling pathway among the most activated pathways in patients with high aGPCRs expression. Overall, our data suggest that particular aGPCRs are frequently upregulated in AML and associated with poor clinical outcome. Future functional and mechanistic analyses are needed to address the role of aGPCRs in AML., (Copyright © 2019 Elsevier Inc. All rights reserved.)

CD97, a member of the adhesion G-protein coupled receptor family, is normally expressed on leukocytes and smooth muscles. CD97 is also expressed in a variety of solid cancers, particularly those with aggressive metastatic phenotypes. Here we characterize the clinical significance of CD97 in acute myeloid leukemia (AML). We analyzed 173 patients from the TCGA AML data set and found that CD97 was higher in cytogenetically normal patients compared with cytogenetically abnormal patients (p = 0.023). High CD97 was also associated with NPM1 mutations (p = 0.0033). Patients with high CD97 expression had shorter overall (median: 7.35 months vs. 24.1 months, p = 0.0015) and disease-free (median DFS: 8.2 months vs. 18.2 months, p = 0.017) survival. Importantly, we identified pathways involved in the leukemia stem cell interaction with the bone marrow niche, such as integrin, CXCR4, and interleukin-8, among the most upregulated signaling pathways in patients with high CD97 expression. Our results suggest that high CD97 expression is associated with poor clinical outcome and indicate a need for future functional and mechanistic studies to investigate the role of CD97 in AML., (Copyright © 2019 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background: Acute myeloid leukemia (AML) is characterized by rapid growth of abnormal blasts that overcrowd normal hematopoiesis. Defective mitochondrial biogenesis has been implicated in AML, which we believe is partly due to the deregulation of the mitochondrial transcription machinery (MTM) genes influencing the expression of mitochondrial genes. Here, we aim to characterize MTM gene upregulation in AML., Methods: Molecular and clinical patient data were retrieved from several public AML datasets. Kaplan-Meier survival curves were used to compare overall survival between patients, while Mann-Whitney U's non-parametric and Fisher's exact test were used for comparing continuous and categorical variables, respectively., Results: The MTM genes TFB1M, TFB2M, TFAM, and POLRMT were upregulated in patients with AML compared with healthy donors. Upregulation of one or more of these genes was associated with higher percentage of peripheral blood blasts (P = 0.002), normal cytogenetic status (P = 0.027) and NPM1 mutations (P = 0.009). Additionally, patients with high expression of MTM genes (Z ≥ 1) had shorter median overall survival compared with low MTM gene expression (Z < 1) (months: 11.8 vs 24.1, P = 0.027; multivariate survival analysis Cox Proportional Hazards model, HR: 1.82 (1.22-2.70); p-value: 0.003)., Conclusion: The mitochondrial transcriptional machinery is upregulated and associated with worse clinical outcome in patients with AML and may present a viable therapeutic target., (© 2019 The Authors.)

Background: Caspase activation and recruitment domain 11 (CARD11) encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nuclear factor κB, c-Jun N-terminal kinase, and mechanistic target of rapamycin complex 1. Germline CARD11 mutations cause several distinct primary immune disorders in human subjects, including severe combined immune deficiency (biallelic null mutations), B-cell expansion with nuclear factor κB and T-cell anergy (heterozygous, gain-of-function mutations), and severe atopic disease (loss-of-function, heterozygous, dominant interfering mutations), which has focused attention on CARD11 mutations discovered by using whole-exome sequencing., Objectives: We sought to determine the molecular actions of an extended allelic series of CARD11 and to characterize the expanding range of clinical phenotypes associated with heterozygous CARD11 loss-of-function alleles., Methods: Cell transfections and primary T-cell assays were used to evaluate signaling and function of CARD11 variants., Results: Here we report on an expanded cohort of patients harboring novel heterozygous CARD11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with CARD11 mutations. In addition to (and sometimes excluding) severe atopy, heterozygous missense and indel mutations in CARD11 presented with immunologic phenotypes similar to those observed in signal transducer and activator of transcription 3 loss of function, dedicator of cytokinesis 8 deficiency, common variable immunodeficiency, neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome. Pathogenic variants exhibited dominant negative activity and were largely confined to the CARD or coiled-coil domains of the CARD11 protein., Conclusion: These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity., (Published by Elsevier Inc.)

