T-cell Acute Lymphoblastic Leukemia Progression Research Articles

The DMT1 isoform lacking the iron-response element regulates normal and malignant hematopoiesis via NOTCH pathway activation.

Natural resistance-associated macrophage protein 2 (NRAMP 2; also known as DMT1 and encoded by SLC11A2) is mainly known for its iron transport activity. Recently, the DMT1 isoform lacking the iron-response element (nonIRE) was associated with aberrant NOTCH pathway activity. In this report, we investigated the function of DMT1 nonIRE in normal and malignant hematopoiesis. Knockdown of Dmt1 nonIRE in mice showed that it has non-canonical functions in hematopoietic stem cell differentiation: its knockdown suppressed development along the myeloid and lymphoid lineages, while promoting the production of platelets. These phenotypic effects on the hematopoietic system induced by Dmt1 nonIRE knockdown were linked to suppression of Notch/Myc pathway activity. Conversely, our data indicate a non-canonical function for DMT1 nonIRE overexpression in boosting NOTCH pathway activity in T-cell leukemia homeobox protein 1 (TLX1)-defective leukemia. This work sets the stage for future investigation using a multiple-hit T-cell acute lymphoblastic leukemia (T-ALL) model to further investigate the function of DMT1 nonIRE in T-ALL disease development and progression.

The BET inhibitor GNE-987 effectively induces anti-cancer effects in T-cell acute lymphoblastic leukemia by targeting enhancer regulated genes.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy originating from T progenitor cells. It accounts for 15% of childhood and 25% of adult ALL cases. GNE-987 is a novel chimeric molecule developed using proteolysis-targeting chimeras (PROTAC) technology for targeted therapy. It consists of a potent inhibitor of the bromodomain and extraterminal (BET) protein, as well as the E3 ubiquitin ligase Von Hippel-Lindau (VHL), which enables the effective induction of proteasomal degradation of BRD4. Although GNE-987 has shown persistent inhibition of cell proliferation and apoptosis, its specific antitumor activity in T-ALL remains unclear. In this study, we aimed to investigate the molecular mechanisms underlying the antitumor effect of GNE-987 in T-ALL. To achieve this, we employed technologies including RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and CUT&Tag. The degradation of BET proteins, specifically BRD4, by GNE-987 has a profound impact on T-ALL cell. In in vivo experiments, sh-BRD4 lentivirus reduced T-ALL cell proliferation and invasion, extending the survival time of mice. The RNA-seq and CUT&Tag analyses provided further insights into the mechanism of action of GNE-987 in T-ALL. These analyses revealed that GNE-987 possesses the ability to suppress the expression of various genes associated with super-enhancers (SEs), including lymphoblastic leukemia 1 (LCK). By targeting these SE-associated genes, GNE-987 effectively inhibits the progression of T-ALL. Importantly, SE-related oncogenes like LCK were identified as critical targets of GNE-987. Based on these findings, GNE-987 holds promise as a potential novel candidate drug for the treatment of T-ALL.

CXCR3/CXCL10 Signaling Pathway Drives T-ALL into the CNS/Meningeal Microenvironment

