Treatment Of T-cell Acute Lymphoblastic Leukemia Research Articles

NKG2D-CAR memory T cells target pediatric T-cell acute lymphoblastic leukemia in vitro and in vivo but fail to eliminate leukemia initiating cells

IntroductionRefractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation.MethodsIn this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA-) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells’ cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality.ResultsIn vitro, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo, they exhibited functional properties of leukemia initiating cells.DiscussionThe data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.

A CH2CH3 hinge region enhances the cytotoxicity of anti-CD5 CAR-T cells targeting T cell acute lymphoblastic leukemia

Chimeric antigen receptor T cell (CAR-T) therapies show considerable clinical efficacy in patients with B cell malignancies, but their efficacy is limited in patients with T cell acute lymphoblastic leukemia (T-ALL). CD5 is expressed on ∼85 % of malignant T cells, and CD5-targeting CAR-T cells can exhibit potent antitumor activity against T-ALL. However, optimization of CAR costimulatory endo-, hinge, and transmembrane domains could further increase their expansion and persistence, thereby enhancing their efficacy following exposure to tumor cells. Here we designed CD5-specific CARs with different molecular structures to generate CAR-T cells and investigated their anti-tumor efficacy in vitro and in vivo. CD5 CARs with a 4-1BB costimulatory domain (BB.z) or a CD28 costimulatory domain (28.z) exhibited specific cytotoxicity against CD5+ malignant cells in vitro. However, both failed to prolong the survival of T-ALL xenograft mice. Subsequently, we substituted the 28.z CAR hinge region with CH2CH3, which enhanced the ability of CH2CH3-CD5 CAR-T cells to specifically eradicate T-ALL cells in vitro and in vivo. Furthermore, patient-derived CH2CH3-CD5 CAR-T cells were generated which showed a marked killing effect of CD5-positive acute T-ALL cells in vitro. The anti-tumor activity of CD5 CAR-T cells with a CD28 co-stimulation domain and CH2CH3 hinge region was superior to those with BB.z and 28.z domains. These preclinical data provided new insights into the factors dictating efficacy in T-ALL treatment with CAR-T cells and hold promise for clinical translation.

Isoforms of the TAL1 transcription factor have different roles in hematopoiesis and cell growth.

T-cell acute lymphoblastic leukemia (T-ALL) protein 1 (TAL1) is a central transcription factor in hematopoiesis. The timing and level of TAL1 expression orchestrate the differentiation to specialized blood cells and its overexpression is a common cause of T-ALL. Here, we studied the 2 protein isoforms of TAL1, short and long, which are generated by the use of alternative promoters as well as by alternative splicing. We analyzed the expression of each isoform by deleting an enhancer or insulator, or by opening chromatin at the enhancer location. Our results show that each enhancer promotes expression from a specific TAL1 promoter. Expression from a specific promoter gives rise to a unique 5' UTR with differential regulation of translation. Moreover, our study suggests that the enhancers regulate TAL1 exon 3 alternative splicing by inducing changes in the chromatin at the splice site, which we demonstrate is mediated by KMT2B. Furthermore, our results indicate that TAL1-short binds more strongly to TAL1 E-protein partners and functions as a stronger transcription factor than TAL1-long. Specifically TAL1-short has a unique transcription signature promoting apoptosis. Finally, when we expressed both isoforms in mice bone marrow, we found that while overexpression of both isoforms prevents lymphoid differentiation, expression of TAL1-short alone leads to hematopoietic stem cell exhaustion. Furthermore, we found that TAL1-short promoted erythropoiesis and reduced cell survival in the CML cell line K562. While TAL1 and its partners are considered promising therapeutic targets in the treatment of T-ALL, our results show that TAL1-short could act as a tumor suppressor and suggest that altering TAL1 isoform's ratio could be a preferred therapeutic approach.

Apoptosis-mediated inhibition of human T-cell acute lymphoblastic leukemia upon treatment with Staphylococus Aureus enterotoxin-superantigen.