Good's syndrome (thymoma and hypogammaglobulinaemia) is a rare secondary immunodeficiency disease, previously reported in the published literature as mainly individual cases or small case series. We use the national UK-Primary Immune Deficiency (UKPID) registry to identify a large cohort of patients in the UK with this PID to review its clinical course, natural history and prognosis. Clinical information, laboratory data, treatment and outcome were collated and analysed. Seventy-eight patients with a median age of 64 years, 59% of whom were female, were reviewed. Median age of presentation was 54 years. Absolute B cell numbers and serum immunoglobulins were very low in all patients and all received immunoglobulin replacement therapy. All patients had undergone thymectomy and nine (12%) had thymic carcinoma (four locally invasive and five had disseminated disease) requiring adjuvant radiotherapy and/or chemotherapy. CD4 T cells were significantly lower in these patients with malignant thymoma. Seventy-four (95%) presented with infections, 35 (45%) had bronchiectasis, seven (9%) chronic sinusitis, but only eight (10%) had serious invasive fungal or viral infections. Patients with AB-type thymomas were more likely to have bronchiectasis. Twenty (26%) suffered from autoimmune diseases (pure red cell aplasia, hypothyroidism, arthritis, myasthenia gravis, systemic lupus erythematosus, Sjögren's syndrome). There was no association between thymoma type and autoimmunity. Seven (9%) patients had died. Good's syndrome is associated with significant morbidity relating to infectious and autoimmune complications. Prospective studies are required to understand why some patients with thymoma develop persistent hypogammaglobulinaemia., (© 2018 The Authors. Clinical & Experimental Immunology © 2018 British Society for Immunology, Clinical and Experimental Immunology.)

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy in which the only curative approach is allogeneic stem cell transplant (Allo-HSCT). The recognition and elimination of leukemic clones by donor T-cells contribute significantly to Allo-HSCT success. FLT3-ITD, a common mutation in AML, is associated with poor prognosis. Recently, midostaurin became the first FDA approved FLT3-inhibitor for pre-transplant patients with FLT3-ITD in combination with standard therapy. In addition to their multikinase activity which may affect T-cell signaling, FLT3-inhibitors induce apoptosis of malignant cells which may also enhance antigen presentation to activate T-cells. Considering the increased clinical use of these inhibitors in patients with AML, and the limited clinical benefit derived from their use as single agents, understanding how FLT3-inhibitors affect T cell population and function is needed to improve their clinical benefit. We examined the effect of four different FLT3 inhibitors (midostaurin, sorafenib, tandutinib, and quizartenib) on T cell populations in peripheral blood mononuclear cells (PBMC) obtained from healthy donors and from patients with AML. Midostaurin exhibited a significant decrease in CD4 + CD25 + FOXP3+ T cell population and FOXP3 mRNA expression in healthy and AML PBMCs. Similarly, samples collected from patients with AML treated with midostaurin showed a reduction in Tregs markers. Interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α), and IL-10 levels were also reduced following midostaurin treatment. Considering the FDA approval of midostaurin for use in patients with AML in the pre-transplant setting, our finding will have important clinical implication as it provides the rationale for functional investigation of the use of midostaurin in post-transplant patients.

Acute Myeloid Leukemia is a devastating and heterogeneous, hematological malignancy characterized by the uncontrolled proliferation of undifferentiated myeloid progenitor cells-blasts. Mutations in certain mitochondrial proteins, such as IDH2 have been shown to contribute to leukemogenesis. However, the role of mutations in mitochondrial-encoded Electron Transport Chain (ETC) genes have thus far not been well elucidated in AML. Here, we use TCGA data to characterize mutations in the ETC genes and their association with clinical outcomes in AML. We found that mitochondrial ETC mutations-in Complex I, III, IV and/or V (ATP Synthase)-were present in 8% of patients with AML and were significantly more frequent in older patients. Patients with ETC mutations had worse overall survival than ETC wild type patients (OS: 9.3 vs 20.1 months; p-value: 0.007). Additionally, mutations in either or both Complex I and IV were associated with TP53 mutations (p-value: 0.009), and among TP53 mutated patients, mutations in either or both Complex I and IV were significantly associated with worse overall survival (OS: 0.85 vs 9.4 months; p-value: 0.008). Elucidation of the mechanisms by which ETC mutations contribute to AML pathogenesis and progression would facilitate the development of novel therapeutic targets.

Although germline alterations and somatic mutations in disease cells have been extensively analyzed, molecular changes in immune cells associated with disease conditions have not been characterized in depth. It is clear that our immune system has a critical role in various biological and pathological conditions, such as infectious diseases, autoimmune diseases, drug-induced skin and liver toxicity, food allergy, and rejection of transplanted organs. The recent development of cancer immunotherapies, particularly drugs modulating the immune checkpoint molecules, has clearly demonstrated the importance of host immune cells in cancer treatments. However, the molecular mechanisms by which these new therapies kill tumor cells are still not fully understood. In this regard, we have begun to explore the role of newly developed tools such as next-generation sequencing in the genetic characterization of both cancer cells and host immune cells, a field that is called immunogenomics/ immunopharmacogenomics. This new field has enormous potential to help us better understand changes in our immune system during the course of various disease conditions. Here we report the potential of deep sequencing of T-cell and B-cell receptors in capturing the molecular contribution of the immune system, which we believe plays critical roles in the pathogenesis of various human diseases.