Cure rates for T-cell acute lymphoblastic leukemia (T-ALL) have improved significantly, but central nervous system (CNS) involvement leads to a poor prognosis. Understanding the mechanisms underlying CNS involvement could lead to more effective therapies. The CXCR3/CXCL10 signaling pathway mediates T cell movement across the blood-CSF barrier during neuroinflammation, but its role in T-ALL colonization of the CNS/meninges remains unclear. In our study, we hypothesized that T-ALL cells exploit normal T-cell function to infiltrate the meninges. We examined CXCR3 expression in thymic CD4+CD8+ double positive (DP) cells of ∆E-NOTCH1 T-ALL and control mice. Levels of CXCR3 were higher in CD4 +CD8 + DP cells of T-ALL mice compared to controls. Furthermore, leukemic cells in the meninges, bone marrow (BM), and thymus had higher CXCR3 levels compared to other organs ( p < 0.01), suggesting an organ-specific CXCR3 expression in infiltrating T-ALL cells. Additionally, primary T-ALL samples (n = 24) had elevated CXCR3 mRNA levels compared to normal thymocytes. Using CRISPR-Cas9, we deleted CXCR3 in the KOPTK1 and PER117 cell lines and in two primary patient samples. CXCR3 deletion resulted in decreased proliferation, delayed cell cycle progression in S/G2/M phases but did not affect apoptotic cell death. CXCR3 inactivation led to decreased activation of AKT, ERK1/2, JNK, and reduced the expression of cell motility regulators. Notably, T-ALL cells migrated more efficiently to CXCL10 than to CXCL9 and CXCL11, which was reduced upon CXCR3 silencing ( p < 0.01). Mechanistically, we demonstrated that USP7, a ubiquitin-specific protease, regulates and stabilizes the CXCR3 protein via deubiquitination. To explore the role of CXCR3 in T-ALL in vivo, we transplanted CXCR3 KO and control KOPTK1 cells into NSG mice (8 mice/group). Mice with CXCR3 KO had increased survival and significantly lower leukemia burden in the BM and extramedullary tissues compared to the control group ( p < 0.01). We next analyzed the levels of CXCR3 ligands, CXCL9, CXCL10, and CXCL11, in the blood serum and CSF of ∆E-NOTCH1 and control mice (5 mice/group). T-ALL mice had elevated CXCL10 levels in both blood serum and CSF, with higher levels in the CSF, suggesting compartmentalization of the immune response between the CSF and blood ( p < 0.01). CXCL9 and CXCL11 expression were minimal or undetectable. Remarkably, upon injecting ∆E-NOTCH1-transformed hematopoietic progenitors into CXCL10 knockout ( CXCL10 KO) and control B6 mice (5 mice/group), tissue analysis revealed a significant decrease in T-ALL meningeal infiltration in the CXCL10 KO mice, but not in other organs ( p < 0.01). Conversely, control mice exhibited high levels of leukemic cells in all tested organs. We found that meningeal stromal cells (PDGFR+, CD13+, CD31-, CD45-) expressed CXCL10 in ∆E-NOTCH1 mice, unlike hematopoietic (CD45+) and endothelial cells (CD31+, CD45-), compared to control mice. We further observed upregulation of CXCL10 in human meningeal stromal cells co-cultured with KOPTK1, PER117, and primary samples. Treatment with a CXCR3 antagonist or knockout of CXCR3 reduced leukemic cell migration to the meningeal stromal cells compared to controls ( p < 0.01). Conversely, migration of T-ALL cells to the tested stromal cells was reduced upon treatment with a CXCL10 neutralizing antibody or by CXCL10 deletion in stromal cells. Additionally, we identified that the CXCR3/CXCL10 signaling pathway regulated the expression of VLA-4 and VCAM-1, promoting T-ALL cell adhesion to the meningeal stroma. Genetic inactivation or pharmacological inhibition of CXCR3 in T-ALL cells or CXCL10 in meningeal stromal cells reduced cell-cell adhesion and the expression of VLA-4 and VCAM-1 in T-ALL and meningeal stroma, respectively, further supporting the role of CXCL10-CXCR3 in regulating T-ALL adhesion. Lastly, we investigated whether inflammatory cytokines derived from T-ALL induce CXCL10 in the meningeal microenvironment. We found elevated expression of Ifng, Tnf and Il27 in T-ALL cells infiltrating the meninges, but not other organs of ΔE-NOTCH1 mice ( p < 0.01). Silencing these genes in human T-ALL cells resulted in reduced CXCL10 expression and secretion, along with reduced leukemic cell migration to the tested meningeal stromal cells. In summary, our study emphasizes the key role of CXCR3-CXCL10 signaling in T-ALL progression and meningeal infiltration.

Therapeutic Effects and Metabolic Rewiring upon Glutaminase Loss in T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid neoplasm that requires intensified chemotherapy for treatment. Despite significant improvements in clinical outcomes with these chemotherapy regimens, still 20-25% of pediatric cases relapse, and the prognosis of these refractory T-ALLs remains extremely poor, underscoring the urgent need to explore novel targets for more effective approaches. The identification of highly prevalent (~60% patients) activating mutations in NOTCH1 has brought considerable interest in developing targeted anti-NOTCH1 therapies, offering promising alternatives to chemotherapy. However, early efforts in the clinic with ϒ-secretase inhibitors (which block a proteolytical cleavage critical for the activation of NOTCH1) have been hampered by limited and heterogenous therapeutic responses. Therefore, further advances in treatment of T-ALL require a deeper understanding of the basic mechanisms driving this disease. We have previously discovered a NOTCH1-controlled metabolic pathway that determines response to anti-NOTCH1 therapies. Specifically, glutaminolysis, which replenishes TCA cycle intermediates to maintain metabolism, is a critical pathway for leukemia cell growth downstream of NOTCH1. As such, inhibition of glutaminolysis pharmacologically or genetically (via deletion of glutaminase, Gls) is highly synergistic with anti-NOTCH1 therapies. However, Gls-deficient T-ALLs eventually progress, highlighting the need for improved therapeutic strategies. Moreover, the glutamine fate and the compensatory mechanisms to overcome Gls loss in leukemic cells in vivo are still poorly understood. To investigate the underlying mechanisms mediating the resistance to Gls loss, we systematically traced the nutrient utilization in isogenic Gls-positive and Gls-negative T-ALLs by intravenously infusing 13C-lactate, 13C-glucose and 13C-glutamine in leukemic mice. Quantitative analysis indicates that Gls-positive T-ALL cells use primarily glutamine as a carbon source to feed the TCA cycle. However, TCA labeling from glutamine decreases in Gls-deficient T-ALL cells, while lactate contribution to label TCA intermediates significantly increases, suggesting a potential compensatory metabolic rewiring mechanism to overcome Gls loss uncoupling TCA from glycolysis. In addition, we observed a reduction of the levels of palmitate, palmitoleate and some polyunsaturated fatty acids, together with a significant downregulation of the expression of Fasn, a central regulator of fatty acid anabolism, in Gls-deficient T-ALL cells. These results suggest a switch to lipid catabolic processes to support leukemic cell growth in absence of Gls in vivo. Gene expression profiling and proteomics analyses of these leukemias with isogenic loss of Gls revealed a strong enrichment in heme/porphyrin-biosynthesis signatures as heme-biosynthetic genes were systematically upregulated in Gls-deficient T-ALL cells as compared to T-ALL control cells. Indeed, using a loss-of function CRISPR-based genetic screening in combination with BPTES, a potent glutaminase inhibitor, in a human T-ALL cell line in vitro, we identified heme-biosynthesis pathway as a potential modulator, whose disruption could sensitize T-ALL cells to glutaminase inhibition. Overall, our findings formally show that while glutaminase plays a key role in T-ALL progression, still cells rapidly rewire their metabolism to compensate for Gls loss demonstrating a marked metabolic flexibility. Moreover, our results have unveiled previously unknown metabolic vulnerabilities that could be exploited in combination with glutaminase inhibitors in the clinical setting with the potential to improve the efficacy of T-ALL therapies and lead to better outcomes for patients.