Patients with relapsed T cell acute lymphoblastic leukemia (T-ALL) have limited therapeutic options and poor prognosis. The finding of efficient strategies against this refractory neoplasm is a medical priority. Superantigens (SAgs) are viral and bacterial proteins that bind to major histocompatibility complex class II molecules as unprocessed proteins and subsequently interact with a high number of T cells expressing particular T cell receptor Vβ chains. Although on mature T cells, SAgs usually trigger massive cell proliferation producing deleterious effects on the organism, in contrast, on immature T cells, they may trigger their death by apoptosis. On this basis, it was hypothesized that SAgs could also induce apoptosis in neoplastic T cells that are usually immature cells that probably conserve their particular Vβ chains. In this work, we investigated the effect of the SAg Staphylococcus aureus enterotoxin E (SEE) (that specifically interacts with cells that express Vβ8 chain), on human Jurkat T- leukemia line, that expresses Vβ8 in its T receptor and it is a model of the highly aggressive recurrent T-ALL. Our results demonstrated that SEE could induce apoptosis in Jurkat cells in vitro. The induction of apoptosis was specific, correlated to the down regulation of surface Vβ8 TCR expression and was triggered, at least in part, through the Fas/FasL extrinsic pathway. The apoptotic effect induced by SEE on Jurkat cells was therapeutically relevant. In effect, upon transplantation of Jurkat cells in the highly immunodeficient NSG mice, SEE treatment reduced dramatically tumor growth, decreased the infiltration of neoplastic cells in the bloodstream, spleen and lymph nodes and, most importantly, increased significantly the survival of mice. Taken together, these results raise the possibility that this strategy can be, in the future, a useful option for the treatment of recurrent T-ALL.

WDR54 exerts oncogenic roles in T-cell acute lymphoblastic leukemia.

WDR54 has been recently identified as a novel oncogene in colorectal and bladder cancers. However, the expression and function of WDR54 in T-cell acute lymphoblastic leukemia (T-ALL) were not reported. In this study, we investigated the expression of WDR54 in T-ALL, as well as its function in T-ALL pathogenesis using cell lines and T-ALL xenograft. Bioinformatics analysis indicated high mRNA expression of WDR54 in T-ALL. We further confirmed that the expression of WDR54 was significantly elevated in T-ALL. Depletion of WDR54 dramatically inhibited cell viability and induced apoptosis and cell cycle arrest at S phase in T-ALL cells in vitro. Moreover, knockdown of WDR54 impeded the process of leukemogenesis in a Jurkat xenograft model in vivo. Mechanistically, the expression of PDPK1, phospho-AKT (p-AKT), total AKT, phospho-ERK (p-ERK), Bcl-2 and Bcl-xL were downregulated, while cleaved caspase-3 and cleaved caspase-9 were upregulated in T-ALL cells with WDR54 knockdown. Additionally, RNA-seq analysis indicated that WDR54 might regulate the expression of some oncogenic genes involved in multiple signaling pathways. Taken together, these findings suggest that WDR54 may be involved in the pathogenesis of T-ALL and serve as a potential therapeutic target for the treatment of T-ALL.

Ginsenoside 24-OH-PD from red ginseng inhibits acute T-lymphocytic leukaemia by activating the mitochondrial pathway.

Ginsenoside 24-hydroxy-ginsengdiol (24-OH-PD), extracted from red ginseng, is a novel diol-type ginsenoside, strongly inhibits the growth of human T-cell acute lymphoblastic leukaemia (T-ALL) CCRF-CEM cells. Our research aimed at investigating the mechanism underlying this inhibition. Cell viability was determined using the cell counting kit-8 (CCK-8) assay, and NOD/SCID mice bearing CCRF-CEM cells were used to verify the therapeutic effect of 24-OH-PD on T-ALL in vivo. We equally analysed pathways related to 24-OH-PD in CCRF-CEM cells using RNA-Seq analysis. Cell apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and mitochondrial permeability transition pore (mPTP) levels were detected by flow cytometry. The activity of caspase3 and caspase9 was detected by enzyme activity detection kits. The expression levels of apoptosis-related proteins and mRNA were determined through western blotting and quantitative reverse-transcription PCR assays (qRT-PCR). CCK-8 assay and animal xenograft experiments confirmed that 24-OH-PD significantly inhibited T-ALL in a dose-dependent manner, both in vivo and in vitro. RNA-Seq results suggest that mitochondria-mediated apoptosis pathway plays an important role in this process. Furthermore, intracellular ROS levels increased, mPTP opened, and ΔΨm decreased following 24-OH-PD treatment. Pretreatment with the antioxidant, NAC, reversed the effects of 24-OH-PD on apoptosis and ROS generation. Moreover, 24-OH-PD treatment increased the expression of Bax and caspase family members, thereby releasing cytochrome c (Cytc) and inducing apoptosis. Our findings showed that, 24-OH-PD induces apoptosis in CCRF-CEM cells by activating the mitochondrial-dependent apoptosis pathway through ROS accumulation. This inhibitory effect implies that 24-OH-PD could be further developed as treatment of T-ALL.

Bioinformatics and Raman spectroscopy-based identification of key pathways and genes enabling differentiation between acute myeloid leukemia and T cell acute lymphoblastic leukemia.