Background: Vimentin (VIM) is a type III intermediate filament that maintains cell integrity, and is involved in cell migration, motility and adhesion. When overexpressed in solid cancers, vimentin drives epithelial to mesenchymal transition (EMT) and ultimately, metastasis. The effects of its overexpression in AML are unclear., Methods: In this study, we analyzed the TCGA data of 173 AML patients for which complete clinical and expression data were available. In this analysis, we assessed the association between VIM mRNA expression and patient's clinical and molecular characteristics including clinical outcome., Results: VIM overexpression was associated with higher white blood count (< p = 0.0001). Patients with high VIM expression have worse overall survival (OS) and disease-free survival (DFS) compared with patients with low VIM expression (median OS; 7.95 months vs 19.2 months; p = 0.029). After age-stratification, high VIM expression was significantly associated with worse overall survival in older patients (age ≥ 60; median OS: 5.4 vs 9.9 months: p = 0.0257) but not in younger patients (age < 60). In stratification analysis according to cytogenetic status, high VIM expression was significantly associated with shorter OS (7.95 vs 24.6 months: p = 0.0102) in cytogenetically normal, but not in cytogenetic abnormal AML., Conclusions: Collectively, the data indicate that overexpression of the EMT marker vimentin is associated with poor clinical outcome in older patients with cytogenetically normal AML; and therefore may play a role in this disease.

This is the second report of the United Kingdom Primary Immunodeficiency (UKPID) registry. The registry will be a decade old in 2018 and, as of August 2017, had recruited 4758 patients encompassing 97% of immunology centres within the United Kingdom. This represents a doubling of recruitment into the registry since we reported on 2229 patients included in our first report of 2013. Minimum PID prevalence in the United Kingdom is currently 5·90/100 000 and an average incidence of PID between 1980 and 2000 of 7·6 cases per 100 000 UK live births. Data are presented on the frequency of diseases recorded, disease prevalence, diagnostic delay and treatment modality, including haematopoietic stem cell transplantation (HSCT) and gene therapy. The registry provides valuable information to clinicians, researchers, service commissioners and industry alike on PID within the United Kingdom, which may not otherwise be available without the existence of a well-established registry., (© 2018 British Society for Immunology.)

Immunoglobulin replacement therapy enhances survival and reduces infection risk in patients with agammaglobulinaemia. We hypothesized that despite regular immunoglobulin therapy, some patients will experience ongoing respiratory infections and develop progressive bronchiectasis with deteriorating lung function. One hundred and thirty-nine (70%) of 199 patients aged 1-80 years from nine cities in the United Kingdom with agammaglobulinaemia currently listed on the UK Primary Immune Deficiency (UKPID) registry were recruited into this retrospective case study and their clinical and laboratory features analysed; 94% were male, 78% of whom had Bruton tyrosine kinase (BTK) gene mutations. All patients were on immunoglobulin replacement therapy and 52% had commenced therapy by the time they were 2 years old. Sixty per cent were also taking prophylactic oral antibiotics; 56% of patients had radiological evidence of bronchiectasis, which developed between the ages of 7 and 45 years. Multivariate analysis showed that three factors were associated significantly with bronchiectasis: reaching 18 years old [relative risk (RR) = 14·2, 95% confidence interval (CI) = 2·7-74·6], history of pneumonia (RR = 3·9, 95% CI = 1·1-13·8) and intravenous immunoglobulin (IVIG) rather than subcutaneous immunoglobulin (SCIG) = (RR = 3·5, 95% CI = 1·2-10·1), while starting immunoglobulin replacement after reaching 2 years of age, gender and recent serum IgG concentration were not associated significantly. Independent of age, patients with bronchiectasis had significantly poorer lung function [predicted forced expiratory volume in 1 s 74% (50-91)] than those without this complication [92% (84-101)] (P < 0·001). We conclude that despite immunoglobulin replacement therapy, many patients with agammaglobulinaemia can develop chronic lung disease and progressive impairment of lung function., (© 2017 British Society for Immunology.)

Asparaginase, which depletes asparagine and glutamine, activates amino-acid stress response. Oxidative stress mediated by excessive reactive oxygen species (ROS) causes enhanced mitochondrial permeabilization and subsequent cell apoptosis and is considered as a plausible mechanism for drug-induced hepatotoxicity, a common toxicity of asparaginase in adults with acute lymphoblastic leukemia (ALL). Studies investigating the pharmacogenetics of asparaginase in ALL are limited and focused on asparaginase-induced allergic reaction common in pediatric patients. Here, we sought to determine a potential association between the variant rs4880 in SOD2 gene, a key mitochondrial enzyme that protects cells against ROS, and hepatotoxicity during asparaginase-based therapy in 224 patients enrolled on CALGB-10102, a treatment trial for adults with ALL. We report that the CC genotype of rs4880 is associated with increased hepatotoxicity following asparaginase-based treatment. Thus, rs4880 likely contributes to asparaginase-induced hepatotoxicity, and functional studies investigating this single-nucleotide polymorphism (SNP) are needed to develop therapeutic approaches that mitigate this toxicity.