Vinpocetine Mediates Therapeutic Activity Alone and in Combination with Chemotherapy in T-Cell Acute Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive cancer affecting immature T-cells and constitutes about 15% of pediatric acute lymphoblastic leukemia (ALL) cases. Despite advances in treatment, 20% of patients have or develop treatment-resistant disease, and their survival rates are only 15-25% even with additional intensive chemotherapy and hematopoietic stem cell transplant. Furthermore, the current intensive treatment regimens have long-term side effects in most patients. Therefore, there is a pressing need for new, more targeted, and less toxic treatment options for children with T-ALL. Genomic analysis of leukemia samples has enabled identification of chromosomal aberrations and gene mutations associated with T-ALL leukemia development and progression. Single cell analysis of genes expressed in pediatric T-ALL bone marrow samples at diagnosis identified blast-associated signature genes that correlated with poorer event-free survival, suggesting their involvement in leukemia progression and highlighting the potential for further investigation of the signature as an avenue to develop novel T-ALL therapies. A non-conventional genome-oriented approach was applied to the data utilizing the Library of Integrated Network-Based Cellular Signatures (LINCS) resource to identify drugs that are predicted to reverse the T-ALL blast signature. Vinpocetine, a vinca alkaloid derivative, was identified as a potential therapeutic candidate. Methods: Leukemia cell cultures were treated with incremental concentrations of vinpocetine, alone or in combination with a standard chemotherapeutic agent (vincristine or doxorubicin) for 72 hours and relative cell densities were determined by luminescent viability assay. Interactions between drugs were evaluated by mathematical modeling using the fractional product method. Orthotopic T-ALL patient-derived xenografts (PDXs) were established in immune-compromised NSG mice by intravenous injection and peripheral blood samples were collected at intervals for detection of leukemic blasts (human CD45+) by flow cytometry. Mice with low disease burden (<5% peripheral blasts) were randomized to groups and treated every third day with 10 or 20 mg/kg vinpocetine or an equivalent volume of vehicle administered intraperitoneally. Mice were weighed and their health status was evaluated at intervals to monitor for toxicity. Results: Treatment with vinpocetine resulted in a dose-dependent reduction in cell density in T-ALL cell line (Jurkat, HPB, and PEER) cultures relative to vehicle treatment, with IC50 values between 62.6 -64.4 uM, supporting the therapeutic potential of vinpocetine in T-ALL. Vinpocetine also interacted synergistically with vincristine or doxorubicin to provide enhanced therapeutic efficacy against Jurkat cells, indicated by a greater reduction in cell number compared to either single agent. This promising finding underscores the potential of vinpocetine as a supplemental therapy to improve responses or allow for dose reduction of existing chemotherapeutic agents for T-ALL therapy. In a preliminary dose-finding study, treatment with 20 mg/kg vinpocetine was well-tolerated with no overt evidence of toxicity and there was a slight trend toward decreased disease burden. Conclusion: Our study explores the therapeutic potential of vinpocetine for treatment of T-ALL. The observed inhibitory effects on leukemia cell expansion and enhanced efficacy with standard chemotherapies identify vinpocetine as a promising novel treatment approach for T-ALL. Dose escalation studies are needed to evaluate therapeutic effects of vinpocetine in mouse models.