Acute myeloid leukemia (AML) and T cell acute lymphoblastic leukemia (T-ALL) are two of the most prevalent hematological malignancies diagnosed among adult leukemia patients, with both being difficult to treat and associated with high rates of recurrence and mortality. In the present study, bioinformatics approaches were used to analyze both of these types of leukemia in an effort to identify characteristic gene expression patterns that were subsequently validated via Raman spectroscopy. For these analyses, four Gene Expression Omnibus datasets (GSE13204, GSE51082, GSE89565, and GSE131184) pertaining to acute leukemia were downloaded, and differentially expressed genes (DEGs) were then identified through comparisons of AML and T-ALL patient samples using the R Bioconductor package. Shared DEGs were then subjected to Gene Ontology (GO) enrichment analyses and were used to establish a protein-protein interaction (PPI) network analysis. In total, 43 and 129 upregulated and downregulated DEGs were respectively identified. Enrichment analyses indicated that these DEGs were closely tied to immune function, collagen synthesis and decomposition, inflammation, the synthesis and decomposition of lipopolysaccharide, and antigen presentation. PPI network module clustering analyses further led to the identification of the top 10 significantly upregulated and downregulated genes associated with disease incidence. These key genes were then validated in patient samples via Raman spectroscopy, ultimately confirming the value of these genes as tools that may aid the differential diagnosis and treatment of AML and T-ALL. Overall, these results thus highlight a range of novel pathways and genes that are linked to the incidence and progression of AML and T-ALL, providing a list of important diagnostic and prognostic molecular markers that have the potential to aid in the clinical diagnosis and treatment of these devastating malignancies.

Abstract 4797: β3-AR blockade induces differentiation of malignant leukemia T cells in mature T lymphocytes

Abstract Acute lymphoblastic leukemia (ALL) is a genetically heterogeneous cancer that arises from diverse genetic alterations during early lymphocyte development. About 10-15% of T-cell acute lymphoblastic leukemia (T-ALL) cases occur in childhood; despite having an event-free survival (EFS) rate of 85%, relapse is the main problem of this type of cancer. Mitochondria metabolic reprogramming plays a crucial role in the development of lymphocytes both in physiological and pathological processes such as differentiation and neoplastic transformation respectively. Mitochondria are the main actors of cellular homeostasis. They use glycolysis products exploiting them in the Krebs cycle and in oxidative phosphorylation to produce energy and plays key roles in reactive oxygen species (ROS) generation, Ca2+ signaling, cell death induction, and cellular differentiation. In particular, the increase of ROS is important to reduce tumor cell viability and at the same time is involved in induction of hematopoiesis. β3-adrenoreceptors (β3-Ars) is a class of receptors associated with pro-tumoral signaling in different tumors. In this study, we first have observed the expression of β3-Ars in leukemia T cells and then we studied how the modulation of this receptors affected tumor-related pathways. The treatment with an antagonist of β3-Ars (SR59230A) induced an increase of ROS leading to lymphoblastic cells differentiation, decrease of proliferation and enhanced induction of apoptosis signaling. Overall, these results suggest that β3-Ars could be a possible target for treatment of T-ALL. Citation Format: Francesco Carrozzo, Gennaro Bruno, Claudia Masi, Alessia Boaretto, Claudio Favre, Maura Calvani. β3-AR blockade induces differentiation of malignant leukemia T cells in mature T lymphocytes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4797.

Synthetic oleanane triterpenoids suppress MYB oncogene activity and sensitize T-cell acute lymphoblastic leukemia cells to chemotherapy.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with poor prognosis. The MYB oncogene encodes a master transcription factor that is activated in the majority of human T-ALLs. In the present study, we have performed a large-scale screening with small-molecule drugs to find clinically useful inhibitors of MYB gene expression in T-ALL. We identified several pharmacological agents that potentially could be used to treat MYB-driven malignancies. In particular, treatment with the synthetic oleanane triterpenoids (OTs) bardoxolone methyl and omaveloxolone decreased MYB gene activity and expression of MYB downstream target genes in T-ALL cells with constitutive MYB gene activation. Notably, treatment with bardoxolone methyl and omaveloxolone led to a dose-dependent reduction in cell viability and induction of apoptosis at low nanomolar concentrations. In contrast, normal bone marrow-derived cells were unaffected at these concentrations. Bardoxolone methyl and omaveloxolone treatment downregulated the expression of DNA repair genes and sensitized T-ALL cells to doxorubicin, a drug that is part of the standard therapy of T-ALL. OT treatment may thus potentiate DNA-damaging chemotherapy through attenuation of DNA repair. Taken together, our results indicate that synthetic OTs may be useful in the treatment of T-ALL and potentially also in other MYB-driven malignancies.

IgD/FcδR is involved in T-cell acute lymphoblastic leukemia and regulated by IgD-Fc-Ig fusion protein

T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis as a result of severe immunosuppression and rapid tumor progression with resistance to conventional chemotherapy. Excessive IgD may play a role in T cell activation via IgD Fc receptor (FcδR). Here we aimed to investigate the effects of IgD in T-ALL and demonstrated the potential benefit by targeting IgD/FcδR in T-ALL patients with IgD-Fc-Ig fusion protein. In T-ALL patients’ blood samples and cell lines, the level of IgD, the percentage of FcδR expressing cells and the binding affinity were determined by flow cytometry. T cell viability, proliferation and apoptosis were analyzed. A mouse xenograft model was used to evaluate the in vivo effect of IgD-Fc-Ig, an IgD-FcδR blocker. The levels of serum IgD and FcδR were abnormally increased in part of T-ALL patients and IgD could induce over-proliferation and inhibit apoptosis of T-ALL cells in vitro. FcδR was constitutively expressed on T-ALL cells. IgD-Fc-Ig showed similar binding affinity to FcδR and selectively blocked the stimulation effect of IgD on T-ALL cells in vitro. In vivo study exhibited that IgD-Fc-Ig may also have therapeutic benefit. IgD-Fc-Ig administration inhibited human T-ALL growth and extended survival in xenograft T-ALL mice. In conclusion, this work supports the idea of targeting IgD/FcδR in T-ALL patients with excessive IgD. IgD-Fc-Ig fusion protein might be a potential biological drug with high selectivity for T-ALL treatment.

Exploring the pharmacological mechanisms of Shuanghuanglian against T-cell acute lymphoblastic leukaemia through network pharmacology combined with molecular docking and experimental validation

Context Due to the poor prognosis of T-cell acute lymphoblastic leukaemia (T-ALL), there is an urgent need to identify safer and more cost-effective drugs. Objective This study evaluated the antitumour activity of Shuanghuanglian (SHL) on T-ALL cells and elucidated the mechanism. Materials and methods Jurkat and Molt4 cells were treated with SHL (0.1, 0.2 and 0.4 mg/mL) for 24 and 48 h. The controls were treated with RPMI 1640 containing 10% foetal bovine serum. Cell viability was evaluated through Cell Counting Kit-8 assay. Patterns of death and signalling pathway alterations caused by SHL were identified by network pharmacology combined with GO enrichment analysis and then were verified by Hoechst 33342 staining, Annexin V-FITC/PI staining and Western blotting. Interactions of the active ingredients with targets were analysed by molecular docking. Results The IC50 values of SHL in Jurkat and Molt4 cells were 0.30 ± 0.10 and 0.48 ± 0.07 mg/mL, respectively, at 24 h and 0.27 ± 0.05 and 0.30 ± 0.03 mg/mL at 48 h. In T-ALL, 117 target genes of SHL were mainly enriched in the apoptosis and NOTCH signalling pathways. SHL induced apoptosis was confirmed by Hoechst 33342 staining and flow cytometry. The protein levels of cleaved caspase-7 and cleaved PARP were significantly increased but those of cleaved NOTCH1 and MYC were reduced. The active ingredients of SHL can interact with γ-secretase. Discussion and conclusions: SHL induces apoptosis in T-ALL cells via the NOTCH1-MYC pathway and may be a potential drug for the treatment of T-ALL.

Targeting Leukemia-Initiating Cells and Leukemic Niches: The Next Therapy Station for T-Cell Acute Lymphoblastic Leukemia?

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of hematological malignancy characterized by its high heterogeneity and potentially life-threatening clinical features. Despite the advances in risk stratification and therapeutic management of T-ALL, patients often suffer from treatment failure and chemotherapy-induced toxicity, calling for greater efforts to improve therapeutic efficacy and safety in the treatment of T-ALL. During the past decades, increasing evidence has shown the indispensable effects of leukemia-initiating cells (LICs) and leukemic niches on T-ALL initiation and progression. These milestones greatly facilitate precision medicine by interfering with the pathways that are associated with LICs and leukemic niches or by targeting themselves directly. Most of these novel agents, either alone or in combination with conventional chemotherapy, have shown promising preclinical results, facilitating them to be further evaluated under clinical trials. In this review, we summarize the latest discoveries in LICs and leukemic niches in terms of T-ALL, with a particular highlight on the current precision medicine. The challenges and future prospects are also discussed.