Clonal Evolution Dissection Reveals High MSI2 Level Promotes Chemo-Resistance in T-Cell Acute Lymphoblastic Leukemia

Occurrences of T-cell acute lymphoblastic leukemia (T-ALL) arise in approximately 10-15% of pediatric ALL cases and the outcomes for high-risk T-ALL are inferior. Currently, the precise dissection of clonal evolution with intra-tumoral heterogeneity and to delineate targetable molecular events driving relapse in T-ALL subtypes have become area of interest. We carried out high-throughput droplet-based 5′-single-cell RNA-sequencing (scRNA-seq) and paired T-cell receptor sequencing (scTCR-seq) of isolated CD7 + BMMCs from five patients' Dx_Rel paired samples. To investigate the gene signature variation underlying leukemic progression, we observed two distinct patterns of evolutionary trajectories in the four Dx_Rel nonETP-ALL pairs. From T956 and T723, a significant outgrowth in TCR initially negligible diagnostic subclusters at relapse, to which surrogate TCR repertoires were correspondingly linked and enumerated, suggestive robust dominant “clonal shifts” under continuous chemotherapeutic pressure. Whereas in Dx_Rel pairs T593 and T856, which contained subclusters with distinct gene signatures at diagnosis or relapse respectively, shared an identical TCR genotype (TCR Dx-Rel major clone). The clonal architecture clues gained from simultaneous scRNA-seq and scTCR-seq are strongly indicative of the leukemic “clonal drift” that commonly occurs under chemotherapeutic stress, during which somehow, hypothetically, molecular signatures within the same TCR subclones intrinsically progressed over time ( Fig. A-H). Next, we designed a targeted scDNA-seq library within a custom panel covering 127 amplicons and 102 genes via Tapastri platform to validate these two patterns of clonal evolution during leukemia progression unambiguously. Likewise, we captured that in persisted subclones in T593 and T856 leukemic cells attained reproductive fitness from maintaining a mutational static status with remarkable transcriptomic variation from diagnosis to relapse. To further interpret the transcriptomic signature drift in the persisted TCR-defined major clones from T593 and T856 pairs, differential expression analysis was employed with RNA-binding protein Musashi-2 (MSI2) showing the most prominent differential expression pattern across drifted subclones catching our attention as a potential indicator of intrinsic clonal progression related to chemo-resistance. Comprehensive analysis of RNA-seq data from the CCCG-ALL-2015 cohort (n=72) and TARGET T-ALL cohort (n = 264) revealed that higher levels of MSI2 mRNA expression in pediatric T-ALL were correlated with higher measurable residual disease (MRD) after induction, and significant correlation between elevated MIS2 mRNA and resistance to prednisone, daunorubicin, and cytarabine were observed from ex vivo drug response assay. RNA immunoprecipitation and high-throughput mRNA sequencing from T-ALL cell lines detected a consistent significant enrichment of MYC transcript peaks in three T-ALL cell lines, indicating the molecular mechanism of MSI2 upregulation on MYC in the progression of T-ALL. Finally, In vitro and in vivo pharmacotyping analysis further validated the MSI2 inhibition significantly sensitized T-ALL to the chemotherapeutic drugs. Collectively, our study leveraging single-cell multi-omics profiling has provided a snapshot of the clonal architecture evolution that occurs from diagnosis to relapse in T-ALL, proving the coexistence of “clonal shift” and “clonal drift” in leukemic progression. Systemic analysis of drifted T-ALL clones has provided compelling evidence of the posttranscriptional regulatory effect of MSI2 on MYC and its potential correlation with leukemic reoccurrence, pinpointing MSI2 as a promising drug target of T-ALL in the future practice.

Integrin signaling is critical for myeloid-mediated support of T-cell acute lymphoblastic leukemia

We previously found that T-cell acute lymphoblastic leukemia (T-ALL) requires support from tumor-associated myeloid cells, which activate Insulin Like Growth Factor 1 Receptor (IGF1R) signaling in leukemic blasts. However, IGF1 is not sufficient to sustain T-ALL in vitro, implicating additional myeloid-mediated signals in leukemia progression. Here, we find that T-ALL cells require close contact with myeloid cells to survive. Transcriptional profiling and in vitro assays demonstrate that integrin-mediated cell adhesion activates downstream focal adhesion kinase (FAK)/ proline-rich tyrosine kinase 2 (PYK2), which are required for myeloid-mediated T-ALL support, partly through activation of IGF1R. Blocking integrin ligands or inhibiting FAK/PYK2 signaling diminishes leukemia burden in multiple organs and confers a survival advantage in a mouse model of T-ALL. Inhibiting integrin-mediated adhesion or FAK/PYK2 also reduces survival of primary patient T-ALL cells co-cultured with myeloid cells. Furthermore, elevated integrin pathway gene signatures correlate with higher FAK signaling and myeloid gene signatures and are associated with an inferior prognosis in pediatric T-ALL patients. Together, these findings demonstrate that integrin activation and downstream FAK/PYK2 signaling are important mechanisms underlying myeloid-mediated support of T-ALL progression.

CCR9 overexpression promotes T-ALL progression by enhancing cholesterol biosynthesis.