A System-Level Study Reveals That a Defective Thymic Niche Accelerates the T-ALL Initiation

The ontogeny of T-cell acute lymphoblastic leukemia (T-ALL) is unknown. In humans and mice, T-ALL is diagnosed when malignant T cells are detectable in the peripheral blood, i.e., at a stage when disease development is complete. Therefore, the malignant processes in the thymus - where the T cells develop - are an unexplored "black box". We took an interdisciplinary approach combining in vivo experiments with computational modeling to investigate the role of the thymic niche in disease manifestation. We focused on the role of interleukin 7 (IL-7) in the thymus, as approximately 70-80% of T-ALL patients have either increased IL-7 receptor (IL-7R) expression or gain-of-function mutations in this gene. In addition, autocrine production of IL-7 by T-ALL blasts has been detected in some patients. Using our recently developed "virtual thymus" model (Aghaallaei et al., Science Advances, 2021), we first simulated more than 200 scenarios to find out the conditions that lead to massive expansion of a single clone in the thymus. We then genetically engineered thymocytes and thymic epithelial cells (TECs) in medaka fish based on in silico predictions. Consistent with our computational model, we found that manipulation of thymocytes or TECs alone resulted in only 25% (N=36) and 10% (N=29) T-ALL development, respectively, in the thymus of Medaka larvae after five days; however, this frequency was accelerated to 54% (N=45) when both cell types were manipulated simultaneously. Our results strongly suggest that defective thymic niche has a strong impact on T-ALL development. Taken together, we propose a previously unknown mechanism that may trigger T-ALL initiation in the thymus. In addition, we introduce the "virtual thymus" as a new versatile in silico model that can predict the outcome of conditions for the development or treatment of T-ALL.

Preclinical Evaluation of ISB 1442, a First-in-Class CD38 and CD47 Bispecific Antibody Innate Cell Modulator for the Treatment of AML and T-ALL

ISB 1442 is a bispecific antibody (BsAb) using Ichnos' proprietary Bispecific Engagement by Antibodies based on the TCR (BEAT®) platform. A fully human BsAb with anti-CD38 and CD47 binding arms, ISB 1442 was developed for the treatment of hematologic malignancies. The CD38 binding arm consists of two bi-paratopic Fabs that strongly bind to CD38 through avidity-induced interactions. The anti-CD47 arm comprises a single Fab designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes. With this design, the CD38 Fab arm preferentially drives binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. Hence, ISB 1442 is anticipated to induce minimal effects on red blood cells (RBC) compared to benchmark anti-CD47 monoclonal antibodies (mAb), such as magrolimab. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Preclinical activity of ISB 1442 for the treatment of relapsed/refractory multiple myeloma (rrMM), including increased killing potency relative to daratumumab and magrolimab benchmarks as well as more favorable off tumor/on target specificity, was reported previously (Sammicheli et al. ASH 2021). ISB 1442 is currently in a Phase 1 clinical trial for rrMM. Beyond MM, CD38 and CD47 are expressed in other hematologic cancers, including acute myeloid leukemia (AML) and T cell acute lymphoblastic leukemia (T-ALL). In AML, unleashing tumor cell phagocytosis using magrolimab shows clinical benefit in combination with demethylating agents and BCL-2 inhibitors. T-ALL is characterized with high CD38 expression, and daratumumab in combination with chemotherapy is under investigation (NCT03384654) for this indication. ISB 1442 has the potential to treat AML and T-ALL patients with its first-in-class molecular attributes of co-targeting CD38 and CD47 by a single antibody with enhanced Fc effector functions. ISB 1442 induced potent phagocytosis of both AML and T-ALL cell lines in vitro. The induction of phagocytosis was more prominent in cell lines expressing high level of CD38. ISB 1442 also induced higher phagocytosis of both AML and T-ALL tumor cells as compared to daratumumab and anti-CD47 (5F9) in cell lines expressing low level of CD38 and CD47. In addition to phagocytosis, ISB 1442 induced potent ADCC of both AML and T-ALL cell lines. To characterize the complex mechanisms of action of ISB 1442 in a single assay, a multiple mode of action of killing (MMoAK) in vitro assay was established where macrophages and PBMCs are incubated with AML or T-ALL tumor cells and human serum. With this approach, tumor cells can be targeted simultaneously by NK cells from PBMCs (ADCC Mechanism of Action (MOA)), macrophages (ADCP MOA), and complement from human serum (CDC MOA). ISB 1442 induced higher killing of AML and T-ALL cell lines in MMoAK assays as compared to daratumumab or anti-CD47 (5F9). ISB 1442 induced potent killing of primary tumor samples from both AML and T-ALL patients in a heterologous assay using macrophages from healthy donors as effector cells, suggesting that target cancer cells do not have intrinsic mechanisms protecting them from the immune cell-mediated MOA of ISB 1442 killing . In patients' samples having detectable levels of myeloid or NK cells in bone marrow aspirates, ISB 1442 induced killing of tumor cells in the autologous setting in a direct killing assay ex vivo. In summary, these data support the clinical development of 1442 in AML and T-ALL. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to have antitumor activity in AML and T-ALL patients in a single antibody relative to anti-CD38 or anti-CD47 monoclonal antibodies as well as their combination.