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy of the lymphoid progenitor cells, contributing to ∼ 20% of the total ALL cases, with a higher prevalence in adults than children. Despite the important role of human T-ALL cell lines in understanding the pathobiology of the disease, a detailed comparison of the tumorigenic potentials of two commonly used T-ALL cell lines, MOLT4 and JURKAT cells, is still lacking. Methodology: In the present study, NOD-Prkdc scid IL2rgd ull (NTG) mice were intravenously injected with MOLT4, JURKAT cells, and PBS as a control. The leukemiac cell homing/infiltration into the bone marrow, blood, liver and spleen was investigated for bioluminescence imaging, flow cytometry, and immunohistochemistry staining. Gene expression profiling of the two cell lines was performed via RNA-seq to identify the differentially expressed genes (DEGs). CCR9 identified as a DEG, was further screened for its role in invasion and metastasis in both cell lines in vitro. Moreover, a JURKAT cell line with overexpressed CCR9 (Jurkat-OeCCR9) was investigated for T-ALL formation in the NTG mice as compared to the GFP control. Jurkat-OeCCR9 cells were then subjected to transcriptome analysis to identify the genes and pathways associated with the upregulation of CCR9 leading to enhanced tumirogenesis. The DEGs of the CCR9-associated upregulation were validated both at mRNA and protein levels. Simvastatin was used to assess the effect of cholesterol biosynthesis inhibition on the aggressiveness of T-ALL cells. Results: Comparison of the leukemogenic potentials of the two T-ALL cell lines showed the relatively higher leukemogenic potential of MOLT4 cells, characterized by their enhanced tissue infiltration in NOD-PrkdcscidIL2rgdull (NTG) mice. Transcriptmoe analysis of the two cell lines revealed numerous DEGs, including CCR9, enriched in vital signaling pathways associated with growth and proliferation. Notably, the upregulation of CCR9 also promoted the tissue infiltration of JURKAT cells in vitro and in NTG mice. Transcriptome analysis revealed that CCR9 overexpression facilitated cholesterol production by upregulating the expression of the transcriptional factor SREBF2, and the downstream genes: MSMO1, MVD, HMGCS1, and HMGCR, which was then corroborated at the protein levels. Notably, simvastatin treatment reduced the migration of the CCR9-overexpressing JURKAT cells, suggesting the importance of cholesterol in T-ALL progression. Conclusions: This study highlights the distinct tumorigenic potentials of two T-ALL cell lines and reveals CCR9-regulated enhanced cholesterol biosynthesis in T-ALL.

ATF4 renders human T-cell acute lymphoblastic leukemia cell resistance to FGFR1 inhibitors through amino acid metabolic reprogramming.

Abnormalities of FGFR1 have been reported in multiple malignancies, suggesting FGFR1 as a potential target for precision treatment, but drug resistance remains a formidable obstacle. In this study, we explored whether FGFR1 acted a therapeutic target in human T-cell acute lymphoblastic leukemia (T-ALL) and the molecular mechanisms underlying T-ALL cell resistance to FGFR1 inhibitors. We showed that FGFR1 was significantly upregulated in human T-ALL and inversely correlated with the prognosis of patients. Knockdown of FGFR1 suppressed T-ALL growth and progression both in vitro and in vivo. However, the T-ALL cells were resistant to FGFR1 inhibitors AZD4547 and PD-166866 even though FGFR1 signaling was specifically inhibited in the early stage. Mechanistically, we found that FGFR1 inhibitors markedly increased the expression of ATF4, which was a major initiator for T-ALL resistance to FGFR1 inhibitors. We further revealed that FGFR1 inhibitors induced expression of ATF4 through enhancing chromatin accessibility combined with translational activation via the GCN2-eIF2α pathway. Subsequently, ATF4 remodeled the amino acid metabolism by stimulating the expression of multiple metabolic genes ASNS, ASS1, PHGDH and SLC1A5, maintaining the activation of mTORC1, which contributed to the drug resistance in T-ALL cells. Targeting FGFR1 and mTOR exhibited synergistically anti-leukemic efficacy. These results reveal that FGFR1 is a potential therapeutic target in human T-ALL, and ATF4-mediated amino acid metabolic reprogramming contributes to the FGFR1 inhibitor resistance. Synergistically inhibiting FGFR1 and mTOR can overcome this obstacle in T-ALL therapy.

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.

Withdrawal: Down-regulation of circRNA_0000143 regulates miR-142-3p/GRα axis to facilitate the progression of T-ALL.

Circular RNAs (circRNAs) regulate gene expression in eukaryotes and participate in the progression of malignancy. Instead, the function and underlying mechanisms of circ_0000143 in T-cell acute lymphoblastic leukemia (T-ALL) remain unclear. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was executed to probe circ_0000143 expression in bone marrow samples from T-ALL patients. T-ALL cell lines (MOLT4 and CCRF-CEM) were used to construct cell models. The biological functions of circ_0000143 were studied by CCK-8 assay, Transwell assay, flow cytometry and Western blotting in vitro. Bioinformatics analysis, dual-luciferase reporter gene assay and RIP assay were applied to predict and validate the interaction between circ_0000143 and miR-142-3p, as well as miR-142-3p and cyclic glucocorticoid receptor alpha (GRα). It was revealed that circ_0000143 expression level was significantly reduced in T-ALL samples compared with healthy control. Circ_0000143 overexpression suppressed the viability, migration and invasion, and induced apoptosis of T-ALL cell line. Furthermore, circ_0000143 acted as a molecular sponge to the expression of miR-142-3p. GRα was validated as a downstream target of miR-142-3p, and GRα was positively modulated by circ_0000143. Taken together, these findings reveal that circ_0000143 / miR-142-3p / GRα axis is implicated in the progression of T-ALL , which is expected to become a novel therapeutic target in T-ALL.