Single-Cell Multi-Omics Reveals Immune Microenvironment Alterations in T-Cell Acute Lymphoblastic Leukemia

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that predominantly occurs in children and young adults. Although intensified multi-agent chemotherapy improved overall survival rates, relapsed/refractory T-ALL remains difficult to treat. In addition, these intensive chemotherapies carry a risk for short- and long-term side effects, especially in children. There is an urgent need for novel therapeutic strategies to improve patient outcome. Targeting the tumor microenvironment has been recognized as a promising strategy in many adult tumors with high neoantigen load, but it is thought to be less effective in pediatric and hematologic malignancies as these carry few genetic mutations. However, comprehensive studies of the immune microenvironment are lacking in many pediatric malignancies, including T-ALL, but are urgently required to identify novel therapeutic targets. The aim of this study is to unravel mechanisms of immune evasion in T-ALL and to establish novel immunotherapeutic targets for the treatment of T-ALL. Methods We performed 5’ single cell transcriptome, surface proteome of ~200 proteins and immune repertoire analyses of ~54,000 cells from 10 primary T-ALL pediatric patient samples at initial diagnosis and 4 normal donors using the 10x Genomics platform. Sequencing data was processed using Cell Ranger (reference genome hg38). Quality control was performed using Scrublet (doublet removal), DropletQC (quality control damaged cells) and Seurat (percentage mtDNA < 10% and genes expressed > 200). RNA counts were normalized with sctransform and surface protein antibody counts were centered log-ratio normalized using Seurat. Non-linear dimensional reduction (UMAP) and differential gene/protein expression was done using Seurat and cell type annotation was done using Azimuth in combination with known marker genes. In addition, we analyzed differential cell type abundance (MiloR), the immune repertoire (Immunarch), transcription factor activity (pyScenic) and ligand-receptor activity (NicheNet). Results Our multi-omics analysis revealed changes in the immune cell type composition in T-ALL associated with an immunosuppressive microenvironment. Specifically, we identified a significant enrichment of non-classical monocytes, atypical memory B-cells and double-negative T-cells (CD3+, CD4-, CD8-) in the T-ALL immune microenvironment compared to the normal immune microenvironment. These CD21-, CD11c+ atypical memory B-cells showed high expression of multiple inhibitory receptors (including PD-1, FCRL3/5 and ILT2), suggesting muted BCR-signaling. Transcription factor analysis showed transcriptional activity of known B-cell regulators SOX5, TBX21 and POU2F2, of which SOX5 is known to be highly expressed during late B-cell differentiation stages including atypical memory B-cells. Double-negative (DN) T-cells, which were oligoclonal, were also overrepresented in the T-ALL microenvironment. Correspondingly, immune repertoire analysis of normal T-cells showed decreased TCR diversity (Chao1 index, p = 0.016, Wilcoxon rank sum test) compared to normal donors. Surprisingly, these DN T-cells displayed features of CD8+ T-cell dysfunction, such as high expression of PD-1, TIM3 and LAG3, as well as features of regulatory T-cells, such as high expression of IL10, ICOS and CTLA4. These cells also demonstrated high transcription factor activity of EOMES, NFATC1 and ETV1, which are all associated with a CD8+ T-cell dysfunctional transcriptional network, as well as FOXP3 transcription factor activity, the master transcription factor in regulatory T-cells. Conclusions Multi-omics profiling of the immune microenvironment in T-ALL revealed the presence of several populations of dysfunctional cells associated with an immunosuppressive microenvironment. Studying ligand-receptor interactions analysis between malignant and immune cells will provide the basis for future (pre)clinical studies leading to the development of novel immunotherapeutic strategies for the treatment of T-ALL.

Inhibition of the m6A reader IGF2BP2 as a strategy against T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant leukemia with extremely limited treatment for relapsed patients. N6‐methyladenosine (m6A) reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) participates in the initiation and growth of cancers by communicating with various targets. Here, we found IGF2BP2 was highly expressed in T-ALL. Gain and loss of IGF2BP2 demonstrated IGF2BP2 was essential for T-ALL cell proliferation in vitro and loss of IGF2BP2 prolonged animal survival in a human T-ALL xenograft model. Mechanistically, IGF2BP2 directly bound to T-ALL oncogene NOTCH1 via an m6A dependent manner. Furthermore, we identified a small-molecule IGF2BP2 inhibitor JX5 and treatment of T-ALL with JX5 showed similar functions as knockdown of IGF2BP2. These findings not only shed light on the role of IGF2BP2 in T-ALL, but also provide an alternative γ‑Secretase inhibitors (GSI) therapy to treat T-ALL.