LncRNA15691 promotes T-ALL infiltration by upregulating CCR9 via increased MATR3 stability.

Our previous studies demonstrated that CCR9 plays an important role in several aspects of T-cell acute lymphoblastic leukemia progression and that CCR9 is a potential therapeutic target. However, the underlying mechanism that regulates CCR9 expression remains incompletely understood. In this study, bioinformatics analysis and validation in clinical samples revealed the lncRNA15691 to be positively correlated with CCR9 mRNA expression and significantly upregulated in T-cell acute lymphoblastic leukemia samples and CCR9high T-cell acute lymphoblastic leukemia cell lines. LncRNA15691, a previously uncharacterized lncRNA, was found to be located in both the cytoplasm and the nucleus via fluorescence in situ hybridization assay. In addition, lncRNA15691 upregulated the expression of CCR9 and was involved in T-cell acute lymphoblastic leukemia cell invasion. In vivo experiments showed that lncRNA15691 promoted leukemia cell homing/infiltration into the bone marrow, blood, and spleen, whereas the CCR9 ligand, CCL25, augmented the extramedullary infiltration of CCR9low leukemia cells overexpressing lncRNA15691 into blood, spleen, and liver. Subsequently, RNA protein pull-down assays, coupled with liquid chromatography-tandem mass spectrometry, were used to uncover potential lncRNA15691-interacting proteins, which were then validated by RNA immunoprecipitation. These mechanistic studies revealed that lncRNA15691 upregulated CCR9 expression via directly binding to and stabilizing MATR3 by inhibiting its nuclear degradation mediated by PKA. Collectively, our study revealed a novel mechanism of regulating CCR9 expression and implicated lncRNA15691 as a potential novel biomarker for T-cell acute lymphoblastic leukemia infiltration.

Notch Partners in the Long Journey of T-ALL Pathogenesis

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological disease that arises from the oncogenic transformation of developing T cells during T-lymphopoiesis. Although T-ALL prognosis has improved markedly in recent years, relapsing and refractory patients with dismal outcomes still represent a major clinical issue. Consequently, understanding the pathological mechanisms that lead to the appearance of this malignancy and developing novel and more effective targeted therapies is an urgent need. Since the discovery in 2004 that a major proportion of T-ALL patients carry activating mutations that turn NOTCH1 into an oncogene, great efforts have been made to decipher the mechanisms underlying constitutive NOTCH1 activation, with the aim of understanding how NOTCH1 dysregulation converts the physiological NOTCH1-dependent T-cell developmental program into a pathological T-cell transformation process. Several molecular players have so far been shown to cooperate with NOTCH1 in this oncogenic process, and different therapeutic strategies have been developed to specifically target NOTCH1-dependent T-ALLs. Here, we comprehensively analyze the molecular bases of the cross-talk between NOTCH1 and cooperating partners critically involved in the generation and/or maintenance and progression of T-ALL and discuss novel opportunities and therapeutic approaches that current knowledge may open for future treatment of T-ALL patients.

Semisynthetic aurones A14 protects against T-cell acute lymphoblastic leukemia via suppressing proliferation and inducing cell cycle arrest with apoptosis

BackgroundAcute lymphoblastic leukemia is an aggressive neoplasm and seriously threatens human health. A14 is one kind of semisynthetic aurone that exhibits the capability to inhibit prostate cancer, but little is known about the role of A14 on T-cell acute lymphoblastic leukemia.MethodsFirstly, the effects of A14 on the ability of leukemia cells to proliferate were measured by Vi-cell counter. Then, we detected the cell cycle and apoptosis by flow cytometry and characterized the related protein expression using immunoblotting. In addition, we constructed stable luciferase expressing cell lines for use in a cell derived xenograft mouse model to measure the effect of A14 on T-cell acute lymphoblastic leukemia.ResultsResults exhibited that A14 markedly suppressed cell proliferation and induced G2/M phase arrest along with cell cycles regulating proteins changes. A14 led to apoptosis in leukemia cells, at least partly, through the cytochrome c signaling pathway. Experiments in cell derived xenograft mouse model also showed that A14 markedly ameliorated the survival rate.ConclusionsThe present study revealed that semisynthetic aurones A14 can effectively protect against T-cell acute lymphoblastic leukemia progression both in vitro and in vivo, indicating the capability of A14 as a promising drug for the treatment of T-cell acute lymphoblastic leukemia.