Characteristics and prognostic effects of NOTCH1/FBXW7 gene mutations in T-cell acute lymphoblastic leukemia patients

Objective: To explore the characteristics, clinical features and prognostic effects of NOTCH1/FBXW7 gene mutations in T-cell acute lymphoblastic leukemia (T-ALL) patients. Methods: The clinical data of 61 T-ALL patients who underwent second-generation gene sequencing in Henan Provincial People's Hospital from March 2016 to March 2021 were retrospectively analyzed. There were 46 males and 15 females, with a median age [M (Q1, Q3)] of 18 (11, 30) years. The relationship between NOTCH1/FBXW7 gene mutation characteristics, clinical and laboratory parameters and their impact on event free survival (EFS) and overall survival (OS) were analyzed. Results: NOTCH1 gene mutations were found in 34 cases (55.7%, 34/61), including 22 cases of heterodimer domain (HD) mutations (64.7%), 7 cases of proline/glutamate/serine/threonine (PEST) mutations (20.6%), and 5 cases of both HD and PEST mutations (14.7%). FBXW7 gene mutations were detected in 9 cases (14.8%, 9/61), of which 5 cases had both NOTCH1 and FBXW7 gene mutations. Twenty-three (37.7%, 23/61) cases were wild type. The median white blood cell count of patients in NOTCH1/FBXW7 gene mutations group and wild-type group was 76.4×109/L (8.3×109/L, 149.2×109/L), 54.1×109/L (5.3×109/L, 156.6×109/L), respectively. Moreover, the hemoglobin was (89.1±27.1) g/L and (99.5±23.1) g/L, respectively, and the median proportion of bone marrow primordial cells was 84.5% (69.0%, 91.3%) and 60.0%(35.0%, 80.0%), respectively. The gene expression rate of SIL-TAL1, Hox11 and Hox11L2 was 7.9% (3/38) vs 17.4% (4/23), 18.4% (7/38) vs 4.3% (1/23), 5.3% (2/38) vs 13.0% (3/23), respectively (all P>0.05). However, the median platelet level in the NOTCH1/FBXW7 gene mutations group was 60.5×109/L (36.8×109/L, 100.3×109/L), which was lower than that in the wild-type group [116.0×109/L (63.0×109/L, 178.0×109/L)] (P=0.018). The median number of gene mutations in the group with NOTCH1/FBXW7 gene mutations group was 2.5 (1.8, 4.0), which was more than that in the group without NOTCH1/FBXW7 gene mutations group [0 (0, 1.0)] (P<0.001). The median EFS and OS of adult NOTCH1/FBXW7 gene mutations group were 28.0 (95%CI: 7.3-48.7) months and 30.0 (95%CI: 8.9-51.1) months, respectively, which were better than those of adult wild-type group [4.5 (95%CI: 0-11.6) months and 9.0 (95%CI: 0-19.1) months] (P=0.008 and 0.014).The median EFS and OS of children NOTCH1/FBXW7 gene mutations group were 12.0 (95%CI: 10.4-13.6) months and 19.0 (95%CI: 13.6-24.4) months, respectively, and those of wild-type group were 10.0 (95%CI: 8.9-11.1) months and 21.0 (95%CI: 0-51.4) months, respectively (P=0.673 and 0.434). Conclusions: The mutation rate of NOTCH1/FBXW7 gene is higher in T-ALL patients. Patients with NOTCH1/FBXW7 gene mutations group have lower platelet count and better EFS and OS. NOTCH1/FBXW7 gene mutation may be used as a hierarchical basis for individualized treatment of adult T-ALL patients.

Inhibition of DEK Enhances Doxorubicin-Induced Apoptosis and Cell Cycle Arrest in T-Cell Acute Lymphoblastic Leukemia Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a serious hematological tumor derived from early T-cell progenitors, which is extremely resistant to chemotherapy. Classically, doxorubicin (DOX) is an effective first-line drug for the treatment of T-ALL; however, DOX resistance limits its clinical effect. The DEK proto-oncogene (DEK) has been involved in neoplasms but remains unexplored in T-ALL. We silenced DEK on Jurkat cells and detected cell proliferation with cell counting and colony formation assay. Then, we detected DEK's drug sensitivity to DOX with CCK-8, cell cycle, and apoptosis with DOX treatment. Western blot analysis was performed to determine protein expression of apoptosis and cell cycle-related genes, including BCL2L1, caspase-3, and cyclin-dependent kinases (CDK). Finally, the tumorigenic ability of DEK was analyzed using a BALB/C nude mouse model. In this study, DEK was highly expressed in Jurkat cells. Inhibition of DEK can lead to decreased cell proliferation and proportion of S-phase cells in the cell cycle and more cell apoptosis, and the effect is more obvious after DOX treatment. Western blot results showed that DOX treatment leads to cell cycle arrest, reduction of cyclin-dependent kinase 6 (CDK6) protein, accumulation of CDKN1A protein, and DOX-induced apoptosis accompanied by reductions in protein levels of BCL2L1, as well as increases in protein level of caspase-3. Furthermore, DEK-silenced Jurkat cells generated a significantly smaller tumor mass in mice. Our study found that DEK is a novel, potential therapeutic target for overcoming DOX resistance in T-ALL.