Silencing of IRF8 Mediated by m6A Modification Promotes the Progression of T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a poor prognosis, urging for novel therapeutic targets and treatment strategies. N6-methyladenosine (m6A) is a crucial methylation modification that affects the pathogenesis of leukemia by regulating the mRNA of key genes. Interferon regulatory factor 8 (IRF8) is a crucial transcription factor for hematological lineage commitment, but its role in T-ALL is unclear. Here, IRF8 is shown to suppress T-ALL. The expression of IRF8 is abnormally silenced in patients with T-ALL. Knockout of Irf8 significantly hastens the progression of Notch1-induced T-ALL in vivo. Overexpression of IRF8 suppresses the proliferation and invasion of T-ALL cells by inhibiting the phosphatidylinositol 3-kinase/AKT signaling pathway. The fat mass- and obesity-associated protein (FTO), an m6A demethylase, is responsible for directly binding to m6A sites in 3' untranslated region of IRF8 messenger RNA (mRNA) and inducing mRNA degradation via m6A modification. Targeting the FTO-IRF8 axis is used as a proof of concept therapy; inhibition of FTO's demethylase activity drastically alleviates the proliferation of leukemic cells and prolongs the survival of T-ALL mice by restoring IRF8 expression. This study elucidates the pathogenesis of T-ALL from the perspective of epitranscriptomics and provides new insight into the genetic mechanisms and targeted therapy 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.

P338: BRANCHED CHAIN AMINO ACID TRANSAMINASE 1 ASSOCIATES WITH THE KU70/KU86 HETERODIMER AND MODULATES THE DNA DAMAGE RESPONSE IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a biologically and clinically heterogeneous disease mostly associated with NOTCH1 mutations that characterize over 60% of cases. Therapy-resistant or refractory T-ALL remains a major clinical challenge. Branched Chain Amino Acid Transferase 1 (BCAT1), a cytosolic aminotransferase converts BCAAs into their corresponding branched-chain α-keto acids and vice versa. Aberrant expression of BCAT1 has been demonstrated in different tumor models, including acute leukemia. However, its biological role in T-ALL remains to be elucidated. Aims: The purpose of this study was to dissect the functional role of BCAT1 in T-ALL, with particular emphasis on a putative link between BCAT1 and NOTCH1 in promoting T-ALL initiation and progression. Methods: We evaluated BCAT1 expression using RNAseq data from 264 T-ALL samples (ALL0434 protocol). BCAT1 transcript and protein levels were determined in T-ALL cell lines (n=13) and patient derived xenografts (PDX; n=12). Metabolomics analysis (using 13C6 labeled Leucine) was performed following NOTCH1 inhibition. Further, five PDX samples were treated with a γ secretase inhibitor (DBZ) to dampen NOTCH signaling. BCAT1 promoter occupancy by NOTCH1 was determined by Chromatin Immunoprecipitation (ChIP) coupled with qPCR. Tandem affinity purification (TAP) and mass spectrometry (MS) analysis were used to identify BCAT1 interacting proteins from CUTLL1 T-ALL cells. Kinetics of the DNA damage response (DDR) was evaluated in BCAT1 knock-down cells. Drug combination experiments using the DNA damaging agent (etoposide) and a specific BCAT inhibitor (ERG245) were performed in vitro and in vivo in PDX T-ALL models. Results: We found variable BCAT1 expression levels amongst the different T-ALL molecular subgroups, with higher expression levels in TLX1 (p<0.001), HOXA-TLX3 (p<0.05) and NKX2-1 (p<0.01) subtypes. Considering recurrent genetic alterations, we found BCAT1 expression to be significantly higher (p<0.01) in NOTCH1 mutated cases compared to un-mutated samples. Interestingly, NOTCH1 mutated PDX samples also showed higher BCAT1 expression compared to un-mutated cases. Metabolic studies disclosed that BCAAs oxidation is decreased following NOTCH1 inhibition (reduction in 3-methyl-2 oxovaleric acid). Further, inhibition of NOTCH1 signaling with DBZ decreased BCAT1 expression in numerous T-ALL models. ChIP analysis and luciferase reporter assays suggest that NOTCH1 may directly regulate BCAT1 expression. Functionally, TAP followed by MS analysis of BCAT1 interacting proteins disclosed that BCAT1 may be implicated in non-metabolic processes such as DNA replication and repair and rRNA processing. Indeed, we found Ku70/Ku86 proteins to interact with BCAT1. Evaluation of the DDR following DNA damage, disclosed that BCAT1 deficient cells present an accentuated but defective DDR which translates into marked DNA damage leading to pronounced cell death. Consistently, a marked synergistic therapeutic response was found between the DNA damaging drug etoposide and the BCAT specific inhibitor ERG245 in numerous T-ALL models. Summary/Conclusion: NOTCH1 may directly regulate BCAT1 expression in T-ALL cells. BCAT1 is involved in sustaining genomic integrity following DNA damage through its interaction with the Ku70/Ku86 heterodimer, known to mediate classical non-homologous end-joining. Our results identify BCAT1 as a novel therapeutic target and suggest that the combination between DNA double stand break inducing agents (such as etoposide) and a BCAT inhibitor could be particularly useful in NOTCH1-mutant T-ALL cases.