CircFBXW7 inhibits the tumorigenesis of T-cell acute lymphoblastic leukemia through modulating miR-494-3p/SOX1 axis

T-cell acute lymphoblastic leukemia (T-ALL) is a type of leukemia with high malignant behaviors, which seriously threatens the health of people. It has been reported that circFBXW7 is downregulated in lymphoblastic leukemia. Nevertheless, the exact role of circFBXW7 in T-ALL remains elusive. MTT assay was used to assess the cell viability. Cell apoptosis was assessed by flow cytometry. In addition, mRNA expressions were assessed by RT-qPCR, and a western blot was applied to investigate the protein levels. Meanwhile, the correlation among circFBXW7, miR-494-3p, and SOX1 was explored by RNA pull-down and dual-luciferase reporter assays. Furthermore, a xenograft mice model was conducted to verify the function of circFBXW7 in T-ALL in vivo. CircFBXW7 was significantly downregulated in T-ALL, of which overexpression inhibited the cell viability and induced the apoptosis of Jurkat cells. Moreover, miR-494-3p was identified to be a functional downstream effector to be involved in circFBXW7-mediated T-ALL cell proliferation. Besides, SOX1 was a direct target of miR-494-3p, and the impact of miR-494-3p mimics on T-ALL cell growth was inhibited in the presence of SOX1 overexpression. Furthermore, overexpression of circFBXW7 dramatically inhibited T-ALL tumor growth. In summary, circFBXW7 attenuated the tumorigenesis of T-ALL through the mediation of the miR-494-3p/SOX1 axis, which might be novel targets for T-ALL treatment.

Novel Compound, ND-17, Regulates the JAK/STAT, PI3K/AKT, and MAPK Pathways and Restrains Human T-lymphoid Leukemia Development.

T cell acute lymphoblastic leukemia (T-ALL) is an invasive hematological malignant disorder of T cell progenitors. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway plays an important role in the development of T-ALL and in the inhibition of the key molecule, JAK2, and could suppress T-ALL cell proliferation. The objective of this study was to investigate the in vitro anti-tumor effects of a novel nilotinib derivative, ND-17, on cancer cell lines via its interactions with JAK2. The effects of ND-17 on cell proliferation and on cell cycle and apoptosis were evaluated using the tetrazolium assay and flow cytometry, respectively. In addition, the ND-17/JAK2 binding interactions were evaluated using surface plasmon resonance and western blot analyses. ND-17 exerted the greatest inhibitory effects on T-ALL cells amongst all hematological cancer cell lines tested. Flow cytometric analysis indicated that ND-17 blocked the cell cycle at the S phase in T-ALL cells. Nilotinib did not significantly inhibit T-ALL cell growth or regulate the cell cycle. Preliminary investigations revealed that the regulation of cyclin-dependent kinases/cyclins was attributed to ND-17-induced cell cycle arrest. Furthermore, ND-17 could bind to JAK2 with strong affinity, and more importantly, ND-17 bound to the ATP pocket of JAK2 in a manner similar to the potent inhibitor. Thus, ND-17 treatment exhibited a prominent effect in inhibiting the phosphorylation of JAK2 in T-ALL cells. An increase in the phosphorylation of JAK2 was observed in interleukin-6- stimulated Jurkat cells, which was reversed by ND-17 treatment. Meanwhile, the combination of TG- 101348 and ND-17 led to further improvement in inhibiting the phosphorylation of JAK2. Moreover, the transfection and knockdown of JAK2 altered the inhibitory effect of ND-17 on Jurkat cell viability. In addition, ND-17 treatment suppressed the JAK/STAT, phosphatidylinositol-3-kinase/protein kinase B/mechanistic target of rapamycin, and mitogen-activated protein kinase/extracellular signal-regulated protein kinases 1 and 2 signaling pathways. These findings suggest that ND-17 could be a promising JAK2 inhibitor for the treatment of T-ALL.