Abstract LB015: CCR7-expressing T-cell acute lymphoblastic leukemia mouse model provides a platform for developing novel anti-CCR7 therapies to prevent CNS invasion

Abstract C-C chemokine receptor 7 (CCR7) plays a pivotal role in promoting the migration of T-cell acute lymphoblastic leukemia (T-ALL) cells into the central nervous system during leukemogenesis. Overexpression of CCR7 has also been linked to developing central nervous system (CNS) lymphoma. CCR7 and its ligand, CCL19 are important mediators in the development of these cancers. Thus there is an urgent need for a translational syngeneic CCR7-expressing T-ALL model that can speed up the discovery of drugs that can be used to block CCR7 function during the progression of T-ALL. Using the established ROSA26 floxed-stop Prdm14 (R26PR)/Mx-1 Cre mouse T-ALL model we have initiated studies using a CCR7/CCL19 antagonist, ELC8-83 to examine the molecular mechanisms that mediate CCR7-directed migration to, invasion of, proliferation within and exit from the CNS. mRNA expression of CCR7 in the T-ALL was confirmed by RT-PCR and protein expression on the cell surface was confirmed by flow cytometry. Chemotaxis and calcium mobilization of T-ALL in response to CCL19 was confirmed using transwell-migration assays and FURA-4. ELC8-83 was used to block this signaling. These studies demonstrate the efficacy of ELC8-83 in blocking signaling in T-ALL and provide a platform for testing ELC8-83 in the mouse model of T-ALL. Citation Format: Angel Torres, Aaron Vazquez, Sean Glenn, Zenaida Fuentes, jessica Chacon, Colin Bill, Charlotte M. Vines. CCR7-expressing T-cell acute lymphoblastic leukemia mouse model provides a platform for developing novel anti-CCR7 therapies to prevent CNS invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB015.

Abstract 825: Evasion of apoptosis in MYC dependent T-ALL through epigenetic control

Abstract Cancer is a complex landscape of aberrant cell signaling programs, often initiated by oncogenes. The causal oncogene drives the preponderance of accumulated mutations that enable tumorigenesis. Cancers arising this way can be reliant on the initiating oncogene, a term known as oncogene addiction. The proto-oncogene c-MYC is a transcription factor that regulates much of the genome and is deregulated in many cancers. The transgenic Eµ-tTA/Tet-O-MYC mouse model of T-cell acute lymphoblastic leukemia (T-ALL), allows for modulation of c-MYC expression (ON vs OFF). When c-MYC is expressed, T-ALL progression is observed; and when c-MYC expression is revoked, tumor regression occurs. Using cells and microarray data from this model we employed a nested effects model (NEM) that infers hierarchical relationships anchored to master transcription factors that govern critical aspects of cell biology. We identified a critical node governed by a class of histone demethylases influencing transcriptional availability across the genome. KDM5B/JARID1b is known as a transcriptional repressor, and is downregulated in the T-ALL model when c-MYC is overexpressed. Using CRISPR/Cas9 mediated mutagenesis to disrupt KDM5B expression, we observed a potent reduction in cell death when c-MYC expression is abrogated; suggesting KDM5B mediates cell death responses in T-ALL. KDM5B is downregulated in the primary murine model when c-MYC is overexpressed, and upregulated concordantly with the loss of MYC expression. Human lymphoma cell lines that are sensitive to the bromodomain inhibitor JQ1 show downregulation of c-MYC and KDM5B upregulation. Xenografts of KDM5B knockout cells in NOD-SCIDIL-2Rg-/- (NSG) mice show increased tumor progression and burden, and more aggressive time to relapse after c-MYC expression is revoked. Future studies examining the KDM5B/c-MYC axis and its influence on chromatin architecture are ongoing. These studies aim to demonstrate a novel paradigm in understanding how c-MYC promotes tumor development through repression of a tumor suppressive epigenetic landscape regulated by KDM5B and identify therapeutic options for treating c-MYC-induced T-ALL. Citation Format: Gabrielle Dewson, Benedict Anchang, Rosalie Sears, Daniel F. Liefwalker. Evasion of apoptosis in MYC dependent T-ALL through epigenetic control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 825.

CD147-specific chimeric antigen receptor T cells effectively inhibit T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is invasive and heterogeneous, and existing therapies are sometimes unsuccessful. Chimeric antigen receptor (CAR) T cell therapy is a breakthrough tumor treatment method, particularly for B cell acute lymphoblastic leukemia. We found that CD147 was highly expressed in tumor T cells of T-ALL patients and T cell lymphoma. Therefore, CD147-CAR T cells that contain a humanized single-chain variable fragment targeting human CD147 and a second-generation CAR frame were constructed for treating T-ALL. CD147-CAR T cells were able to maintain a healthy proliferation rate, preserving a subset of CD62L+/CCR7+ memory T cells. CD147-CAR T cells showed a potent anti-tumor activity against human T-ALL cell line and T-ALL blasts, releasing high level of cytokines in the process. However, CD147-CAR T cells exhibited potential safety toward human normal cells and CD147-deficent cells. NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mice were used to establish a T-ALL xenograft model and CD147-CAR T cells conferred robust protection against T-ALL progression and significantly improved survival in mice. Overall, we found that CD147 is a potential antigen target of CAR T cell therapy for T-ALL.